General Biochemistry MODULE

Biochemistry

1
Notes
GENERAL
BIOCHEMISTRY

1.1 INTRODUCTION
Solutions of chemical reagents are a big part of biochemistry, biological and
chemical based work. For a beginner of experimental procedure making
solutions can also be the most frustrating part. Preparation and handling
solutions are essential part of experimental biochemistry. Thus any of new
science graduates should be clear in preparing reagents, buffers, and accuracy
in pipeting.

The concentration of a dissolved salt in water refers to the amount of salt (solute)
that is dissolved in water (solvent). Solutes are the substance of interest to be
dissolved and the term solvent denotes the material in which the solute is
dissolved.

Solution is a mixture that contains solute and a solvent. Solute can be denoted
as the component of a solution present in the lesser amount and the solvent is
the component of a solution present in the greater amount. Concentration can
be written as the amount of a solute present in a solution per amount of solvent.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the importance of solution preparation in biochemistry
z explain different concentration units
z describe different terms of percent solutions

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Biochemistry
1.2 UNITS OF CONCENTRATION
z There are many ways to express concentrations. Concentration may be
expressed several different ways and some of the more common concentration
units are:
1. Equivalent weight
2. Molarity
Notes 3. Molality
4. Normality
5. Percent solution (weight/weight)
6. Percent solution (weight/volume)
7. Percent solution (volume/volume)

1.2.1 Equivalent Weight

The equivalent weight is determined by dividing the atomic or molecular weight
by the valence. A major use of the concept of equivalents is that one equivalent
of an ion or molecule is chemically equivalent to one equivalent of a different
ion or molecule. The mass of a substance especially in grams is chemically
equivalent to eight grams of oxygen or one gram of hydrogen : the atomic or
molecular weight divided by the valence
Valance could be determined as
1. The absolute value of ion charge
2. The number of H+ or OH– that a species can react with
3. The absolute value of change in charge on a species when undergoing a
chemical reaction.

1.2.1.1 Preparation of NaOH

Solutions of NaOH can be prepared by either dissolving solid NaOH pellets in
water or by diluting a concentrated solution of NaOH. However, the exact
concentration of the solution prepared by these methods cannot be calculated
from the weighed mass or using the dilution equation for two reasons:
1. Solid sodium hydroxide is hygroscopic (“water-loving”). Pellets of NaOH
exposed to air will increase in mass as they become hydrated so the actual
mass of pure NaOH is not accurately known.
2. Sodium hydroxide in solution reacts with carbonic acid and its concentration
decreases over time. The acid is formed when small amounts of CO2 gas
(which is always present in air) dissolves in solution.

ZZX H2O + Na+(aq) + HCO3–(aq)

H2CO3(aq) + NaOH(aq) YZZ

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The water used to make the NaOH solution can be boiled to expel the dissolved Biochemistry
CO2 gas but this time-consuming procedure is often not possible in a short
laboratory period. A stock solution of NaOH can be made in advance with boiled
water but will re-absorb CO2 over a period of time unless stored in airtight
containers. Therefore, if we want to know the exact concentration of a freshly
made NaOH solution, we need to “standardize” it. That is, determine its exact
concentration by titrating it with a known mass of a primary standard acid.
A “primary standard” is a substance that is used to determine the concentration Notes
of a solution. A primary standard should have the following properties: It should
be available in very pure form at reasonable cost and should have a high
equivalent weight to minimize weighing errors. It should be stable at room
temperature, easy to dry, and should not easily absorb water when exposed to
air (hygrophobic).
Potassium hydrogen phthalate (“KHP”) is the primary standard reagent commonly
used to standardize NaOH. It is a monoprotic acid whose formula is KHC8H4O4
and molecular weight is 204.22 g/mol.
KHC8H4O4(aq) + NaOH(aq) ⎯→ H2O + Na+(aq) + K+(aq) + C8H4O4(aq)
The white powdery acid is normally heated at 110°C for one hour to remove any
loosely bound waters of hydration and then cooled in a desiccator before use.
The exact mass (and number of moles of acid) is determined by weighing the
dried acid on an analytical balance. The acid is then dissolved in water and NaOH
is added until an endpoint (the point at which an indicator changes color) is
reached. The phenophthalein indicator used in this experiment is colorless in
acid and pink in base. Therefore, the solution containing KHP will remain
colorless as long as some KHP is still present. Once the last of the KHP has
reacted, the solution will turn pink with one excess drop of base.
The exact concentration of NaOH is calculated by using the stoichiometry from
reaction to convert the number of moles of KHP used to moles of NaOH and
then dividing by the volume of NaOH used to reach the endpoint of the reaction.

1.2.1.2 Equivalent Weight of an Acid

In an acid base titration, the equivalence point is the volume of added base where
the moles of –OH added (from the base) equal the moles of H+ initially present
(from the acid). (i.e) moles of H+ initially present = moles –OH added (at
equivalence point)
To approximate the equivalence point, an indicator with an endpoint close to the
equivalence point is added to the analyte solution. A balanced equation can be
written describing the chemical reaction occurring between the titrant (the base
in this experiment) and analyte (the acid in this experiment) if the identity of
both is known.

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Biochemistry For example, the titration of hydrochloric acid with potassium hydroxide can be
written:
HCl(aq) + KOH(aq) ⎯→ H2O + KCl(aq)
In the case if the identity of the acid is unknown, but the number of acidic
hydrogens (H+) carried by the acid is known, a balanced equation can still be
written.
Notes For example, the titration of a triprotic acid (an acid with 3 H+) with sodium
hydroxide can be written:
H3X(aq) + 3KOH(aq) ⎯→ 3H2O(l) + K3X(aq)
(X : the unknown anion of the acid)
The formula weight of this unknown acid can be calculated by using dimensional
analysis. First, the base’s concentration is used to convert the base’s volume at
the endpoint to moles. Then, multiplying by the mole ratio between acid and base
from the balanced chemical equation allows for the calculation of the moles of
the acid. Now the mass of acid titrated must be divided by the moles of acid
calculated giving a result with the units of g/mol.

1.2.1.3 Equivalent Weight of an Oxidizing Agent

The concept of equivalents and equivalent mass is not restricted to acid-base
reactions alone. Unlike acid-base reactions in redox reactions, the electrons are
the active units (the equivalents) and the equivalent weights are the masses of
oxidizing or reducing agent that deliver or accept 1 mole of electrons. But in
case of acid and base the hydrogen or hydroxide ions plays key role in
determination of equivalent weight.

INTEXT QUESTIONS 1.1

1 The equivalent weight is determined by dividing the atomic or molecular
weight by the ...................
2 The mass of a substance especially in grams is chemically equivalent to
................... grams of oxygen
(a) 2 (b) 5 (c) 8 (d) 10
3 ................... is the primary standard reagent commonly used to standardize
NaOH3 Molarity

1.2.2 Molarity
Molarity is based on the volume of solution containing the solute. Since density
is a temperature dependent property a solution’s volume, and thus its molar

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concentration, changes with temperature. By using the solvent’s mass in place Biochemistry
of the solution’s volume, the resulting concentration becomes independent of
temperature.
Molarity is the common way of referring to concentrations of solutions. The goal
of most basic molarity problems shall be to get the moles from grams by dividing
the molecular weight and then dividing by the total number of liters or by given
the molarity find the number of grams of the solution by multiplying the volume Notes
then the molecular weight. Molarity might give you the density of the solution,
from which you can obtain the mass by multiplying the density by the volume.
Although there are several ways in which the concentration of a solution can be
quantified, molarity is one of the most basic and widely used. Molarity (M) is
defined as the number of moles of solute dissolved in one liter of solution. The
higher the molarity, the more concentrated or strong the solution is. For example,
a 12 M (which is said “twelve molar”) solution of HCl (ie. hydrochloric acid)
is much more concentrated than a 0.10 M solution! The basic formula for
calculating molarity is:
Molarity (M) = moles of solute (mol) per liters of solution (L)
To solve for moles of solvent, we can use algebra to manipulate the above
equation producing the following derived formulas:
moles of solute (mol) = Molarity (M) × liters of solution (L)
In simple terms, the following formula could be used for preparation of molar
solutions for lab solutions preparation
For preparation of molar solution

Molecular weight of the compound (A)

1000
× Required morality (B) × Required volume of solution (C) = D gram
In the above equation for preparation of solution of ‘B’ molarity, ‘D’ grams of
the solute could be dissolved in ‘C’ ml of solvent.

INTEXT QUESTIONS 1.2

1. .................... is based on the volume of solution containing the solute
(a) Normality (b) Molarity (c) Molality (d) Percent
2. Molarity is the common way of referring to .................... of solutions

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Biochemistry 3. .................... is defined as the number of moles of solute dissolved in one

liter of solution
4. The higher the ...................., the more concentration of the solution

1.2.3 Molality
The molal unit is not used nearly as frequently as the molar unit and is used in
Notes thermodynamic calculations where a temperature independent unit of
concentration is needed. A molality is the number of moles of solute dissolved
in one kilogram of solvent. The term molality and molarity should not be
confused. While expressing the Molality it is represented by a small “m,”
whereas molarity is represented by an upper case “M.”
In case of preparation of molar solution except water all other solvent must be
weighed. The water is exempted from weighing because; one liter of water has
a specific gravity of 1.0 and weighs one kilogram. So one can measure out one
liter of water and the solute could be directly added to it. But other solvents might
have a specific gravity greater than or less than one. Therefore, one liter of any
solvent other than water is not likely to occupy a liter of space.
For example to make a one molal aqueous (water) solution of sodium chloride
(NaCl), measure out one kilogram of water and add one mole of the solute, NaCl
to it. The atomic weight of sodium is 23 and the atomic weight of chlorine is
35. Therefore the formula weight for NaCl is 58. So 58 grams of NaCl could
be dissolved in 1kg water for preparation of 1 molal solution of NaCl.

INTEXT QUESTIONS 1.3

1. .................. is the unit used in thermodynamic calculations where a
temperature independent unit of concentration is needed.
2. A molality is the number of moles of solute dissolved in ..................
kilogram of solvent
(a) 3 (b) 2 (c) 1 (d) 6
3. Molality it is represented by a ..................
(a) Small ‘m’ (b) Capital ‘M’ (c) Small ‘n’ (d) Capital ‘N’

1.2.4 Normality
The concentration of a solution could also be expressed in terms of Normality.
It is based on an alternate chemical unit of mass called the equivalent weight.
The normality of a solution is the concentration expressed as the number of

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equivalent weights (equivalents) of solute per liter of solution. In a chemical Biochemistry
mixture 1 normal (1 N) solution contains 1 equivalent weight of solute per liter
of solution. Since normality simplifies the calculations required for chemical
concentration, it is widely used in analytical chemistry.
Every substance may be assigned an equivalent weight. The equivalent weight
may be equal to the formula weight (molecular weight, mole weight) of the
substance or equal to an integral fraction of the formula weight (i.e., molecular Notes
weight divided by 2, 3, 4, and so on).
The above phenomenon could be better explained with the following example
to gain an understanding of the meaning of equivalent weight:
HCl(aq) + NaOH(aq) ⎯→ NaCl(aq) + H2O
1 mole 1 mole
(36.5 grams) (40.0 grams)
H2SO4(aq) + 2NaOH(aq) ⎯→ Na2SO4(aq) + 2H2O
1 mole 1 mole
(98.1 grams) (80.0 grams)
In the above chemical reaction 1 mole of hydrochloric acid (HCl) reacts with
1 mole of sodium hydroxide (NaOH) and 1 mole of sulfuric acid (H2SO4) reacts
with 2 moles of NaOH. If you made 1 molar solutions of these substances, 1
liter of 1 M HCl will react with 1 liter of 1 M NaOH and 1 liter of 1 M H2SO4
will react with 2 liters of 1 M NaOH. Therefore, H2SO4 has twice the chemical
capacity of HCl when reacting with NaOH. The equivalent weight of HCl is
equal to its molecular weight, but that of H2SO4 is ½ its molecular weight.
Expressions for normality are shown below. Notice the similarity to molar
solution definition.

Number of equivalents of solute Equivalents

Normality (N) = =
1 liter of solution liter
grams of solute
where Number of equivalents of solute =
equivalent weight of solute

grams of solute grams

finally N = =
eq wt solute × L solution eq wt × L
So, 1 liter of solution containing 36.5 grams of HCl would be 1 N, and 1 liter
of solution containing 49.0 grams of H2SO4 would also be 1 N. A solution
containing 98.1 grams of H2SO4 (1 mole) per liter would be 2 N.

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Biochemistry

INTEXT QUESTIONS 1.4

1. ...................... based on an alternate chemical unit of mass called the
equivalent weight
(a) Normality (b) Molarity (c) Percent (d) Molality
Notes 2. One mole of ...................... reacts with 1 mole of sodium hydroxide (NaOH)
3. One mole of sulfuric acid (H2SO4) reacts with ...................... moles of
NaOH
4. The equivalent weight of HCl is equal to its ......................

1.2.5 Percentage Solutions

The percentage solution could be expressed in terms of weight percent (% w/
w), volume percent (% v/v) and weight-to-volume percent (% w/v) units of
solute present in 100 units of solution. For example a solution of 1.5% w/v
NH4NO3, contains 1.5 gram of NH4NO3 in 100 mL of solution.

1.2.5.1 Percent by weight (% w/w)

In case of preparing a solution based on percentage by weight, one would simply
determine what percentage was required (for example, a 20% by weight aqueous
solution of sodium chloride) and the total quantity to be prepared.
If the total quantity needed is 1 kg, then it would simply be a matter of calculating
20% of 1 kg which, of course is:
20 /100 * 1000 g/kg = 200 g NaCl/kg.
Thus finally to bring the total quantity to 1 kg, it would be necessary to add 800g
water.

1.2.5.2 Percent by volume (% w/v)

Preparation of solutions based on percent by volume it requires the calculation
same as for percent by weight, except that calculations are based on volume. In
simple terms one should plan that what percentage was desired (for example,
a 20% by volume aqueous solution of sodium chloride) and the total quantity
to be prepared in terms of volume.
For example if 20 % is to be prepared for a total quantity of 1 liter, then it would
simply be a matter of calculating 20% of NaCl in 1 liter, the formula can be
written as:
20/100 * 1000 ml/l = 200 g NaCl/l.

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1.2.5.3 Percent by volume (% v/v) Biochemistry

Volume percent or volume/volume percent most often is used when preparing

solutions of liquids. This is typically only used for mixtures of liquids. Volume
percent is relative to volume of solution, not volume of solvent. The advantage
of volume/volume units is that gaseous concentrations reported in these units
do not change as a gas is compressed or expanded.
For example, 70% v/v rubbing alcohol may be prepared by taking 700 ml of Notes
isopropyl alcohol and adding sufficient water to obtain 1000 ml of solution
(which will not be 300 ml).
Table 1.1 Common Units for Reporting Concentration
Sl.No Name Units Symbol
1. Molarity moles solute M
liters solution
2. Normality equivalents solute N
liters solution
3. Molality moles solute m
kilograms solvent
4. Weight percent grams solute % w/w
100 grams solution
5. Volume percent mL solute % v/v
100 mL solution
6. weight-to-volume percent grams solute % w/v
100 mL solution

INTEXT QUESTIONS 1.5

1. Percent by weight could be expressed as ................
2. For preparation of 20 % NaCl by (w/v) ................ grams of NaCl is to be
dissolved in 1 L of water.
(a) 200 (b) 100 (c) 400 (d) 50
3. volume/volume percent most often is used when preparing solutions of
................
4. Match the following:
(i) Molarity (a) N
(ii) Normality (b) m
(iii) Molality (c) M

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Biochemistry

WHAT HAVE YOU LEARNT

z Preparation solutions of chemical reagents are very important task for the
beginners of biochemistry.
z Concentration of a biological solution could be expressed in terms of
equivalent weight, normality, molarity, molality, percent solution (weight/
Notes
weight, weight/ volume, volume /volume).
z The equivalent weight is determined by dividing the atomic or molecular
weight by the valence.
z Molarity could be simply termed as “M” and are units of moles of solute
per liter of solution.
z Molality is units of moles of solute per kilogram of solution and is termed
as “m”. While the normality is termed as “N” and are units of equivalent
of solute per liter of solution.
z The percentage solution could be expressed in terms of weight percent (%
w/w), volume percent (% v/v) and weight-to-volume percent (% w/v) units
of solute present in 100 units of solution.

TERMINAL QUESTIONS
1. Write a brief note about the importance of solution preparation in
biochemistry.
2. Write a note on Equivalent weight.
3. Describe about molarity and molality.
4. Write a short note on normality.
5. Write about different terms of percent solutions.
6. Tabulate the units and symbols of different modes of concentrations.

ANSWERS TO INTEXT QUESTIONS

1.1
1. Valence
2. 8
3. Potassium hydrogen phthalate

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1.2 Biochemistry

1. Molarity
2. Concentrations
3. Molarity
4. Molarity
Notes
1.3
1. Molality
2. 1
3. Small ‘n’

1.4
1. Normality
2. Hydrochloric acid
3. Two
4. Molecular weight

1.5
1. (% w/w)
2. 200
3. Liquids
4. Match the following:
(i) (b)
(ii) (a)
(iii) (c)

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 MODULE Carbohydrate

Biochemistry

2
Notes
CARBOHYDRATES

2.1 INTRODUCTION
A carbohydrate is a large biological molecule, or macromolecule, consisting
only of carbon (C), hydrogen (H), and oxygen (O), usually with a
hydrogen:oxygen atom ratio of 2:1. Carbohydrates are technically hydrates of
carbon, structurally it is more accurate to view them as polyhydroxy
aldehydes and ketones.

OBJECTIVES
After reading this lesson, you will be able to:

z define carbohydrates

z classify Common Carbohydrates

z describe the sources and composition of carbohydrates

z explain the Digestion and absorption of carbohydrates.

2.2 DEFINITION
Carbohydrates are polyhydroxy aldehydes or ketones, or compounds that can be
hydrolyzed to them. Carbohydrate is an organic compound comprising
only carbon, hydrogen, and oxygen, usually with a hydrogen: oxygen atom ratio
of 2:1 (as in water). Carbohydrates are technically hydrates of carbon.
The empirical formula is Cn(H2O)n .

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Biochemistry
2.3 SOURCES OF CARBOHYDRATES
Baked goods commonly contain dietary starch and added sugar. Most dietary
carbohydrates come from plants. Sugars and starches are nutritive carbohydrates,
meaning they are broken down and utilized by the body, primarily to generate
energy. Although dietary fiber is also a carbohydrate, it contributes no calories
because it is not digested or absorbed.
Notes
2.3.1 Grain Products
Grain products are the leading source of carbohydrates in the diet. Grains
naturally contain high concentrations of starch, which our gastrointestinal
system breaks down into sugars. Common grains in diet include wheat, oats,
rice, barley and cornmeal. Any food that includes grain or grain flour as a
primary ingredient contains carbohydrates, such as bread and other baked goods,
pasta, cereal, crackers. Choosing whole-grain products instead of those made
from refined grains boosts your dietary fiber intake, which supports your heart
and digestive health.

2.3.2 Starchy Vegetables and Beans

Beans and starchy vegetables, such as potatoes, yams, green peas, and corn,
contain high levels of complex carbohydrates that our body digests into sugars.
In addition, starchy vegetables and beans contribute vitamins, minerals and fiber
to our diet. Dry beans also serve as a good source of lean dietary protein.

2.3.3 Fruits
All fruit and fruit juices contain carbohydrates in the form of natural sugars, such
as glucose and fructose. Fruit sugars contribute nearly all of the calories
contained in these foods. With persistently low consumption rates, fruit
contributes less than 8 percent of the average daily calories in the diet. Fresh fruit
is a healthier option than fruit juice because it provides more dietary fiber and
less carbohydrate by volume.

2.3.4 Beverages
Dairy milk is the only significant source of dietary carbohydrates not derived
from plants. A cup of unflavored milk contains about 11 to 12 grams of
carbohydrate in the form of milk sugar, or lactose. Chocolate milk contains more
than twice the amount of carbohydrate per cup compared to plain milk because
sugar is added to sweeten the flavor. Sugar-sweetened soda, fruit drinks and
sports and energy drinks substantially contribute to dietary carbohydrate intake.
Wine, beer and liqueurs also contain carbohydrates. Dessert wines typically
contain roughly four times the amount of carbohydrates found in table wines.

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Biochemistry 2.3.5 Sweets and added Sugars

Eating candy and desserts markedly boosts the number of carbohydrates in our
diet. Indulging in a 1.6-ounce milk chocolate bar adds more than 26 grams of
carbohydrates to our daily intake; a slice of cherry pie adds approximately 47
to 69 grams. Sugar added to processed foods that you may not consider sweet
can be an unrecognized source of carbohydrates in our diet. Commercial pasta
Notes sauces, salad dressings, sandwich bread, energy and nutrition bars, cereals, heat-
and-eat meals and other convenience foods commonly contain high-fructose
corn syrup or another form of sugar for added flavor. Opting for whole, fresh
foods rather than processed foods helps us avoid hidden carbohydrates.

2.4 CLASSIFICATION
Carbohydrates are classified into following classes depending upon whether
these undergo hydrolysis and if so on the number of products form:

Monosaccharides, Disaccharides, Trisaccharides, Oligosaccharides, Polysaccha-

rides

2.4.1 Monosaccharides
Molecules having only one actual or potential sugar group are called
monosaccharides. They are simple carbohydrates that cannot be hydrolyzed
further into polyhydroxy aldehydes or ketone unit

Sugars having aldehyde group are called aldoses and sugars with keto group are
called ketoses. Depending on the number of carbon atoms monosaccharides are
named as triose (C3), tetrose (C4), pentose (C5), hexose (C6), heptose (C7) and so
on.

Monosaccharide classifications based on the number of carbons

Number of Category Examples

Carbons Name
4 Tetrose Erythrose, Threose
5 Pentose Arabinose, Ribose, Ribulose, Xylose,
Xylulose, Lyxose
6 Hexose Allose, Altrose, Fructose, Galactose,
Glucose, Gulose, Idose, Mannose,
Sorbose, Talose, Tagatose
7 Heptose Sedoheptulose, Mannoheptulose

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Biochemistry

INTEXT QUESTIONS 2.1

1. Carbohydrate consists of ...................., .................... & .................... molecule
2. Hydrogen Oxygen atom ratio in Carbohydrate is ....................
3. Molecules having only one actual sugar group is called as ....................
4. Sugars having aldehyde group is called as .................... Notes
5. Sugars with keto group is called as ....................
6. Match the following
1. Monosaccharides with six carbons (a) Tetrose
2. Monosaccharides with four carbons (b) Heptose
3. Monosaccharides with five carbons (c) Hexose
4. Monosaccharides with seven carbons (d) Pentose
7. Match the following
1. Hexose (a) Erythrose
2. Heptose (b) Ribose
3. Tetrose (c) Mannoheptulose
4. Pentose (d) Fructose

2.4.2 Sterioisomers
Compounds having same structural formula but differing in spatial configuration
are known as sterioisomers. While writing the molecular formula of
monosaccharides, the spatial arrangements of H and OH groups are important,
since they contain asymmetric carbon atoms. Asymmetric carbon means four
different groups are attached to the same carbon. The reference molecule is
glyceraldehyde which has a single asymmetric carbon.

H O H O
C C

H C OH HO C H
D (+) L (–)
CH2OH CH2OH

glyceraldehyde

The number of possible sterioisomers depends on the number of asymmetric

carbon atoms by the formula 2n where n is the number of asymmetric carbon
atoms

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Biochemistry Reference carbon atom of sugars

The configuration of H and OH at the second carbon of glyceraldehyde will form
two mirror images, they are denoted as D and L varieties. All monosaccharides
are molecules derived from glyceraldehyde by successive addition of carbon
atoms. Therefore the penultimate carbon atom is the reference carbon atom for
naming the mirror images. This is also referred to as absolute configuration.

Notes Optical activity

The presence of asymmetrical carbon atom causes optical activity. When a beam
of plane polarized light is passed through a solution of carbohydrates, it will
rotate the light either to right or to left. Depending on the rotation, molecules
are called dextrorotatory (+) (d) or levorotatory (–).

Racemic mixture
Equimolecular mixture of optical isomers has no net rotation and hence it is
called as racemic mixture.

Epimers
When sugars are different from one another, only in configuration with regard
to the single carbon atom, other than the reference carbon atom, they are called
Epimers. Example Glucose and Mannose are an epimeric pair which differ only
with respect to C2. Similarly galactose is the fourth epimer of glucose
H 1 O H O H O
C C C
2
H C OH HO C H H C OH
3
HO C H HO C H HO C H
4
H C OH H C OH HO C H
5
H C OH H C OH H C OH
6
CH2OH CH2OH CH2OH
D-Glucose D-Mannose D-Galactose

Anomers
When D glucose is crystallized at room temperature and a fresh solution is
prepared, its specific rotation of polarized light is +112°; but after 12-18 hours
it changes to +52.5°. If initial crystallization is taking place at 98° C and then
solubilized, the specific rotation is found to be +19°, which also changes
to+52.5° within few hours. This change in rotation with time is called
mutarotation.
This is explained by the fact that D-glucose has two anomers, alpha and beta
varieties. These anomers are produced by the spatial configuration with
reference to the first carbon atom in aldoses and second carbon in ketoses. Hence

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these carbons are called anomeric carbon atoms. Thus α-D-glucose has specific Biochemistry
rotation of +112° and β=D-glucose has specific rotation of +19°. Both undergo
mutarotation and at equilibrium one-third molecules are alpha type and two-third
are beta variety to get the specific rotation +52.5°
H 1 O
C
2 6 CH OH CH2OH
H C OH 2 CHO
3 5 O O Notes
HO C H H H H H H OH H OH
4 4 1 HO H
H C OH
5 HO OH H OH HO OH H H H OH
H C OH
H OH
6 3 2
CH2OH H OH H OH CH2OH
D-Glucose α-D-Glucose β-D-Glucose Cyclation of Glucose

2.5 REACTIONS OF MONOSACCHARIDES

2.5.1 Enediol formation
In mild alkaline solutions, carbohydrates containing free sugar group (aldehyde
or keto group) will tautomerise to form enediols, where two hydroxyl groups
are attached to the double-bonded carbon, in mild alkaline conditions, glucose
is converted into fructose and mannose.
H
C O HO C H CH2OH
H C OH H C OH C O
HO C H HO C H HO C H
H C OH H C OH H C OH
H C OH H C OH H C OH
CH2OH CH2OH CH2OH
aldose enediol ketose
D-Glucose D-fructose

Benedict’s Reaction
Benedict’s reagent contains sodium carbonate, copper sulphate and sodium
citrate. In alkaline medium, sugars form enediols which will reduce cupric ions
and correspondingly the sugar is oxidized. Any sugar with free aldehyde or keto
group will reduce benedict’s reagent and called as reducing sugars.
H O –O O
C
+ Cu(citrate)22– C
+ Cu2O(s)
R
R
An aldose Benedict¢s reagent Carboxylate Brick-red
(blue solution) anion precipitate

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Biochemistry Osazone Formation

All reducing sugars will form osazone with excess of phenylhydrazine when
kept at boiling temperature. Osazones are insoluable. Each sugar will have
characteristic crystal form of osazone

CHO CH NNHC6H5
C6H5NHNH2
H C OH HCL
H C OH
Notes
(CHOH)4 (CHOH)4
CH2OH CH2OH
D-glucose D-glucose phenyl hydrazone

2 C6H5NHNH2

CH NNHC6H5
NH3 + C6H5NH2 + C NNHC6H5
(CHOH)4
CH2OH
D-Clucosazone

Oxidation of Sugars
Under mild oxidation conditions the aldehyde group is oxidized to carboxyl
group to produce aldonic acid. Ex: Glucose to Gluconic acid
When aldehyde group is protected and the molecule is oxidized the last carbon
becomes COOH group to produce uronic acid. Ex: Glucose to Glucuronic acid.

COOH CHO
H C OH H C OH
HO C H HO C H
H C OH H C OH
H C OH H C OH
CH2OH COOH
D-gluconic acid D-glucuronic acid

Under strong oxidation conditions the first and last carbon atoms are
simultaneously oxidized to form dicarboxyllic acids, known as saccharic acids.
Ex: Glucose to Glucosaccharic acid.

Furfural Derivatives
Monosaccharides when treated with concentrated sulphuric acid undergo
dehydration with removal of 3 molecules of water. Therefore hexoses give

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hydroxymethyl furfural and pentoses give furfural. These furfural derivatives Biochemistry
can condense with phenolic compounds to give coloured products. This forms
the basis of Molisch test, a general test for carbohydrates.

Reduction to form Alcohols

When treated with reducing agents such as sodium amalgam, hydrogen can
reduce sugars. Aldose yields corresponding alcohols. But ketose forms two Notes
alcohols, because of appearance of a new asymmetric carbon atom. Ex: Glucose
forms sorbitol and fructose forms sorbitol and mannitol.

CH2OH
H C OH
HO C H
H C OH
H C OH
CH2OH
Glucitol or Sorbitol
(a sugar alcohol)

Glycosides: When the hemi-acatal group of a monosaccharide is condensed

with an alcohol or phenol group, it is called glycoside.

Formation of esters: Hydroxyl groups of sugars can be esterified to form

acetates, propionates, benzoates, phosphates, etc

Amino sugars
Amino groups may be substituted for hydroxyl groups of sugars to give rise to
amino sugars. Ex: Glucose to Glucosamine. Generally the amino group is added
to the second carbon atom of hexoses. The amino group may be further
acetylated to form N-acetylated sugars such N-acetyl-glucosamine.

CH2OH
O
H H OH

HO OH H H

H NH2
Glucosamine
(an amino sugar)

Deoxy sugars: Oxygen of the hydroxyl group may be removed to form deoxy
sugars.

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Biochemistry 5 5
HOCH2 HOCH2
O OH O OH
4 1 4 1
H H H H H H H H

3 2 3 2
HO OH HO OH
Ribose Deoxyribose
Notes
Disaccharides
When two monosaccharides are combined together with elimination of a water
molecule it is called disaccharide. Monosaccharides are combined by glycosidic
bond.
Disaccharide Description Component monosaccharides

Sucrose common table sugar glucose α1→2 fructose

Maltose product of starch hydrolysis glucose α1→4 glucose
Trehalose found in fungi glucose α1→1 glucose
Lactose main sugar in milk galactose β1→4 glucose
Melibiose found in legumes galactose β1→6 glucose

CH2OH
O O
H H H HOCH2 H

H
HO OH H HO
CH2OH
O

H OH OH H

Sucrose

CH2OH

CH2OH O
H OH
H
O
H H O OH H
H

HO OH H
H H OH

H OH

Lactose

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Biochemistry
6 CH OH 6 CH OH
2 2
5 O 5 O
H H H H H
H
4 1 4 1
OH
HO OH H
O
H
HO
3 2 3 2
H OH H OH
Notes
Maltose

Sucrose
Sucrose also called saccharose, is ordinary table sugar refined from sugar cane
or sugar beets. Sucrose is not a reducing sugar. This is because the glycosidic
linkage inolves first carbon of glucose and second carbon of fructose, and hence
there is no free reducing groups. When sucrose is hydrolyzed the resulting
products have reducing property. Hydrolysis of sucrose (optical rotation +66.5°)
will produce one molecule of glucose (+52.5°) and one molecule of fructose (-
92°). Therefore the products will change the dextrorotation to levorotation, or
the plane of rotation is inverted. Equimolecular mixture of glucose and fructose
thus formed is called invert sugar.

Lactose
Lactose is the sugar present in milk. It is reducing disaccharide.

Maltose
Maltose consists of two α-D-glucose molecules. It is a reducing disaccharide

Polysaccharides
Polysaccharides are polymerized products of many monosaccharide units. They
may be homo or hetero polysaccharides. Many polysaccharides, unlike sugars,
are insoluble in water. Dietary fiber includes polysaccharides and oligosaccharides
that are resistant to digestion and absorption in the human small intestine but
which are completely or partially fermented by microorganisms in the large
intestine.

Homopolysaccharides
They have only one type of monosaccharide units.

Starch
Starch is the major form of stored carbohydrate in plants. Starch is composed
of a mixture of two substances:amylose, an essentially linear polysaccharide,

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Biochemistry and amylopectin, a highly branched polysaccharide. Both forms of starch are
polymers of α-D-Glucose. Natural starches contain 10-20% amylose and 80-
90% amylopectin. Amylose forms a colloidal dispersion in hot water (which
helps to thicken gravies) whereas amylopectin is completely insoluble.
z Amylose molecules consist typically of 200 to 20,000 glucose units which
form a helix as a result of the bond angles between the glucose units.
Notes CH2OH CH2OH CH2OH CH2OH CH2OH
H O H O H O H O H H O H
H H H
H H H H H
OH H OH H OH H OH H O OH H O
O O O

H OH H OH H OH H OH H OH
Amylose

z Amylopectin differs from amylose in being highly branched. Short side

chains of about 30 glucose units are attached with 1α→6 linkages
approximately every twenty to thirty glucose units along the chain.
Amylopectin molecules may contain up to two million glucose units.

CH2OH CH2OH
H O H H O H
H H

O OH H O OH H
O

H OH H OH
CH2OH CH2OH CH2 CH2OH CH2OH
H O H O O O H H O H
H H H H H
H H H H H
OH H OH H OH H OH H O OH H O
O O O

H OH H OH H OH H OH H OH

Amylopectin

The side branching chains are clustered together within the amylopectin
molecule

Glycogen
Glucose is stored as glycogen in animal tissues by the process of glycogenesis.
When glucose cannot be stored as glycogen or used immediately for energy, it
is converted to fat. Glycogen is a polymer of α-D-Glucose identical to
amylopectin, but the branches in glycogen tend to be shorter (about 13 glucose
units) and more frequent. The glucose chains are organized globularly like

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branches of a tree originating from a pair of molecules of glycogenin, a protein Biochemistry
with a molecular weight of 38,000 that acts as a primer at the core of the
structure. Glycogen is easily converted back to glucose to provide energy.

Notes

Dextran
Dextran is a polysaccharide similar to amylopectin, but the main chains are
formed by 1α→6 glycosidic linkages and the side branches are attached by
1α→3 or 1α→4 linkages. Dextran is an oral bacterial product that adheres to
the teeth, creating a film called plaque. It is also used commercially in
confections, in lacquers, as food additives, and as plasma volume expanders.

CH2
CH2
H O H
H O H
H
H
OH H
HO O OH H
HO O

H HO
H HO
CH2 6
CH2
H O
H 5 O H
H H
H
OH H 4 1
HO O H
HO O
3 2
H HO
H OH
CH2

H O H
H

OH H
HO O

H OH

Dextran

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Biochemistry Inulin: Inulins, also called fructans, are polymers consisting of fructose units
that typically have a terminal glucose. Oligofructose has the same structure as
inulin, but the chains consist of 10 or fewer fructose units. Oligofructose has
approximately 30 to 50 percent of the sweetness of table sugar. Inulin is less
soluble than oligofructose and has a smooth creamy texture that provides a fat-
like mouthfeel. Inulin and oligofructose are nondigestible by human intestinal
enzymes, but they are totally fermented by colonic microflora. The short-chain
Notes fatty acids and lactate produced by fermentation contribute 1.5 kcal per gram
of inulin or oligofructose. Inulin and oligofructose are used to replace fat or sugar
and reduce the calories of foods like ice cream, dairy products, confections and
baked goods.
HOCH2 O
H

H HO
CH2OH

OH H
O

O
CH2 H

H HO
CH2OH

O OH H
CH2OH
O O
H H H CH2 H

H HO
HO OH H
CH2OH
O

H OH OH H
Inulin n = approx. 35

Cellulose
Cellulose is a polymer of β-D-Glucose, which in contrast to starch, is oriented
with –CH2OH groups alternating above and below the plane of the cellulose
molecule thus producing long, unbranched chains. The absence of side chains
allows cellulose molecules to lie close together and form rigid structures.
Cellulose is the major structural material of plants. Wood is largely cellulose,
and cotton is almost pure cellulose. Cellulose can be hydrolyzed to its constituent
glucose units by microorganisms that inhabit the digestive tract of termites and
ruminants. Cellulose may be modified in the laboratory by treating it with nitric

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acid (HNO3) to replace all the hydroxyl groups with nitrate groups (-ONO2) to Biochemistry
produce cellulose nitrate (nitrocellulose or guncotton) which is an explosive
component of smokeless powder. Partially nitrated cellulose, known as pyroxylin,
is used in the manufacture of collodion, plastics, lacquers, and nail polish.
H OH H OH
CH2OH CH2OH

H O O H
H H O
H O OH H H OH H
Notes
OH H H OH H H O
O H H
H H O
O

H OH CH2OH H OH CH2OH
Cellulose

Chitin
Chitin is an unbranched polymer of N-Acetyl-D-glucosamine. It is found in
fungi and is the principal component of arthropod and lower animal exoskeletons,
e.g., insect, crab, and shrimp shells. It may be regarded as a derivative of
cellulose, in which the hydroxyl groups of the second carbon of each glucose
unit have been replaced with acetamido (–NH(C=O)CH3) groups.

H NHCOCH3 H NHCOCH3
CH2OH CH2OH

H O O H
H H O
H O OH H H OH H

OH H H OH H H O
O H H
H H O
O

H CH2OH H NHCOCH3 CH2OH

NHCOCH3
Chitin

2.6 HETEROPOLYSACCHARIDES
Heteropolysaccarides contain two or more different kind of monosaccharides.
Usually they provide extracellular support for organisms of all kingdoms: the
bacteria cell envelope, or the matrix that holds individual cells together in animal
tissues, and provides protection, shape and support to cells, tissues and organs.
Heteropolysaccharides provide extracellular support to very different organisms,
from bacteria to humans; together with fibrous proteins, like collagen, elastin,
fibronectin, laminin and others, heteropolysaccharides are the most important
components of the extracellular matrix. Hyaluronic acid, condroitin sulfates
and dermatan sulfates are important heteropolysaccharides in the extracellular
matrix. These heteropolysaccharides usually are formed by the repetition of a
disaccharide unit of an aminosugar and an acid sugar.

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Biochemistry Other common constituents are sulfate groups linked to certain monosaccharides.
Usually heteropolysaccharides are associated with proteins forming proteoglycans,
glycosaminoglycans or mucopolysaccharides (since they are abundant in
mucous secretions). As a group, they perform diverse functions: structural,
water metabolism regulation (as a reservoir of water), cellular cement, biological
sieve, biological lubricant, docking sites for growth factors, among other
functions.
Notes
Established specific functions of some glycosaminoglycans are:
Hyaluronic Acid (Hyaluronate): It is a lubricant in the synovial fluid of joints,
give consistency to vitreous humor, contributes to tensile strength and elasticity
of cartilages and tendons (Answer to C-O6)
Chondroitin Sulfates: contributes to tensile strength and elasticity of cartilages,
tendons, ligaments and walls of aorta.
Dermatan sulfate (former chondroitin sulfate B) is found mainly in skin, but
also is in vessels, heart, lungs. It may be related to coagulation and vascular
diseases and other conditions.
Keratan sulfate: Present in cornea, cartilage bone and a variety of other
structures as nails and hair.

Digestion and absorption

All carbohydrates absorbed in the small intestine must be hydrolyzed to
monosaccharides prior to absorption. The digestion of starch begins with the
action of salivary alpha-amylase/ptyalin, although its activity is slight in
comparison with that of pancreatic amylase in the small intestine. Amylase
hydrolyzes starch to alpha-dextrin, which are then digested by gluco-amylase
(alpha-dextrinases) to maltose and maltotriose. The products of digestion of
alpha-amylase and alpha-dextrinase, along with dietary disaccharides are
hydrolyzed to their corresponding monosaccharides by enzymes (maltase,
isomaltase, sucrase and lactase) present in the brush border of small intestine.
In the typical Western diet, digestion and absorption of carbohydrates is fast and
takes place usually in the upper small intestine. However, when the diet contains
carbohydrates not easily digestible, digestion and absorption take place mainly
in the ileal portion of the intestine.
Digestion of food continues while simplest elements are absorbed. The
absorption of most digested food occurs in the small intestine through the brush
border of the epithelium covering the villi(small hair-like structure). It is not a
simple diffusion of substances, but is active and requires energy use by the
epithelial cells.

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During the phase of carbohydrate absorption, fructose is transported into the Biochemistry
intestinal cell's cytosol, glucose and galactose competes with other Na +
transporter required for operation. From the cytosol, monosaccharides pass into
the capillaries by simple or facilitated diffusion.

Carbohydrates that are not digested in the small intestine, including resistant
starch foods such as potatoes, beans, oats, wheat flour, as well as several non-
polysaccharides oligosaccharides and starch, are digested in a variable when Notes
they reach the large intestine. The bacterial flora metabolize these compounds
in the absence of oxygen. This produces gases (hydrogen, carbon dioxide and
methane) and short-chain fatty acids (acetate, propionate, butyrate). The gases
are absorbed and excreted by breathing or through the anus. Fatty acids are
rapidly metabolized. Thus butyrate, used mainly in the colonic, is an important
nutritional source for these cells and regulates their growth, acetate into the
blood and taken up by the liver, muscle and other tissues, and propionate, which
is an important precursor of glucose in animals, it is not so in humans.

Are polymers made up of two to ten monosaccharide units joined together by

glycosidic linkages. Oligosaccharides can be classified as di-, tri-, tetra-
depending upon the number of monosaccharides present. Among these the most
abundant are the disaccharides, with two monosaccharide units.

INTEXT QUESTIONS 2.2

1. Compounds having same structural formula but differing in spatial
configuration are known as ..................
2. This change in rotation with time is called ..................
3. Equimolecular mixture of glucose and fructose thus formed is called
..................
4. Types of Polysaccharides are .................. & ..................

WHAT HAVE YOU LEARNT

z Carbohydrates are polyhydroxy aldehydes or ketones, or compounds that
can be hydrolyzed to Monosacchrides.
z Carbohydrate is an organic compound comprising only carbon, hydrogen,
and oxygen, usually with a hydrogen: oxygen atom ratio of 2:1.

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Biochemistry z Baked goods commonly contain dietary starch and added sugar. Most
dietary carbohydrates come from plants
z Carbohydrates are classified depending on hydrolysis as Monosaccharides,
Disaccharides, Trisaccharides, Oligosaccharides, Polysaccharides
z Molecules having only one actual or potential sugar group are called
monosaccharides which cannot be hydrolyzed further into polyhydroxy
Notes aldehydes or ketone unit
z Sugars having aldehyde group are called aldoses and sugars with keto group
are called ketoses. Depending on the number of carbon atoms
monosaccharides are named as triose (C3), tetrose (C4), pentose (C5),
hexose (C6), heptose (C7) and so on.
z Compounds having same structural formula but differing in spatial
configuration are known as sterioisomers
z The number of possible sterioisomers depends on the number of asymmetric
carbon atoms by the formula 2n where n is the number of asymmetric carbon
atoms
z The presence of asymmetrical carbon atom causes optical activity. Depending
on the rotation molecules are called dextrorotatory (+) (d) or levorotatory(-
)
z When sugars are different from one another, only in configuration with
regard to the single carbon atom are called Epimers
z When two monosaccharides are combined together with elimination of a
water molecule it is called disaccharide. Monosaccharides are combined by
glycosidic bond
z Sucrose, Maltose, Trehalose, Lactose and Melibose are disaccharide
z Polysaccharides are polymerized products of many monosaccharide units.
They may be homo or hetero polysaccharides.
z Homopolysaccharides have only one type of monosaccharide units.
z Homopolysaccharides are starch, Glycogen, Dextran, Inulin, Cellulose,
chitin
z Heteropolysaccarides contain two or more different kind of monosaccharides
z Carbohydrates absorbed in the small intestine and are hydrolyzed to
monosaccharides prior to absorption.
z The digestion of starch begins with the action of salivary alpha-amylase/
ptyalin, although its activity is slight in comparison with that of pancreatic
amylase in the small intestine.

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Biochemistry

TERMINAL QUESTIONS
1. Classify Monosaccharides with examples
2. Classify Polysaccharides with examples
3. Write short note on absorption of Carbohydrates
Notes

ANSWERS TO INTEXT QUESTIONS

2.1
1. Carbon, Hydrogen & Oxygen
2. 2 : 1
3. Monosaccharides
4. Aldoses
5. Ketoses
6. 1. (c)
2. (a)
3. (d)
4. (b)
7. 1. (d)
2. (c)
3. (a)
4. (b)

2.2
1. Sterioisomers
2. Mutarotation
3. Invert sugar
4. Homopolysaccharide & Heteropolysaccharide

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Biochemistry

3
Notes
CARBOHYDRATE
METABOLISM

3.1 INTRODUCTION
All living cells require energy to carry out various cellular activities. This energy
is stored in the chemical bonds of organic molecules (e.g. carbohydrates, fats,
proteins) that we eat as food. These organic molecules are broken down by
enzymatic reactions in cells to generate energy in the form of adenosine
triphosphate (ATP). The ATP generated by these pathways in cells is used to
drive fundamental cellular processes. The food we consume is mainly comprised
of proteins, polysaccharides (carbohydrates) and fats. These are first broken
down into smaller units: proteins into amino acids, polysaccharides into sugars,
and fats into fatty acids and glycerol. This process of digestion occurs outside
the cell. The amino acids, simple sugars and fatty acids then enter the cell and
undergo oxidation by glycolysis (in the cytosol) and the citric acid cycle (in the
mitochondria) to generate ATP (from ADP and Pi).

OBJECTIVES
After reading this lesson you will be able to
z describe glycolysis, Citric Acid Cycle

z explain Glycogenesis and Glycogenolysis

z describe the Hormonal regulation of blood sugar level

3.2 GLYCOLYSIS (EMBDEN-MEYERHOF PATHWAY)

Definition
In glycolysis pathway glucose is converted to pyruvate (aerobic condition) or
lactate (anaerobic condition), along with production of a small quantity of energy.

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Site of reaction: All the reaction steps take place in the cytoplasm. Biochemistry

Importance of the glycolysis pathway:

z It is the only pathway that is taking place in all the cells of the body.
z Glycolysis is the only source of energy in erythrocytes.
z In strenuous exercise, when muscle tissue lacks enough oxygen, anaerobic
glycolysis forms the major source of energy for muscles.
Notes
z The glycolytic pathway may be considered as the preliminary step before
complete oxidation.
z The glycolytic pathway provides carbon skeletons for synthesis of non-
essential amino acids as well as glycerol part of fat.
z Most of the reactions are reversible.

Steps of glycolytic pathway

1. Glucose is phosphorylated to glucose -6-phosphate. The enzyme is
hexokinase, which splits ATP into ADP and the Pi is added on to the glucose.
The energy released by hydrolysis of ATP is utilised for the forward reaction.
Hexokinase is the key glycolytic enzyme and the reaction is irreversible.
2. Glucose-6-phosphate is isomerised to fructose-6-phosphate by
phosphohexose isomerase.
3. Fructose-6-phosphate is further phosphorylated to fructose-1,6-bisphosphate.
The enzyme is phosphofructokinase, it is an important key enzyme and the
reaction is irreversible.
4. Fructose-1, 6-bisphosphate is cleaved into two 3 carbon atoms; one
glyceraldehyde-3-phosphate and another molecule of dihydroxyacetone
phosphate. The enzyme is aldolase. Dihydroxyacetone phosphate is
isomerised to glyceraldehyde-3-phosphate by the enzyme phophotriose
isomerase.
5. Glyceraldehyde-3-phosphate is dehydrogenated and simultaneously
phosphorylated to 1,3-bis-phosphoglycerate with the help of NAD+. The
enzyme is glyceraldehyde-3-phosphate dehydrogenase.
6. 1, 3-bis-phosphoglycerate is converted to 3-phosphoglycerate by the enzyme
1, 3-bis-phosphoglycerate kinase. Here one molecule of ATP is formed and
this reaction is an example for Substrate level phosphorylation.
7. 3-phosphoglycerate is isomerised to 2-phosphoglycerate by shifting the
phosphate group from 3 rd to 2 nd carbon atom. The enzyme is
phosphoglucomutase.
8. 2-phosphoglycerate is converted to phosphoenol pyruvate by the enzyme
enolase. One water molecule is removed. A high energy phosphate bond
is produced. This enzyme requires Mg++and inhibited by fluoride.
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Biochemistry 9. Phosphoenol pyruvate is dephosphorylated to pyruvate, by pyruvate kinase.

One molecule of ATP is generated. This step is irreversible.
10. In anaerobic condition pyruvate is reduced to lactate by lactate
dehydrogenase.In aerobic conditions pyruvate enters citric acid cycle for
complete oxidation. The lactate from anaerobic cycle enters cori’s cycle.
Glucose ATP
Locks glucose
Notes Hexokinase in cell
ADP
Glucose-6-phosphate
Phosphoglucose Frees up
isomerase carbon 1
Fructose-6-phosphate
ATP
Adds phosphate to
Phosphofructokinase
prepare for cleavage
ADP
Fructose-1,6-bisphosphate
Aldolase Cleavage
Dihydroxyacetone Glyceraldehyde-
phosphate Triose phosphate isomerase 3-phosphate
makes single product Pi + NAD
+

Glyceraldehyde-3-phosphate Activates
dehydrogenase phosphate
+
NADH + H
1,3-Bisphosphoglycerate
ADP
First ATP Phosphoglycerate
of glycolysis kinase ATP
3-Phospogiycerate
Moves
Phosphoglyceromutase
phosphate
2-phospoglycerate
Activates
Enolase phosphate

Phosphoenolpyruvate
ADP
Second ATP Pyruvate kinase NAD
of glycolysis NADH
ATP
CO2 Pyruvate Lactate
aerobic Lactate dehydrogenase
metabolism anaerobic metabolism
Fig. 3.1

Energy yield from glycolysis

Aerobic conditions
Number of ATPs gained per glucose molecule is 8 ATPs
Anaerobic conditions
Number of ATPs gained per glucose molecule is 2 ATPs

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Biochemistry
3.3 CITRIC ACID CYCLE: (KREB’S CYCLE)
Under aerobic conditions the end product of glycolysis is pyruvic acid. The next
step is the formation of acetyl coenzyme A (acetyl CoA) - this step is technically
not a part of the citric acid cycle, but is shown on the diagram on the top left.
Acetyl CoA, whether from glycolysis or the fatty acid spiral, is the initiator of
the citric acid cycle. In carbohydrate metabolism, acetyl CoA is the link between
glycolysis and the citric acid cycle. The initiating step of the citric acid cycle Notes
occurs when a four carbon compound (oxaloacetic acid) condenses with acetyl
CoA (2 carbons) to form citric acid (6 carbons).
The whole purpose of a “turn” of the citric acid cycle is to produce two carbon
dioxide molecules. This general oxidation reaction is accompanied by the loss
of hydrogen and electrons at four specific places. These oxidations are connected
to the electron transport chain where many ATP are produced.

Step 1
The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form
a molecule of citrate. The acetyl coenzyme A acts only as a transporter of acetic
acid from one enzyme to another. After Step 1, the coenzyme is released by
hydrolysis so that it may combine with another acetic acid molecule to begin
the Krebs cycle again.

Step 2
The citric acid molecule undergoes an isomerization. A hydroxyl group and a
hydrogen molecule are removed from the citrate structure in the form of water.
The two carbons form a double bond until the water molecule is added back.
Only now, the hydroxyl group and hydrogen molecule are reversed with respect
to the original structure of the citrate molecule. Thus, isocitrate is formed.

Step 3
In this step, the isocitrate molecule is oxidized by a NAD molecule. The NAD
molecule is reduced by the hydrogen atom and the hydroxyl group. The NAD
binds with a hydrogen atom and carries off the other hydrogen atom leaving a
carbonyl group. This structure is very unstable, so a molecule of CO2 is released
creating alpha-ketoglutarate.

Step 4
In this step, coenzyme A, returns to oxidize the alpha-ketoglutarate molecule.
A molecule of NAD is reduced again to form NADH and leaves with another
hydrogen. This instability causes a carbonyl group to be released as carbon

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Biochemistry dioxide and a thioester bond is formed in its place between the former alpha-
ketoglutarate and coenzyme A to create a molecule of succinyl-coenzyme
A complex.

Step 5
A water molecule sheds its hydrogen atoms to coenzyme A. Then, a free-floating
phosphate group displaces coenzyme A and forms a bond with the succinyl
Notes complex. The phosphate is then transferred to a molecule of GDP to produce
an energy molecule of GTP. It leaves behind a molecule of succinate.

Step 6
In this step, succinate is oxidized by a molecule of FAD (Flavin adenine
dinucleotide). The FAD removes two hydrogen atoms from the succinate and
forces a double bond to form between the two carbon atoms, thus
creating fumarate.

Step 7
An enzyme adds water to the fumarate molecule to form malate. The malate
is created by adding one hydrogen atom to a carbon atom and then adding a
hydroxyl group to a carbon next to a terminal carbonyl group.

Step 8
In this final step, the malate molecule is oxidized by a NAD molecule. The
carbon that carried the hydroxyl group is now converted into a carbonyl group.
The end product is oxaloacetate which can then combine with acetyl-coenzyme
A and begin the Krebs cycle all over again.

Summary of Krebs Cycle

In summary, three major events occur during the Krebs cycle. One GTP
(guanosine triphosphate) is produced which eventually donates a phosphate
group to ADP to form one ATP; three molecules of NAD are reduced; and one
molecule of FAD is reduced. Although one molecule of GTP leads to the
production of one ATP, the production of the reduced NAD and FAD are far more
significant in the cell’s energy-generating process. This is because NADH and
FADH2 donate their electrons to an electron transport system that generates large
amounts of energy by forming many molecules of ATP.

Yield of ATP
At this point the yield of ATP is 4 moles per mole of Glucose as it passes through
the Krebs cycle.

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z This is not much more than the 2 moles which would have been produced Biochemistry
from glycolysis.
z However, NADH and FADH2 are energy rich molecules
z Their oxidation is highly exergonic and is coupled with the production of
ATP from ADP
z Oxidation of 1 mole NADH produces 3 moles ATP
Notes
z Oxidation of 1 mole FADH2 produces 2 moles ATP
z Thus total ATP yield = (10 × 3) + (2 × 2) + 4 = 38 moles ATP per mole
Glucose

Fig. 3.2: Glycogenesis and Glycogenolysis

Biosynthesis of Glycogen
The goal of glycolysis, glycogenolysis, and the citric acid cycle is to conserve
energy as ATP from the catabolism of carbohydrates. If the cells have sufficient
supplies of ATP, then these pathways and cycles are inhibited. Under these
conditions of excess ATP, the liver will attempt to convert a variety of excess
molecules into glucose and/or glycogen.

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Biochemistry Glycogenesis
Glycogenesis is the formation of glycogen from glucose. Glycogen is synthesized
depending on the demand for glucose and ATP (energy). If both are present in
relatively high amounts, then the excess of insulin promotes the glucose
conversion into glycogen for storage in liver and muscle cells.
In the synthesis of glycogen, one ATP is required per glucose incorporated into
Notes the polymeric branched structure of glycogen. actually, glucose-6-phosphate is
the cross-roads compound. Glucose-6-phosphate is synthesized directly from
glucose or as the end product of gluconeogenesis.

Glucose
Hexokinase (in muscle)
Glucokinase (in liver)
Glucose 6-phosphate
Phosphoglucomutase
Glucose1-phosphate
UDP- glucose pyrophosphatase
UDP-glucose
Glycogen synthase
a(1®4) glucosyl units
Branching enzyme

Glycogen
[a(1®4) and a (1®6) glucosyl units]

Fig. 3.3: Steps of glycogensis

Glycogenolysis
In glycogenolysis, glycogen stored in the liver and muscles, is converted first to
glucose-1- phosphate and then into glucose-6-phosphate. Two hormones which
control glycogenolysis are a peptide, glucagon from the pancreas and epinephrine
from the adrenal glands.
Glucagon is released from the pancreas in response to low blood glucose and
epinephrine is released in response to a threat or stress. Both hormones act upon
enzymes to stimulate glycogen phosphorylase to begin glycogenolysis and
inhibit glycogen synthetase (to stop glycogenesis).
Glycogen is a highly branched polymeric structure containing glucose as the
basic monomer. First individual glucose molecules are hydrolyzed from the
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chain, followed by the addition of a phosphate group at C-1. In the next step the Biochemistry
phosphate is moved to the C-6 position to give glucose 6-phosphate, a cross road
compound.
Glucose-6-phosphate is the first step of the glycolysis pathway if glycogen is the
carbohydrate source and further energy is needed. If energy is not immediately
needed, the glucose-6-phosphate is converted to glucose for distribution in the
blood to various cells such as brain cells. Notes

Glycogen

Glycogen phosphorylase
a(a®4)®a(1®4)-glucan transferase
Amylo-a(1®6)-glucosidase

Glucose 1-phosphate

Phosphoglucomutase

Glucose 6-phosphate In muscle

Glucose 6-phosphatase

Glucose In liver

Fig. 3.4: Steps of glycogenolysis

Gluconeogenesis
Gluconeogenesis is a metabolic pathway that results in the generation
of glucose from non-carbohydrate carbon substrates such as pyruvate,
lactate, glycerol, and glucogenic amino acids.

The vast majority of gluconeogenesis takes place in the liver and, to a smaller
extent, in the cortex of kidneys. This process occurs during periods
of fasting, starvation, or intense exercise and is highly endergonic.
Gluconeogenesis is often associated with ketosis.

Entering the pathway

Several non-carbohydrate carbon substrates can enter the gluconeogenesis
pathway. One common substrate is lactic acid, formed during anaerobic
respiration in skeletal muscle. Lactate is transported back to the liver where it
is converted into pyruvate by the Cori cycle using the enzyme lactate

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Biochemistry dehydrogenase. Pyruvate, the first designated substrate of the gluconeogenic

pathway, can then be used to generate glucose. All citric acid cycle intermediates,
through conversion to oxaloacetate, amino acids other than lysine or leucine,
and glycerol can also function as substrates for gluconeogenesis. Amino acids
must have their amino group removed by transamination or deamination before
entering the cycle directly (as pyruvate or oxaloacetate), or indirectly via the
citric acid cycle.
Notes

Fig. 3.5

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Fatty acids cannot be converted into glucose in animals, the exception being odd- Biochemistry
chain fatty acids which yield propionyl CoA, a precursor forsuccinyl CoA. In
plants, specifically in seedlings, the glyoxylate cycle can be used to convert fatty
acids (acetate) into the primary carbon source of the organism. The glyoxylate
cycle produces four-carbon dicarboxylic acids that can enter gluconeogenesis.
Glycerol, which is a part of alltriacylglycerols, can also be used in gluconeogenesis.
In organisms in which glycerol is derived from glucose (e.g., humans and other
mammals), glycerol is sometimes not considered a true gluconeogenic substrate, Notes
as it cannot be used to generate new glucose.
Gluconeogenesis is a pathway consisting of eleven enzyme-catalyzed reactions.
The pathway can begin in the mitochondria or cytoplasm, depending on the
substrate being used. Many of the reactions are reversible steps found in
glycolysis.
Gluconeogenesis begins in the mitochondria with the formation of oxaloacetate
through carboxylation of pyruvate at the expense of one molecule of ATP. This
reaction is catalyzed by pyruvate carboxylase, which is stimulated by high levels
of acetyl-CoA(when fatty acid oxidation is high in the liver) and inhibited by
high levels of ADP.
Oxaloacetate must then be reduced into malate using NADH in order to be
transported out of the mitochondria.
In the cytoplasm, malate is oxidized to oxaloacetate using NAD+, where the
remaining steps of gluconeogenesis occur. Oxaloacetate is then decarboxylated
and phosphorylated to produce phosphoenolpyruvate by phosphoenolpyruvate
carboxykinase. One molecule of GTP is hydrolyzed to GDP in the course of this
reaction.
The next steps in the reaction are the same as reversed glycolysis. However,
fructose-1,6-bisphosphatase converts fructose-1,6-bisphosphate to fructose-6-
phosphate. The purpose of this reaction is to overcome the large negative ΔG.
Glucose-6-phosphate is formed from fructose-6-phosphate by phospho gluco-
isomerase. Glucose-6-phosphate can then be used for glucose generation or in
other metabolic pathways. Free glucose is not generated automatically because
glucose, unlike glucose-6-phosphate, tends to freely diffuse out of the cell.
The final reaction of gluconeogenesis, the formation of glucose, is carried out
in the lumen of the endoplasmic reticulum. Glucose-6-phosphate is hydrolyzed
by glucose-6-phosphatase to produce glucose. Glucose is then shuttled into the
cytosol by glucose transporters located in the membrane of the endoplasmic
reticulum.

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Biochemistry Regulation
While most steps in gluconeogenesis are the reverse of those found inglycolysis,
three regulated and strongly exergonic reactions are replaced with more
kinetically favorable reactions. Hexokinase/glucokinase, phosphofructokinase,
and pyruvate kinase enzymes of glycolysis are replaced with glucose-6-
phosphatase, fructose-1,6-bisphosphatase, and PEP carboxykinase. This system
Notes of reciprocal control allow glycolysis and gluconeogenesis to inhibit each other
and prevent the formation of afutile cycle.

The majority of the enzymes responsible for gluconeogenesis are found in

the cytoplasm; the exceptions are mitochondrial pyruvate carboxylase, and, in
animals, phosphoenol-pyruvate carboxykinase. The latter exists as an isozyme
located in both the mitochondrion and the cytosol. As there is no known
mechanism to transport phosphoenolpyruvate from the mitochondrion into the
cytosol, the cytosolic enzyme is believed to be the isozyme important for
gluconeogenesis. The rate of gluconeogenesis is ultimately controlled by the
action of a key enzyme, fructose-1,6-bisphosphatase, which is also regulated
through signal ransduction bycAMP and its phosphorylation.

Most factors that regulate the activity of the gluconeogenesis pathway do so by

inhibiting the activity or expression of key enzymes. However, both acetyl
CoA and citrate activate gluconeogenesis enzymes (pyruvate carboxylase and
fructose-1,6-bisphosphatase, respectively). Due to the reciprocal control of the
cycle, acetyl-CoA and citrate also have inhibitory roles in the activity of pyruvate
kinase.

Insulin and Glucagon: Control of Blood Glucose

One of the most important and tightly regulated responses in the human body
is the concentration of blood glucose (blood sugar). Glucose is the major
breakdown product of cellular metabolism. As such, it is required both as an
energy source and as a source of carbon for making organic molecules. Blood
glucose concentrations are regulated by negative feedback pathways that are
modulated by two separate hormones: insulin and glucagon. Both of these
hormones are produced in special cells called islet cells, or islets of Langerhans
– are found in clusters throughout the pancreas. Islet cells make up a very small
percentage of the pancreas (about 1-2%); the remainder of the organ is an
exocrine gland producing digestive enzymes and bicarbonate ion. This tiny
number of endocrine cells is exceedingly important. Each islet contains two
kinds of cells: alpha cells, which produce glucagon, and beta cells, which
produce insulin.

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Insulin vs. Glucagon Biochemistry

Normally, blood glucose concentrations in human blood should range between

70-110 milligrams (mg/ml). Insulin and glucagon operate in an antagonistic
(opposing) manner. The result is a precise control of blood glucose levels within
this range.

The insulin pathway is activated when blood glucose levels are too high. High
blood glucose levels (e.g., occurring after the stomach has digested a food high Notes
in sugar) stimulate beta cells in the pancreas to release insulin. Insulin causes
an increased uptake of glucose from the blood; promotes conversion of glucose
into triglycerides in the liver, fat and muscle cells; and increases the cellular rate
of glycolysis – breaking glucose into smaller components that can be used for
synthesis of other compounds.

The glucagon pathway is activated when blood glucose levels are too low. Low
blood glucose levels (e.g., due to exercise combined with not eating for several
hours) stimulate the alpha cells in the pancreas to produce glucagon. Glucagon
causes the liver to convert stored glycogen into glucose, then release the glucose
into the blood (a process called glycogenolysis). The two hormones, insulin and
glucagon, each regulate the other. A decrease in insulin (as well as low glucose
levels) stimulates the secretion of glucagon, while an increase in insulin (as well
as an increase in blood glucose) suppresses glucagon secretion. This results in
a continuous cycle, with insulin and glucagon constantly monitoring blood
glucose levels and regulating their secretion to maintain these levels as nearly
constant as possible.

The main function of insulin is removal of excess blood glucose. Because all
cells use glucose as an energy source and as a raw material for making other
organic compounds, all cells except brain cells are targets for insulin. Since the
function of glucagon is opposite that of insulin, it stimulates the addition of
glucose to the bloodstream. Thus, it targets cells with high concentrations of
energy stored as glycogen, including the liver and skeletal muscles. It also
stimulates glucose production from fats, so adipose tissue cells are another target
of glucagon.

Lactose intolerance
Lactose intolerance is a common digestive problem where the body is unable
to digest lactose, a type of sugar mainly found in milk and dairy products. The
body digests lactose by using an enzyme called lactase to break down lactose
into two simpler sugars called glucose and galactose, which can then be easily
absorbed into the bloodstream. Enzymes are proteins that cause chemical
reactions to occur.

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Biochemistry In cases of lactose intolerance, the body does not produce enough of the lactase
enzyme so lactose stays in the digestive system, where it is fermented by bacteria
(in the same way that yeast is fermented to produce beer). It’s this fermentation
process that causes the symptoms associated with lactose intolerance.

Levels of lactase often fall as people grow older and some health conditions can
also reduce the production of lactase.
Notes

Symptoms of lactose intolerance include

z a bloated stomach
z flatulence (wind)
z diarrhoea

Treating lactose intolerance

Limiting intake of food and drink containing lactose is the main treatment for
lactose intolerance.
Depending on a person’s levels of intolerance, they may also require additional
calcium and vitamin D supplements to keep the bones strong and healthy.
Advice from a dietitian may sometimes be helpful in determining the best diet
for a person.
Lactase substitutes are also available. These are drops that you can add to your
meals or drinks to improve your digestion of lactose.

Diabetes Mellitus
Diabetes mellitus (often referred to simply as diabetes) is a group of metabolic
diseases characterized by high blood glucose levels. The term comes from two
Greek words: “diabetes” comes from a verb that means “to pass through” and
refers to the frequent, copious urination that is a characteristic of the disease;
the word “meli” is Greek for “honey” so the term “mellitus” refers to the
presence of high levels of glucose (sugar) in the blood. In addition to urination,
other classic symptoms of diabetes are increased thirst and hunger. The
diabetic’s blood contains more glucose than can be taken up by the cells so this
excess glusose is therefore released in the urine (a diagnostic characteristic of
diabetes is sugar in the urine). The presence of sugar results in more water being
drawn into the urine to balance the osmotic pressure, leading to copious
urination.

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Biochemistry

INTEXT QUESTIONS 3.1

1. In glycolysis pathway glucose is aerobically converted to .................. and
anaerobically ..................
2. All the reaction steps take place in ..................
3. Glycolysis is the only source of energy in .................. cells Notes
4. Number of ATPs gained per glucose molecule in Aerobic conditions of
glycolysis is ..................
5. Number of ATPs gained per glucose molecule in Anaerobic conditions of
glycolysis is ..................
6. Krebs Cycle yield .................. ATP per mole Glucose

WHAT HAVE YOU LEARNT

z In glycolysis pathway glucose is converted to pyruvate in aerobic condition
or lactate in anaerobic condition. All the reaction steps take place in the
cytoplasm.
z Glycolysis is the only pathway that is taking place in all the cells of the
body and is the only source of energy in erythrocytes.
z In Glycolyis, Aerobic conditions yields 8 ATPs and in Anaerobic conditions
yields 2 ATPs per glucose molecule
z Citric acid cycle produces two carbon dioxide molecules. And oxidations
are connected to the electron transport chain where many ATP are produced.
z A total of 38 moles ATP per mole Glucose is yielded in Krebs Cycle
z Glycogenesis is the formation of glycogen from glucose. Glycogen is
synthesized depending on the demand for glucose and ATP (energy).
z In glycogenolysis, glycogen stored in the liver and muscles, is converted
first to glucose-1- phosphate and then into glucose-6-phosphate.
z Two hormones which control glycogenolysis are a peptide, glucagon from
the pancreas and epinephrine from the adrenal glands.
z Gluconeogenesis is a metabolic pathway that results in the generation
of glucose from non-carbohydrate carbon substrates such as pyruvate, lactate,
glycerol, and glucogenic amino acids.
z Blood glucose concentrations in human blood should range between 70-110
milligrams per milliliter (mg/mL). Insulin and glucagon operate in an
antagonistic (opposing) manner.

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Biochemistry

TERMINAL QUESTIONS
1. Explain glycolysis
2. Explain krebs cycle
3. Explain glycogenesis
Notes 4. What is the hormone control of blood sugar

ANSWERS TO INTEXT QUESTIONS

3.1
1. Pyruvate & Lactate
2. Cytoplasm
3. Erythrocytes
4. 8 ATPs
5. 2 ATPs
6. 38 moles

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Proteins MODULE
Biochemistry

4
Notes
PROTEINS

4.1 INTRODUCTION
Proteins are the most abundant biological macromolecules, occurring in all cells
and all parts of cells. Amino acids are the building blocks of proteins. All
proteins, whether from the most ancient lines of bacteria or from the most
complex forms of life, are constructed from the same set of 20 amino acids. What
is most remarkable is that cells can produce proteins with strikingly different
properties and activities by joining the same 20 amino acids in many different
combinations and sequences. From these building blocks different organisms
can make such widely diverse products as enzymes, hormones, antibodies,
transporters, muscle fibers, the lens protein of the eye, feathers, spider webs,
rhinoceros horn, milk proteins, antibiotics, and mushroom poisons and other
substances having distinct biological activities. While proteins contain only
L-α-amino acids, microorganisms elaborate peptides that contain both D- and
L-α-amino acids.

OBJECTIVES
After reading this lesson, you will be able to
z describe amino acids
z explain the structure of amino acids
z classify amino acids
z describe proteins
z describe the structure of protein
z explain the function of proteins
z explain the digestion and absorption of proteins
z describe products of amino acids
z explain transamination, Deamination, Urea Cycle

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Biochemistry
4.2 AMINO ACIDS
Proteins are the essential agents of biological function, and amino acids are the
building blocks of proteins. The diversity of the thousands of proteins found in
nature arises from the commonly occurring 20 amino acids. Proteins are
polymers of amino acids, with each amino acid residue joined to its neighbor
by a specific type of covalent bond. Proteins can be broken down (hydrolyzed)
Notes to their constituent amino acids the free amino acids derived from them. Of the
over 300 naturally occurring amino acids, 20 constitute the monomer units of
proteins. All 20 amino acids (Table 4.1) are biologically essential. Humans can
synthesize 12 (nutritionally nonessential) of the 20 common amino acids from
the amphibolic intermediates of glycolysis and of the citric acid cycle. Of the
12 nutritionally nonessential amino acids, nine are formed from amphibolic
intermediates and three (cysteine, tyrosine and hydroxylysine) from nutritionally
essential amino acids.
Table 4.1 List of essential and nonessential amino acids
Essential Nonessential
Histidine Alanine
Isoleucine Arginine
Leucine Aspartic acid
Lysine Cysteine
Methionine Glutamic acid
Phenylalanine Glutamine
Threonine Glycine
Tryptophan Proline
Valine Serine
Tyrosine
Asparagine
Selenocysteine
Pyrrolysine

Essential amino acids are "essential" not because they are more important to life
than the others, but because the body does not synthesize them. They must be
present in the diet or they will not be present in the body. In addition, the amino
acids arginine, cysteine, glycine, glutamine, histidine, proline, serine and
tyrosine are considered conditionally essential, meaning they are not normally
required in the diet, but must be supplied exogenously to specific populations
that do not synthesize them in adequate amounts.

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Selenocysteine, while not normally considered an amino acid present in proteins, Biochemistry
selenocysteine occurs at the active sites of several enzymes. Examples include
the human enzymes thioredoxin reductase, glutathione peroxidase, and the
deiodinase that converts thyroxine to triiodothyronine. Pyrrolysine sometimes
considered “the 22nd amino acid”, is not listed here as it is not used by humans.

4.2.1 Amino Acids are Chiral Molecules

Notes
An α-amino acid consists of a central carbon atom, called the α carbon, linked
to an amino group, a carboxylic acid group, a hydrogen atom, and a distinctive
R group. For all the common amino acids except glycine, the α carbon is bonded
to four different groups: a carboxyl group, an amino group, an R group, and a
hydrogen atom (Fig. 4.1; in glycine, the R group is another hydrogen atom). The
α-carbon atom is thus a chiral center. Because of the tetrahedral arrangement
of the bonding orbitals around the α-carbon atom, the four different groups can
occupy two unique spatial arrangements, and thus amino acids have two possible
stereoisomers. Since they are nonsuperimposable mirror images of each other
(Fig. 4.2), the two forms represent a class of stereoisomers called enantiomers
(Fig. 4.3). The R group is often referred to as the side chain. Enantiomeric
molecules display a special property called optical activity – the ability to rotate
the plane of polarization of plane-polarized light. Clockwise rotation of incident
light is referred to as dextrorotatory (D) behavior, and counterclockwise
rotation is called levorotatory (L) behavior. Only L amino acids are constituents
of proteins. The magnitude and direction of the optical rotation depend on the
nature of the amino acid side chain.
COO–
H 3N + C H

R
Fig. 4.1: General structure of an amino acid. This structure is common to all but one of
the α-amino acids. (Proline, a cyclic amino acid, is the exception.) The R group or side
chain (red) attached to the α carbon (blue) is different in each amino acid.

Fig. 4.2: The L and D Isomers of Amino Acids. R refers to the side chain. The L and D
isomers are mirror images of each other.

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Biochemistry
COO– COO–
H 3N + C H H C +NH
3
CH3 CH3
L-Alanine D-Alanine

Fig. 4.3: Stereoisomerism in a-amino acids. The two stereoisomers of alanine, L- and
D-alanine, are nonsuperimposable mirror images of each other (enantiomers).
Notes

4.2.2 Structure of a Typical Amino Acid

Amino acids in solution at neutral pH exist predominantly as dipolar ions (also
called zwitterions). Amino acids can exist as zwitterions - substances containing
equal numbers of positive and negative charge due to their carboxyl and amine
groups, which can be negatively and positively charged, respectively. In the
dipolar form, the amino group is protonated (NH3+) and the carboxyl group is
deprotonated (COO–). The ionization state of an amino acid varies with pH
(Figure 4.4). They differ from each other in their side chains, or R groups, which
vary in structure, size, and electric charge, and which influence the solubility of
the amino acids in water.

Fig. 4.4: Ionization State as a Function of pH. The ionization state of amino acids is
altered by a change in pH. The zwitterionic form predominates near physiological pH.

4.2.3 Amino Acids can join via Peptide Bonds

The crucial feature of amino acids that allows them to polymerize to form
peptides and proteins is the existence of their two identifying chemical groups:

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the amino (NH3+) and carboxyl (COO–) groups. The amino and carboxyl groups Biochemistry
of amino acids can react in a head-to-tail fashion, eliminating a water molecule
and forming a covalent amide linkage, which, in the case of peptides and
proteins, is typically referred to as a peptide bond.

4.3 CLASSIFICATION
The structures and abbreviations for the 20 amino acids commonly found in Notes
proteins are shown in Figure 4.5. All the amino acids except proline have both
free amino and free carboxyl groups. The classifications of amino acids is based
on the polarity of the side chains. Thus, the structures shown in Figure 4.5 are
grouped into the following categories: (1) nonpolar or hydrophobic amino acids,
(2) neutral (uncharged) but polar amino acids, (3) acidic amino acids (which
have a net negative charge at pH 7.0), and (4) basic amino acids (which have
a net positive charge at neutral pH).

4.3.1 Nonpolar Amino Acids

The nonpolar amino acids include all those with alkyl chain R groups (alanine,
valine, leucine, and isoleucine), as well as proline (with its unusual cyclic
structure), methionine (one of the two sulfur-containing amino acids), and two
aromatic amino acids, phenylalanine and tryptophan. Tryptophan is sometimes
considered a borderline member of this group because it can interact favorably
with water via the N–H moiety of the indole ring. Proline, strictly speaking, is
not an amino acid but rather an α-imino acid.

4.3.2 Polar, Uncharged Amino Acids

The polar, uncharged amino acids except for glycine contain R groups that can
form hydrogen bonds with water. Thus, these amino acids are usually more
soluble in water than the nonpolar amino acids. Tyrosine displays the lowest
solubility in water of the 20 common amino acids. Glycine, the simplest amino
acid, has only a single hydrogen for an R group, and this hydrogen is not a good
hydrogen bond former. Glycine’s solubility properties are mainly influenced by
its polar amino and carboxyl groups, and thus glycine is best considered a
member of the polar, uncharged group. It should be noted that tyrosine has
significant nonpolar characteristics due to its aromatic ring and could arguably
be placed in the nonpolar group.

4.3.3 Acidic Amino Acids

There are two acidic amino acids – aspartic acid and glutamic acid – whose R
groups contain a carboxyl group. Aspartic acid and glutamic acid thus have a
net negative charge at pH 7. Many proteins that bind metal ions for structural

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Biochemistry or functional purposes possess metal binding sites containing one or more
aspartate and glutamate side chains.

4.3.4 Basic Amino Acids

Three of the common amino acids have side chains with net positive charges
at neutral pH: histidine, arginine, and lysine. The ionized group of histidine is
an imidazolium, that of arginine is a guanidinium, and lysine contains a
Notes protonated alkyl amino group. Arginine and lysine side chains, which are
protonated under physiological conditions, participate in electrostatic interactions
in proteins.
ALIPHATIC AMIND ACIDS CH3 CH3 CH3

CH3 CH3 CH CH2

H CH3 CH CH2 CH3 CH

+ + + + +
H9N C COO– H3N C COO– H3N C COO– H3N C COO– H3N C COO–
H H H H H
Glycine (Gly) G Alanine (Ala) A Valine (Val) V Leucine (Leu) L Isoleucine IIe

AMIND ACIDS WITH HYDROXYL- OR SULFUR- CH3 CYCLIC AMINO

CONTAINING SIDE CHAINS ACID
S
OH SH CH3 CH2 CH2
CH2 CH2 HCOH CH2 CH2 CH2
+ + + + +
H3N C COO– H3N C COO– H3N C COO– H3N C COO– H2N C COO–
H H H H H
Serine (Ser) S Alanine (Ala) A Valine (Val) V Leucine (Leu) L Isoleucine (IIe) I

AROMATIC AMINO ACIDS BASIC AMINO ACIDS

NH2
+ +
NH3 C NH2

OH H CH2 NH

N HN CH2 CH2
+
NH CH2 CH2

CH2 CH2 CH2 CH2 CH2 CH2

+ + + + + +
H 3N C COO– H3N C COO– H3N C COO– H3N C COO– H 3N C COO– H3N C COO–
H H H H H H
Phenylalanine Tyrosine Tryptophan Histidine Lysine Arginine
(Phe) F (Tyr) Y (Tip) W (His) H (Lys) K (Arg) R

ACIDIC AMINO ACIDS AND THEIR AMIDES

O O– O NH2
C C
O O– O NH2
C CH2 C CH2

CH2 CH2 CH2 CH2

+ + + +
H3N C COO– H3N C COO– H3N C COO– H 3N C COO–
H H H H
Aspartic acid Glutamic acid Asparagine Glutamine
(Asp) D (Glu) E (Asn) N (Gln) Q

Fig. 4.5: Classification of amino acids.

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Biochemistry
4.4 PROTEIN
Proteins are a diverse and abundant class of biomolecules, constituting more
than 50% of the dry weight of cells. This diversity and abundance reflect the
central role of proteins in virtually all aspects of cell structure and function.
Biologically occurring polypeptides range in size from small to very large,
consisting of two or three to thousands of linked amino acid residues. Peptides
are chains of amino acids, two amino acid molecules can be covalently joined Notes
through a substituted amide linkage, termed a peptide bond (Figure 4.6), to yield
a dipeptide. Such a linkage is formed by removal of the elements of water
(dehydration) from the α-carboxyl group of one amino acid and the α-amino
group of another. Peptide bond formation is an example of a condensation
reaction, a common class of reactions in living cells. Three amino acids can be
joined by two peptide bonds to form a tripeptide; similarly, amino acids can be
linked to form tetrapeptides, pentapeptides, and so forth. When a few amino
acids are joined in this fashion, the structure is called an oligopeptide. When
many amino acids are joined, the product is called a polypeptide. Proteins may
have thousands of amino acid residues. Although the terms “protein” and
“polypeptide” are sometimes used interchangeably, molecules referred to as
polypeptides generally have molecular weights below 10,000, and those called
proteins have higher molecular weights. Proteins can be assigned to one of three
global classes on the basis of shape and solubility: fibrous, globular, or
membrane.

O O
CH3 CH C O + NH3+ CH COO ¾¾® CH3 CH C N CH COO
+
H
NH3 CH2 NH3+ CH2
OH OH
Alanine Serine Alanine-Serine

Fig. 4.6: Peptide bond formation between two amino acids Alanine and Serine.

Fibrous proteins tend to have relatively simple, regular linear structures. These
proteins often serve structural roles in cells. Typically, they are insoluble in water
or in dilute salt solutions. In contrast, globular proteins are roughly spherical
in shape. The polypeptide chain is compactly folded so that hydrophobic amino
acid side chains are in the interior of the molecule and the hydrophilic side chains
are on the outside exposed to the solvent, water. Membrane proteins are found
in association with the various membrane systems of cells. For interaction with
the nonpolar phase within membranes, membrane proteins have hydrophobic
amino acid side chains oriented outward. As such, membrane proteins are
insoluble in aqueous solutions but can be solubilized in solutions of detergents.

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Biochemistry
4.5 THE LEVELS OF PROTEIN STRUCTURE
The various levels of protein structural organization are defined as follows.

4.5.1 Primary Structure

The amino acid sequence is the primary (1°) structure of a protein, such as that
shown in Figure 4.7.
Notes
4.5.2 Secondary Structure
Through hydrogen bonding interactions between adjacent amino acid residues
the polypeptide chain can arrange itself into characteristic helical or pleated
segments. These segments constitute structural conformities, so-called regular
structures that extend along one dimension, like the coils of a spring. Such
architectural features of a protein are designated secondary (2°) structures
(Figure 4.7). Secondary structures are just one of the higher levels of structure
that represent the three-dimensional arrangement of the polypeptide in space.

4.5.3 Tertiary Structure

When the polypeptide chains of protein molecules bend and fold in order to
assume a more compact three-dimensional shape, a tertiary (3°) level of structure
is generated (Figure 4.7). It is by virtue of their tertiary structure that proteins
adopt a globular shape. A globular conformation gives the lowest surface to-
volume ratio, minimizing interaction of the protein with the surrounding
environment.

Fig. 4.7: Structures of protein.

4.5.4 Quaternary Structure

Many proteins consist of two or more interacting polypeptide chains of
characteristic tertiary structure, each of which is commonly referred to as a
subunit of the protein. Subunit organization constitutes another level in the

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hierarchy of protein structure, defined as the protein’s quaternary (4°) structure Biochemistry
(Figure 4.7). Whereas the primary structure of a protein is determined by the
covalently linked amino acid residues in the polypeptide backbone, secondary
and higher orders of structure are determined principally by noncovalent forces
such as hydrogen bonds and ionic, van der Waals, and hydrophobic interactions.

4.6 FUNCTIONS OF PROTEINS

Notes
Proteins are the agents of biological function. Virtually every cellular activity
is dependent on one or more particular proteins. Thus, a convenient way to
classify the enormous number of proteins is by the biological roles they fill. The
various functions of proteins are as follows.

4.6.1 Enzymes
By far the largest class of proteins is enzymes. More than 3000 different enzymes
are listed in Enzyme Nomenclature, the standard reference volume on enzyme
classification. Enzymes are catalysts that accelerate the rates of biological
reactions. Each enzyme is very specific in its function and acts only in a
particular metabolic reaction. Virtually every step in metabolism is catalyzed by
an enzyme. Enzymes are systematically classified according to the nature of the
reaction that they catalyze, such as the transfer of a phosphate group
(phosphotransferase) or an oxidation–reduction (oxidoreductase). The formal
names of enzymes come from the particular reaction within the class that they
catalyze, as in ATP: D-fructose-6-phosphate 1-phosphotransferase. Often,
enzymes have common names in addition to their formal names. ATP:
D-fructose-6-phosphate 1-phosphotransferase is more commonly known as
phosphofructokinase (kinase is a common name given to ATP-dependent
phosphotransferases).

4.6.2 Regulatory Proteins

A number of proteins do not perform any obvious chemical transformation but
nevertheless can regulate the ability of other proteins to carry out their
physiological functions. Such proteins are referred to as regulatory proteins. A
well-known example is insulin, the hormone regulating glucose metabolism in
animals. Insulin is a relatively small protein and consists of two polypeptide
chains held together by disulfide cross-bridges. Other hormones that are also
proteins include pituitary somatotropin and thyrotropin, which stimulates the
thyroid gland.

4.6.3 Transport Proteins

A third class of proteins is the transport proteins. These proteins function to
transport specific substances from one place to another. One type of transport

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Biochemistry is exemplified by the transport of oxygen from the lungs to the tissues by
haemoglobin or by the transport of fatty acids from adipose tissue to various
organs by the blood protein serum albumin.Membrane transport proteins take
up metabolite molecules on one side of a membrane, transport them across the
membrane, and release them on the other side. Examples include the transport
proteins responsible for the uptake of essential nutrients into the cell, such as
glucose or amino acids.
Notes
4.6.4 Storage Proteins
Proteins whose biological function is to provide a reservoir of an essential
nutrient are called storage proteins. Because proteins are amino acid polymers
and because nitrogen is commonly a limiting nutrient for growth, organisms
have exploited proteins as a means to provide sufficient nitrogen in times of
need. For example, ovalbumin, the protein of egg white, provides the developing
bird embryo with a source of nitrogen during its isolation within the egg. Casein
is the most abundant protein of milk and thus the major nitrogen source for
mammalian infants. The seeds of higher plants often contain as much as 60%
storage protein to make the germinating seed nitrogen-sufficient during this
crucial period of plant development. In corn (Zea mays or maize), a family of
low molecular weight proteins in the kernel called zeins serve this purpose.
Ferritin is a protein found in animal tissues that binds iron, retaining this
essential metal so that it is available for the synthesis of important iron-
containing proteins such as hemoglobin.

4.6.5 Contractile and Motile Proteins

Certain proteins endow cells with unique capabilities for movement. Cell
division, muscle contraction, and cell motility represent some of the ways in
which cells execute motion. Examples include actin and myosin, the filamentous
proteins forming the contractile systems of cells, and tubulin, the major
component of microtubules.

4.6.6 Structural Proteins

An apparently passive but very important role of proteins is their function in
creating and maintaining biological structures. Structural proteins provide
strength and protection to cells and tissues. Monomeric units of structural
proteins typically polymerize to generate long fibers (as in hair). α-Keratins are
insoluble fibrous proteins making up hair, horns, and fingernails. Collagen,
another insoluble fibrous protein, is found in bone, connective tissue, tendons,
and cartilage, where it forms inelastic fibrils of great strength. One-third of the
total protein in a vertebrate animal is collagen. A structural protein having elastic
properties is, appropriately, elastin, an important component of ligaments.

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Certain insects make a structurally useful protein, fibroin (a a-keratin), the major Biochemistry
constituent of cocoons (silk) and spider webs.

4.6.7 Scaffold Proteins (Adapter Proteins)

Some proteins play a recently discovered role in the complex pathways of
cellular response to hormones and growth factors. These proteins, the scaffold
or adapter proteins, have a modular organization in which specific parts
Notes
(modules) of the protein’s structure recognize and bind certain structural
elements in other proteins through protein–protein interactions.

4.6.8 Protective and Exploitive Proteins

In contrast to the passive protective nature of some structural proteins, another
group can be more aptly classified as protective or exploitive proteins because
of their biologically active role in cell defense, protection, or exploitation.
Prominent among the protective proteins are the immunoglobulins or antibodies
produced by the lymphocytes of vertebrates. Antibodies have the remarkable
ability to specifically recognize and neutralize “foreign” molecules resulting
from the invasion of the organism by bacteria, viruses, or other infectious agents.
Another group of protective proteins is the blood-clotting proteins, thrombin and
fibrinogen, which prevent the loss of blood when the circulatory system is
damaged. Arctic and Antarctic fishes have antifreeze proteins to protect their
blood against freezing in the below-zero temperatures of high-latitude seas.
Another class of exploitive proteins includes the toxins produced by bacteria,
such as diphtheria toxin and cholera toxin. It is worth repeating that the great
diversity of function in proteins, as reflected is attained using just 20 amino
acids.

4.7 DIGESTION AND ABSORPTION OF PROTEINS

Several groups of enzymes catalyze the digestion of proteins. There are two main
classes of proteolytic digestive enzymes (proteases), with different specificities
for the amino acids forming the peptide bond to be hydrolyzed. Endopeptidases
hydrolyze peptide bonds between specific amino acids throughout the molecule.
They are the first enzymes to act, yielding a larger number of smaller fragments,
eg, pepsin in the gastric juice and trypsin, chymotrypsin, and elastase secreted
into the small intestine by the pancreas. Exopeptidases catalyze the hydrolysis
of peptide bonds, one at a time, from the ends of polypeptides.
Carboxypeptidases, secreted in the pancreatic juice, release amino acids from
the free carboxyl terminal, and aminopeptidases, secreted by the intestinal
mucosal cells, release amino acids from the amino terminal. Dipeptides, which
are not substrates for exopeptidases, are hydrolyzed in the brush border of
intestinal mucosal cells by dipeptidases. The proteases are secreted as inactive

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Biochemistry zymogens; the active site of the enzyme is masked by a small region of its
peptide chain, which is removed by hydrolysis of a specific peptide bond.
Pepsinogen is activated to pepsin by gastric acid and by activated pepsin
(autocatalysis). In the small intestine, trypsinogen, the precursor of trypsin, is
activated by enteropeptidase, which is secreted by the duodenal epithelial cells;
trypsin can then activate chymotrypsinogen to chymotrypsin, proelastase to
elastase, procarboxypeptidase to carboxypeptidase, and proaminopeptidase
Notes to aminopeptidase.

4.7.1 Free amino acids and small peptides are absorbed by different
mechanisms
The end product of the action of endopeptidases and exopeptidases is a mixture
of free amino acids, di- and tripeptides, and oligopeptides, all of which are
absorbed. Free amino acids are absorbed across the intestinal mucosa by sodium-
dependent active transport. There are several different amino acid transporters,
with specificity for the nature of the amino acid side chain (large or small;
neutral, acidic, or basic). Dipeptides and tripeptides enter the brush border of
the intestinal mucosal cells, where they are hydrolyzed to free amino acids,
which are then transported into the hepatic portal vein.

4.8 SPECIALIZED PRODUCTS OF AMINO ACIDS

PHENYLALANINE, TYROSINE
4.8.1 Phenylalanine
Phenylalanine is first converted to tyrosine. Hyperphenylalaninemias arise
from defects in phenylalanine hydroxylase itself (type I, classic phenylketonuria
or PKU), in dihydrobiopterin reductase (types II and III), or in dihydrobiopterin
biosynthesis (types IV and V). DNA probes facilitate prenatal diagnosis of
defects in phenylalanine hydroxylase or dihydrobiopterin reductase. A diet low
in phenylalanine can prevent the mental retardation of PKU (frequency 1:10,000
births). Elevated blood phenylalanine may be detectable by a less reliable
screening test that employs FeCl3 to detect urinary phenylpyruvate. FeCl3
screening for PKU of the urine of newborn infants is compulsory in the United
States and many other countries.

4.8.2 Tyrosine
The probable metabolic defect in type I tyrosinemia (tyrosinosis) is at
fumarylacetoacetate hydrolase. Therapy employs a diet low in tyrosine and
phenylalanine. Untreated acute and chronic tyrosinosis leads to death from liver
failure. Alternate metabolites of tyrosine are also excreted in type II tyrosinemia

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(Richner-Hanhart syndrome), a defect in tyrosine aminotransferase, and in Biochemistry
neonatal tyrosinemia, due to lowered p-hydroxyphenylpyruvate hydroxylase
activity. Therapy employs a diet low in protein.

4.9 TRANSAMINATION (IMPORTANCE OF

TRANSAMINASES)
The first step in the catabolism of most L-amino acids, once they have reached Notes
the liver, is removal of the α-amino groups, promoted by enzymes called
aminotransferases or transaminases. The effect of transamination reactions is
to collect the amino groups from many different amino acids in the form of L-
glutamate. The glutamate then functions as an amino group donor for biosynthetic
pathways or for excretion pathways that lead to the elimination of nitrogenous
waste products. Cells contain different types of aminotransferases. All
aminotransferases have the same prosthetic group and the same reaction
mechanism. The prosthetic group is pyridoxal phosphate (PLP), the coenzyme
form of pyridoxine, or vitamin B6. Its primary role in cells is in the metabolism
of molecules with amino groups. Pyridoxal phosphate functions as an intermediate
carrier of amino groups at the active site of aminotransferases.

4.10 DEAMINATION
While ammonia, derived mainly from the α-amino nitrogen of amino acids, is
highly toxic, tissues convert ammonia to the amide nitrogen of nontoxic
glutamine. Subsequent deamination of glutamine in the liver releases ammonia,
which is then converted to nontoxic urea. The deamination of amino acids leaves
α-keto acid carbon skeletons. Several of these α-keto acids are citric acid cycle
intermediates. Amino acids are used to synthesize liver and plasma proteins, or
their carbon skeletons are converted to glucose and glycogen by gluconeogenesis;
the ammonia formed by deamination is converted to urea. The α-amino acids
collected in the liver in the form of the amino group of L-glutamate molecules
must be removed from glutamate to prepare them for excretion. In hepatocytes,
glutamate is transported from the cytosol into mitochondria, where it undergoes
oxidative deamination catalyzed by L-glutamate dehydrogenase. In mammals,
this enzyme is present in the mitochondrial matrix.

4.11 UREA CYCLE

Urea is the major end product of nitrogen catabolism in humans. Urea synthesis
is a cyclic process. Synthesis of 1 mol of urea requires 3 mol of ATP plus 1 mol
each of ammonium ion and of the α-amino nitrogen of aspartate. Five enzymes

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Biochemistry catalyze the numbered reactions of Figure 3.8. Of the six participating amino
acids, N-acetylglutamate functions solely as an enzyme activator. The others
serve as carriers of the atoms that ultimately become urea. The major metabolic
role of ornithine, citrulline, and argininosuccinate in mammals is urea
synthesis. Since the ornithine consumed in reaction 2 is regenerated in reaction
5, there is no net loss or gain of ornithine, citrulline, argininosuccinate, or
arginine. Ammonium ion, CO2, ATP, and aspartate are, however, consumed.
Notes
Some reactions of urea synthesis occur in the matrix of the mitochondrion, other
reactions in the cytosol.

4.11.1 Carbamoyl phosphate synthase I initiates Urea biosynthesis

Condensation of CO2, ammonia, and ATP to form carbamoyl phosphate is
catalyzed by mitochondrial carbamoyl phosphate synthase I. Carbamoyl
phosphate synthase I, the rate-limiting enzyme of the urea cycle, is active only
in the presence of its allosteric activator N-acetylglutamate, which enhances the
affinity of the synthase for ATP.

4.11.2 Carbamoyl phosphate plus Ornithine forms Citrulline

L-Ornithine transcarbamoylase catalyzes transfer of the carbamoyl group of
carbamoyl phosphate to ornithine, forming citrulline and orthophosphate. While
the reaction occurs in the mitochondrial matrix, both the formation of ornithine
and the subsequent metabolism of citrulline take place in the cytosol.

4.11.3 Citrulline plus Aspartate forms Argininosuccinate

Argininosuccinate synthase links aspartate and citrulline via the amino group
of aspartate and provides the second nitrogen of urea. The reaction requires ATP
and involves intermediate formation of citrullyl-AMP. Subsequent displacement
of AMP by aspartate then forms citrulline.

4.11.4 Cleavage of Argininosuccinate forms Arginine and Fumarate

Cleavage of argininosuccinate, catalyzed by argininosuccinase, proceeds with
retention of nitrogen in arginine and release of the aspartate skeleton as fumarate.

4.11.5 Cleavage of Arginine releases Urea and re-forms Ornithine

Hydrolytic cleavage of the guanidino group of arginine, catalyzed by liver
arginase, releases urea. The other product, ornithine, reenters liver mitochondria
for additional rounds of urea synthesis.

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4.11.6 Carbamoyl phosphate synthase I is the pacemaker enzyme of the Biochemistry

Urea cycle
The activity of carbamoyl phosphate synthase I is determined by N-
acetylglutamate, whose steady-state level is dictated by its rate of synthesis from
acetyl-CoA and glutamate and its rate of hydrolysis to acetate and glutamate.
These reactions are catalyzed by N-acetylglutamate synthase and N-
acetylglutamate hydrolase, respectively. Major changes in diet can increase the Notes
concentrations of individual urea cycle enzymes 10-fold to 20-fold. Starvation,
for example, elevates enzyme levels, presumably to cope with the increased
production of ammonia that accompanies enhanced protein degradation.

NH4+
CO2

O
CO2+NH4+ H2N C NH2
Urea
CH2NH2+ H2O NH2+
Carbamoyl
2Mg-ATP CH2 C NH
Phosphate
N-Acetyl- Synthase I Arginase
CH2 CH2 NH
glutamate 1 5
H C NH2+ CH2
2 Mg-ADP+M
COO– CH2
L-Ornithine H C NH2+
O O
H2N C O P O– COO– HC COO–
L-Arglnine
O– Ornithine –OOC CH
Carbomoylphosphate Transecarbamoylase Fumarate
P1 2

NH2
Argininosuccinate
C O
4
CH2 NH
CH2
CH2
H C NH2+ NH COO–
COO– 3 C NH CH
L-Citrullline Argininosuccinate CH2
CH2 NH
Sunthase
CH2 COO–

Mg-ATP AMP+Mg-PP1 CH2

COO–
H C NH2+
H2N C H
COO–
CH2 Argininosuccinate
COO–
L-Aspartate

Fig. 4.8: Reactions and intermediates of urea biosynthesis. Reactions 1 and 2 occur
in the matrix of liver mitochondria and reactions 3, 4, and 5 in liver cytosol. CO2
(as bicarbonate), ammonium ion, ornithine, and citrulline enter the
mitochondrial matrix via specific carriers present in the
inner membrane of liver mitochondria.

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Biochemistry

INTEXT QUESTIONS 4.1

I. Choose the best answer
1. The simplest amino acid having only a single hydrogen for an R group
is
Notes (a) Valine (b) Glycine
(c) Histidine (d) Alanine
2. Peptide bond formation is an example of the reaction
(a) Condensation (b) Hydrolysis
(c) Deamination (d) Transformation
3. The characteristic helical or pleated segments formed through hydrogen
bonding interactions between adjacent amino acid residues in the
polypeptide chain of protein is seen in
(a) Primary structure (b) Tertiary structure
(c) Secondary structure (d) Quaternary structure
4. For the synthesis of hemoglobin, the following protein binds iron
(a) Ferritin (b) Myosin
(c) Keratin (d) Tubulin
5. The blood clotting proteins thrombin and fibrinogen comes under
(a) Adapter proteins (b) Structural proteins
(c) Protective proteins (d) Transport proteins
II. Fill in the blanks
6. Trypsinogen, the precursor of trypsin, is activated by ............... in the
small intestine
7. Pyridoxal phosphate (PLP), the coenzyme form of ...............
8. The oxidative deamination of glutamate catalysed by L-glutamate
dehydrogenase takes place in ............... of hepatocytes
9. The rate-limiting enzyme of the urea cycle is ...............
10. Some reactions of urea synthesis occur in the matrix of mitochondria,
other reactions in the ...............
III. Match the following
11. Essential amino acid (a) Ovalbumin
12. Non-essential amino acid (b) Leucine

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13. Storage protein (c) L-glutamate dehydrogenase Biochemistry

14. Oxidative deamination (d) proteases

15. Proteolytic enzymes (e) Alanine

WHAT HAVE YOU LEARNT

Notes
z Both D-amino acids and non-α-amino acids occur in nature, but only L-
α-amino acids are present in proteins.
z The 20 amino acids commonly found as residues in proteins contain an α-
carboxyl group, an a-amino group, and a distinctive R group substituted on
the a-carbon atom. The a-carbon atom of all amino acids except glycine
is asymmetric, and thus amino acids can exist in at least two stereoisomeric
forms.
z Amino acids can be joined covalently through peptide bonds to form
peptides and proteins.
z Cells generally contain thousands of different proteins, each with a different
biological activity.
z Differences in protein function result from differences in amino acid
composition and sequence.
z Every protein has a three-dimensional structure that reflects its function.
Tertiary structure is the complete three dimensional structure of a polypeptide
chain. There are two general classes of proteins based on tertiary structure:
fibrous and globular.
z Proteins are the agents of biological functions, virtually occurring in every
cellular activity in the form of enzymes, regulatory proteins, transport
proteins, storage proteins, contractile and motile proteins, structural proteins,
adapter proteins, and protective and exploitive proteins.
z Proteins are digested at the intestinal region by several groups of proteolytic
digestive enzymes (proteases), with different specificities for the amino
acids forming the peptide bond to be hydrolyzed.
z Phenylalanine is first converted to tyrosine. The defects in phenylalanine
hydroxylase leads to Hyperphenylalaninemias (type I, classic phenylketonuria
or PKU). The metabolic defect in fumarylacetoacetate hydrolase leads to
type I tyrosinemia (tyrosinosis). Alternate metabolites of tyrosine are also
excreted in type II tyrosinemia (Richner-Hanhart syndrome), a defect in
tyrosine aminotransferase.

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Biochemistry z The catabolism of most L-amino acids in the liver is promoted by

transamination and deaminations reactions. Glutamate functions as an
amino group donor for biosynthetic pathways or for excretion pathways that
lead to the elimination of nitrogenous waste products.
z Urea is the major end product of nitrogen catabolism in humans. Urea
synthesis is a cyclic process. Some reactions of urea synthesis occur in the
Notes matrix of the mitochondrion, other reactions in the cytosol.

ANSWERS TO INTEXT QUESTIONS

4.1
I. 1. (b) 2. (a) 3. (c) 4. (a) 5. (c)
II. 6. Enteropeptidase
7. Pyridoxine
8. Mitochondrial matrix
9. Carbamoyl phosphate synthase I
10. Cytosol
III. 11. (b) 12. (e) 13. (a) 14. (c) 15. (d)

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Lipids MODULE
Biochemistry

5
Notes
LIPIDS

5.1 INTRODUCTION
The lipids are a heterogeneous group of compounds, including fats, oils,
steroids, waxes, and related compounds, which are related more by their physical
than by their chemical properties. Lipids are a class of compounds distinguished
by their insolubility in water and solubility in nonpolar solvents. Lipids are
important in biological systems because they form the cell membrane, a
mechanical barrier that divides a cell from the external environment. Lipids also
provide energy for life and several essential vitamins are lipids. Lipids can be
divided in two major classes, nonsaponifiable lipids and saponifiable lipids. A
nonsaponifiable lipid cannot be broken up into smaller molecules by hydrolysis,
which includes triglycerides, waxes, phospholipids, and sphingolipids. A
saponifiable lipid contains one or more ester groups allowing it to undergo
hydrolysis in the presence of an acid, base, or enzyme. Nonsaponifiable lipids
include steroids, prostaglandins, and terpenes. Within these two major classes
of lipids, there are several specific types of lipids important to life, including
fatty acids, triglycerides, glycerophospholipids, sphingolipids, and steroids.
Each of these categories can be further broken down. Nonpolar lipids, such as
triglycerides, are used for energy storage and fuel. Polar lipids, which can form
a barrier with an external water environment, are used in membranes. Polar
lipids include glycerophospholipids and sphingolipids. Fatty acids are important
components of all of these lipids.

OBJECTIVES
After reading this lesson, you will be able to:
z classify lipids
z describe fatty acids and classify them
z enlist functions of lipids
z describe cholesterol and its importance

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Biochemistry
5.2 BIOLOGICAL ROLES OF LIPID
Lipids have the common property of being relatively insoluble in water and
soluble in nonpolar solvents such as ether and chloroform. They are important
dietary constituents not only because of their high energy value but also because
of the fat-soluble vitamins and the essential fatty acids contained in the fat of
natural foods. Fat is stored in adipose tissue, where it also serves as a thermal
Notes insulator in the subcutaneous tissues and around certain organs. Nonpolar lipids
act as electrical insulators, allowing rapid propagation of depolarization waves
along myelinated nerves. Combinations of lipid and protein (lipoproteins) are
important cellular constituents, occurring both in the cell membrane and in the
mitochondria, and serving also as the means of transporting lipids in the blood.
Knowledge of lipid biochemistry is necessary in understanding many important
biomedical areas, e.g., obesity, diabetes mellitus, atherosclerosis, and the role
of various polyunsaturated fatty acids in nutrition and health.

5.3 CLASSIFICATION OF LIPIDS

Lipids are classified as follows:

1. Simple lipids: Esters of fatty acids with various alcohols.

(a) Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state.
(b) Waxes: Esters of fatty acids with higher molecular weight monohydric
alcohols.
2. Complex lipids: Esters of fatty acids containing groups in addition to an
alcohol and a fatty acid.
(a) Phospholipids: Lipids containing, in addition to fatty acids and an
alcohol, a phosphoric acid residue. They frequently have nitrogen
containing bases and other substituents, eg, in glycerophospholipids
the alcohol is glycerol and in sphingophospholipids the alcohol is
sphingosine.
(b) Glycolipids (glycosphingolipids): Lipids containing a fatty acid,
sphingosine, and carbohydrate.
(c) Other complex lipids: Lipids such as sulfolipids and aminolipids.
Lipoproteins may also be placed in this category.
3. Precursor and derived lipids: These include fatty acids, glycerol, steroids,
other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid-
soluble vitamins, and hormones. Because they are uncharged, acylglycerols
(glycerides), cholesterol, and cholesteryl esters are termed neutral lipids.

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5.3.1. Fatty Acids Biochemistry

A fatty acid is a molecule characterized by the presence of a carboxyl group

attached to a long hydrocarbon chain. Therefore these are molecules with a
formula R–COOH where R is a hydrocarbon chain. Fatty acids can be said to
be carboxylic acids, and come in two major varieties.

z Saturated fatty acids do not have any double bonds. A fatty acid is saturated
when every carbon atom in the hydrocarbon chain is bonded to as many Notes
hydrogen atoms as possible (the carbon atoms are saturated with hydrogen).
Saturated fatty acids are solids at room temperature. Animal fats are a source
of saturated fatty acids. In addition, fatty acids pack easily and form rigid
structures (e.g., fatty acids are found in membranes).
z Unsaturated fatty acids can have one or more double bonds along its
hydrocarbon chain. A fatty acid with one double bond is called
monounsaturated. If it contains two or more double bonds, we say that the
fatty acid is polyunsaturated. The melting point of a fatty acid is influenced
by the number of double bonds that the molecule contains and by the length
of the hydrocarbon tail. The more double bonds it contains, the lower the
melting point. As the length of the tail increases, the melting point increases.
Plants are the source of unsaturated fatty acids (Figure 5.1).
–CH = CH – CH = CH –
Unsaturated fatty acid chain
–CH – CH – CH –
Saturated fatty acid chain
Fig. 5.1: Aliphatic chain showing structure of unsaturated fatty acid chain with double
bonds and saturated fatty acid chain with single bonds.

5.4 CLASSIFICATION OF FATTY ACIDS

Naturally occurring fatty acids have cis bonds. Trans-fatty acids are created
artificially using a process called hydrogenation. A trans-fatty acid has a trans
configuration rather than cis configuration at each double bond. This causes the
molecule to straighten. These two stereoisomers can be distinguished in the
following way:

z In a cis stereoisomer, two similar groups attached to the carbon double bond
are found on the same side.
z In a trans stereoisomer, two similar groups attached to the carbon double
bond are found on opposite sides.

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Biochemistry
5.5 ESSENTIAL AND NONESSENTIAL FATTY ACIDS
If a fatty acid can only be obtained from the diet (for humans) then the fatty acid
is an essential fatty acid. Two fatty acids cannot be synthesized in the human
body and are therefore essential. These are linoleic and linolenic fatty acids,
which are both unsaturated. Nonessential fatty acids can be made by the human
body and so do not need to be obtained from diet alone. These are made from
Notes carbohydrates and proteins or from other fatty acids. Fatty acids are an important
source of energy. While carbohydrates or proteins only provide 4 kcal/g of
energy, fatty acids provide more than twice the energy per unit weight of 9 kcal/
g. This is one reason why a high-fat diet can lead to obesity.

5.5.1 Fatty acids are aliphatic carboxylic acids

Fatty acids occur mainly as esters in natural fats and oils but do occur in the
unesterified form as free fatty acids, a transport form found in the plasma. Fatty
acids that occur in natural fats are usually straight-chain derivatives containing
an even number of carbon atoms. The chain may be saturated (containing no
double bonds) or unsaturated (containing one or more double bonds).

5.5.2 Most naturally occurring unsaturated fatty acids have cis double
bonds
The carbon chains of saturated fatty acids form a zigzag pattern when extended,
as at low temperatures. At higher temperatures, some bonds rotate, causing chain
shortening, which explains why biomembranes become thinner with increases
in temperature. A type of geometric isomerism occurs in unsaturated fatty acids,
depending on the orientation of atoms or groups around the axes of double
bonds, which do not allow rotation. If the acyl chains are on the same side of
the bond, it is cis-, as in oleic acid; if on opposite sides, it is trans-, as in elaidic
acid, the trans isomer of oleic acid.
Naturally occurring unsaturated long-chain fatty acids are nearly all of cis
configuration. Thus, oleic acid has an L shape, whereas elaidic acid remains
“straight.” Increase in the number of cis double bonds in a fatty acid leads to
a variety of possible spatial configurations of the molecule – e.g., arachidonic
acid, with four cis double bonds, has “kinks” or a U shape. This has profound
significance on molecular packing in membranes and on the positions occupied
by fatty acids in more complex molecules such as phospholipids. Trans double
bonds alter these spatial relationships. Trans fatty acids are present in certain
foods, arising as a by-product of the saturation of fatty acids during hydrogenation,
or “hardening,” of natural oils in the manufacture of margarine. An additional
small contribution comes from the ingestion of ruminant fat that contains trans
fatty acids arising from the action of microorganisms in the rumen.

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Biochemistry
5.6 COMMON LIPIDS AND THEIR FUNCTIONS
5.6.1 Triglycerides
A triglyceride (often called triacylgycerol) is a fatty acid trimester of glycerol.
Triglycerides are important for human health in that they provide most of the
lipids in our diet. Glycerol has three hydroxyl groups. Fatty acids can be attached
at these three sites forming a triglyceride. One important characteristic of a
tryglycerol is its state at room temperature. The degree of saturation and the Notes
length of their chains attached to the glycerol backbone both determine their state
at room temperature.
z Short-chain unsaturated triglycerides are liquid at room temperature.
z Long-chain saturated triglycerides are solid at room temperature.
Animal fats (lard) contain a high amount of saturated triglycerides while plant
oils (vegetable oil) contain a high amount of unsaturated triglycerides. While
neither is healthy when consumed in excess, vegetable oils are far healthier than
lard.

5.6.2 Triglycerides are the main storage forms of fatty acids

The triglycerides are esters of the trihydric alcohol glycerol and fatty acids.
Mono- and diacylglycerols wherein one or two fatty acids are esterified with
glycerol are also found in the tissues. These are of particular significance in the
synthesis and hydrolysis of triacylglycerols.

5.6.3 The role of triglycerides in health

While fat may seem bad, triglycerides play many important roles in the body.
For example, triglycerides can be used for energy storage in animals. This food
reserve can be called upon during periods of starvation, with the high-calorie
content of the fatty acids adding to the value of storing fat and providing much
needed energy. In addition, triglycerides can provide insulation for animals in
the form of body fat, which allows them to survive in colder temperatures. These
two roles played by fat in the body, which arose over eons of evolution, are now
deemed undesirable in modern industrialized society where humans no longer
face starvation or have to deal directly with cold weather.

5.6.4 Phospholipids are the main lipid constituents of membranes

Phospholipids may be regarded as derivatives of phosphatidic acid, in which the
phosphate is esterified with the OH of a suitable alcohol. Phosphatidic acid is
important as an intermediate in the synthesis of triacylglycerols as well as
phosphoglycerols but is not found in any great quantity in tissues. Amphipathic
lipids self-orient at oil:water interfaces. They form Membranes, Micelles,

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Biochemistry Liposomes, & Emulsions (Figure 5.2). In general, lipids are insoluble in water
since they contain a predominance of nonpolar (hydrocarbon) groups. However,
fatty acids, phospholipids, sphingolipids, bile salts, and, to a lesser extent,
cholesterol contain polar groups. Therefore, part of the molecule is hydrophobic,
or water-insoluble; and part is hydrophilic, or water-soluble. Such molecules are
described as amphipathic. They become oriented at oil:water interfaces with the
polar group in the water phase and the nonpolar group in the oil phase.
Notes

Fig. 5.2: Structure of liposome showing aqueous cavity at the centre of fatty acid bilayer.

A bilayer of such amphipathic lipids has been regarded as a basic structure in

biologic membranes. When a critical concentration of these lipids is present in
an aqueous medium, they form micelles. Aggregations of bile salts into micelles
and liposomes and the formation of mixed micelles with the products of fat
digestion are important in facilitating absorption of lipids from the intestine.
Liposomes may be formed by sonicating an amphipathic lipid in an aqueous
medium. They consist of spheres of lipid bilayers that enclose part of the aqueous
medium. They are of potential clinical use – particularly when combined with
tissue specific antibodies – as carriers of drugs in the circulation, targeted to
specific organs, eg, in cancer therapy. In addition, they are being used for gene
transfer into vascular cells and as carriers for topical and transdermal.

5.7 CHOLESTEROL AND ITS IMPORTANCE

Cholesterol is an important lipid found in the cell membrane. It is a sterol, which
means that cholesterol is a combination of a steroid and an alcohol (Figure 5.3).
It is an important component of cell membranes and is also the basis for the
synthesis of other steroids, including the sex hormones estradiol and testosterone,
as well as other steroids such as cortisone and vitamin D. In the cell membrane,
the steroid ring structure of cholesterol provides a rigid hydrophobic structure
that helps boost the rigidity of the cell membrane. Without cholesterol the cell
membrane would be too fluid. In the human body, cholesterol is synthesized in
the liver.

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Biochemistry

H H
HO

Fig. 5.3: Structure of cholesterol. Notes

Cholesterol is insoluble in the blood, so when it is released into the blood stream
it forms complexes with lipoproteins. Cholesterol can bind to two types of
lipoprotein, called high-density lipoprotein (HDL) and low-density lipoprotein
(LDL). A lipoprotein is a spherical molecule with water soluble proteins on the
exterior. Therefore, when cholesterol is bound to a lipoprotein, it becomes blood
soluble and can be transported throughout the body. HDL cholesterol is
transported back to the liver. If HDL levels are low, then the blood level of
cholesterol will increase.
High levels of blood cholesterol are associated with plaque formation in the
arteries, which can lead to heart disease and stroke. While most cholesterol in
the body is synthesized in the liver, dietary cholesterol also adds to the total blood
levels. Cholesterol intake from the diet enters the bloodstream in the LDL form.
This helps explain why consumption of foods with high-cholesterol content can
lead to increased blood levels of cholesterol which is bad for health. So reducing
the cholesterol in the diet can lower the blood level of cholesterol. This can
reduce the amount of plaque formation. Aerobic exercise also contributes to
health by increasing HDL levels in the blood. Hence more cholesterol is returned
to the liver leading to a lower blood level of cholesterol, and reduced plaque
formation.

5.8 DIGESTION AND ABSORPTION OF LIPIDS

The major lipids in the diet are triacylglycerols and, to a lesser extent,
phospholipids. These are hydrophobic molecules and must be hydrolyzed and
emulsified to very small droplets (micelles) before they can be absorbed. The
fat-soluble vitamins – A, D, E, and K – and a variety of other lipids (including
cholesterol) are absorbed dissolved in the lipid micelles. Absorption of the fat-
soluble vitamins is impaired on a very low fat diet. Hydrolysis of triacylglycerols
is initiated by lingual and gastric lipases that attack the sn-3 ester bond, forming
1,2-diacylglycerols and free fatty acids, aiding emulsification. Pancreatic lipase
is secreted into the small intestine and requires a further pancreatic protein,
colipase, for activity. It is specific for the primary ester links – ie, positions 1
and 3 in triacylglycerols – resulting in 2-monoacylglycerols and free fatty acids
as the major end-products of luminal triacylglycerol digestion. Monoacylglycerols

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Biochemistry are hydrolyzed with difficulty to glycerol and free fatty acids, so that less than
25% of ingested triacylglycerol is completely hydrolyzed to glycerol and fatty
acids.
Bile salts, formed in the liver and secreted in the bile, enable emulsification of
the products of lipid digestion into micelles and liposomes together with
phospholipids and cholesterol from the bile. Because the micelles are soluble,
they allow the products of digestion, including the fat soluble vitamins, to be
Notes transported through the aqueous environment of the intestinal lumen and permit
close contact with the brush border of the mucosal cells, allowing uptake into
the epithelium, mainly of the jejunum. The bile salts pass on to the ileum, where
most are absorbed into the enterohepatic circulation. All long-chain fatty acids
absorbed are converted to triacylglycerol in the mucosal cells and, together with
the other products of lipid digestion, secreted as chylomicrons into the
lymphatics, entering the blood stream via the thoracic duct. Free fatty acids are
removed from the blood very rapidly and oxidized (fulfilling 25–50% of energy
requirements in starvation) or esterified to form triacylglycerol in the tissues. In
starvation, esterified lipids from the circulation or in the tissues are oxidized as
well, particularly in heart and skeletal muscle cells, where considerable stores
of lipid are to be found.

Fig. 5.4: Schematic representation of fat digestion at the intestinal region and absorption
and transport of lipids in the form of triacylglycerol, fatty acids, cholesterol by
chylomicrons. Fatty acids enter the blood circulation and stored in the adipose tissue.
The free fatty acid uptake by tissues is related directly to the plasma free fatty
acid concentration, which in turn is determined by the rate of lipolysis in adipose
tissue. After dissociation of the fatty acid-albumin complex at the plasma
membrane, fatty acids bind to a membrane fatty acid transport protein that acts

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as a transmembrane cotransporter with Na+. On entering the cytosol, free fatty Biochemistry
acids are bound by intracellular fatty acid-binding proteins. The role of these
proteins in intracellular transport is thought to be similar to that of serum
albumin in extracellular transport of long chain fatty acids. Triacylglycerol is
transported from the intestines in chylomicrons and from the liver in very low
density lipoproteins. By definition, chylomicrons are found in chyle formed only
by the lymphatic system draining the intestine. They are responsible for the
transport of all dietary lipids into the circulation. Small quantities of VLDL are Notes
also to be found in chyle; however, most of the plasma VLDL is of hepatic origin.
They are the vehicles of transport of triacylglycerol from the liver to the
extrahepatic tissues (Figure 5.4).

5.9 CLASSIFICATION AND FUNCTIONS OF

LIPOPROTEINS
Cholesterol and other lipids are carried on plasma lipoproteins. Cholesterol and
cholesteryl esters, like triacylglycerols and phospholipids, are essentially
insoluble in water, yet must be moved from the tissue of origin to the tissues
in which they will be stored or consumed. They are carried in the blood plasma
as plasma lipoproteins, macromolecular complexes of specific carrier proteins,
apolipoproteins, with various combinations of phospholipids, cholesterol,
cholesteryl esters, and triacylglycerols. Apolipoproteins (“apo” designates the
protein in its lipid-free form) combine with lipids to form several classes of
lipoprotein particles, spherical complexes with hydrophobic lipids in the core
and hydrophilic amino acid side chains at the surface.

Fig. 5.5: Structure of chylomicron.

Different combinations of lipids and proteins produce particles of different

densities, ranging from chylomicrons (Figure 5.5) to high-density lipoproteins.
Each class of lipoprotein has a specific function, determined by its point of
synthesis, lipid composition, and apolipoprotein content. At least nine different

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Biochemistry apolipoproteins are found in the lipoproteins of human plasma (Table),

distinguishable by their size, their reactions with specific antibodies, and their
characteristic distribution in the lipoprotein classes. These protein components
act as signals, targeting lipoproteins to specific tissues or activating enzymes that
act on the lipoproteins. Chylomicrons with the movement of dietary
triacylglycerols from the intestine to other tissues are the largest of the
lipoproteins and the least dense, containing a high proportion of triacylglycerols.
Notes Chylomicrons are synthesized in the ER of epithelial cells that line the small
intestine, then move through the lymphatic system and enter the bloodstream via
the left subclavian vein.
The apolipoproteins of chylomicrons include apoB-48 (unique to this class of
lipoproteins), apoE, and apoC-II. ApoC-II activates lipoprotein lipase in the
capillaries of adipose, heart, skeletal muscle, and lactating mammary tissues,
allowing the release of free fatty acids to these tissues. Chylomicrons thus carry
dietary fatty acids to tissues where they will be consumed or stored as fuel. The
remnants of chylomicrons (depleted of most of their triacylglycerols but still
containing cholesterol, apoE, and apoB-48) move through the bloodstream to
the liver. Receptors in the liver bind to the apoE in the chylomicron remnants
and mediate their uptake by endocytosis. In the liver, the remnants release their
cholesterol and are degraded in lysosomes. When the diet contains more fatty
acids than are needed immediately as fuel, they are converted to triacylglycerols
in the liver and packaged with specific apolipoproteins into very-low-density
lipoprotein (VLDL). Excess carbohydrate in the diet can also be converted to
triacylglycerols in the liver and exported as VLDLs. In addition to triacylglycerols,
VLDLs contain some cholesterol and cholesteryl esters, as well as apoB-100,
apoC-I, apoC-II, apoC-III, and apo-E. These lipoproteins are transported in the
blood from the liver to muscle and adipose tissue, where activation of lipoprotein
lipase by apoC-II causes the release of free fatty acids from the VLDL
triacylglycerols.
Adipocytes take up these fatty acids, reconvert them to triacylglycerols, and store
the products in intracellular lipid droplets; myocytes, in contrast, primarily
oxidize the fatty acids to supply energy. Most VLDL remnants are removed from
the circulation by hepatocytes. The uptake, like that for chylomicrons, is
receptor-mediated and depends on the presence of apoE in the VLDL remnants.
The loss of triacylglycerol converts some VLDL to VLDL remnants (also called
intermediate density lipoprotein, IDL); further removal of triacylglycerol from
VLDL produces low-density lipoprotein (LDL). Very rich in cholesterol and
cholesteryl esters and containing apoB-100 as their major apolipoprotein, LDLs
carry cholesterol to extrahepatic tissues that have specific plasma membrane
receptors that recognize apoB-100 (Table 5.1).

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The fourth major lipoprotein type, high-density lipoprotein (HDL), originates in Biochemistry
the liver and small intestine as small, protein-rich particles that contain relatively
little cholesterol and no cholesteryl esters. HDLs contain apoA-I, apoC-I, apoC-
II, and other apolipoproteins, as well as the enzyme lecithin-cholesterol acyl
transferase (LCAT), which catalyzes the formation of cholesteryl esters from
lecithin (phosphatidylcholine) and cholesterol. LCAT on the surface of nascent
(newly forming) HDL particles converts the cholesterol and phosphatidylcholine
Notes
of chylomicron and VLDL remnants to cholesteryl esters, which begin to form
a core, transforming the disk-shaped nascent HDL to a mature, spherical HDL
particle. This cholesterol-rich lipoprotein then returns to the liver, where the
cholesterol is unloaded; some of this cholesterol is converted to bile salts.
Table 5.1. Lipoproteins source and components and apolipoproteins.
Lipoprotein Source Main lipid Apolipoproteins
components
Chylomicrons Intestine Triacyglycerol A-I, A-II, A-IV, B
48, C-I, C-II, C-III,E
Chylomicron Chylomicrons Triacylglycerol, B-48, E
remnants phospholipids,
cholesterol
VLDL Liver (intestine) Triacylglycerol B-100, C-I, C-II,
C-III
IDL VLDL Triacylglycerol, B-100, E
cholesterol
LDL VLDL Cholesterol B-100
HDL Liver, intestine, Phospholipids, A-I, A-II, A-IV, C-I,
VLDL, cholesterol C-II, C-III, D, E
chylomicrons

5.10 KETONE BODY TYPES AND IMPORTANCE

Ketone bodies formed in liver to free up CoA for b-oxidation metabolized in
other tissues, including brain, as an energy source. The formation of ketone
bodies is a consequence of prolonged metabolism of fat. Their formation in the
liver actually enables liver to metabolize even more fat by freeing up CoA that
would otherwise be tied up as acetyl-CoA waiting to get into the TCA cycle. The
liver exports the ketone bodies; and other tissues, particularly the brain, can
adapt to use them. With increasing metabolism of fat through b-oxidation, much
of the mitochondrial CoA pool may become tied up as acyl- or acetyl-CoA. In
such cases, the supply of free CoA can be diminished, and this may limit the

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Biochemistry rate of b-oxidation. Upon prolonged fasting and heavy reliance on fat for energy,
the liver induces the enzymes required for the formation of ketone bodies and
brain induces enzymes required for their metabolism.
Ketone bodies are formed in the liver mitochondria by the condensation of three
acetyl-CoA units. Two acetyl-CoAs are condensed to form acetoacetyl-CoA.
The acetoacetyl-CoA is condensed with another acetyl-CoA to give
hydroxymethylglutaryl-CoA (HMG-CoA). This is then split by HMG-CoA lyase
Notes to acetyl-CoA and acetoacetate. The hydroxybutyrate arises from acetoacetate
by reduction. The overall sum of ketone body formation is the generation of
acetoacetate (or hydroxybutyrate) and the freeing-up of the 2 CoAs that were
trapped as acetyl-CoA. Ketone bodies are utilized in other tissues (but not the
liver) by converting the acetoacetate to acetoacetyl-CoA and then converting the
acetoacetyl-CoA to 2 acetyl-CoA, which are burned in the muscle mitochondria.
Increased fatty acid oxidation is a characteristic of starvation and of diabetes
mellitus, leading to ketone body production by the liver (ketosis). Ketone bodies
are acidic and when produced in excess over long periods, as in diabetes, cause
ketoacidosis, which is ultimately fatal. Because gluconeogenesis is dependent
upon fatty acid oxidation, any impairment in fatty acid oxidation leads to
hypoglycemia. This occurs in various states of carnitine deficiency or deficiency
of essential enzymes in fatty acid oxidation, eg, carnitine palmitoyltransferase,
or inhibition of fatty acid oxidation by poisons, eg, hypoglycin.

5.10.1 Ketogenesis occurs when there is a high rate of fatty acid oxidation
in the liver
Under metabolic conditions associated with a high rate of fatty acid oxidation,
the liver produces considerable quantities of acetoacetate and β-hydroxybutyrate.
Acetoacetate continually undergoes spontaneous decarboxylation to yield
acetone. These three substances are collectively known as the ketone bodies.
Acetoacetate and 3-hydroxybutyrate are interconverted by the mitochondrial
enzyme D(β)-3-hydroxybutyrate dehydrogenase; the equilibrium is controlled
by the mitochondrial [NAD+]/ [NADH] ratio, ie, the redox state. The concentration
of total ketone bodies in the blood of well-fed mammals does not normally
exceed 0.2 mmol/L. Extrahepatic tissues utilize them as respiratory substrates.
The net flow of ketone bodies from the liver to the extrahepatic tissues results
from active hepatic synthesis coupled with very low utilization. The reverse
situation occurs in extrahepatic tissues.

5.11 ATHEROSCLEROSIS
Unregulated cholesterol production can lead to serious human disease. When the
sum of cholesterol synthesized and cholesterol obtained in the diet exceeds the
amount required for the synthesis of membranes, bile salts, and steroids,

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pathological accumulations of cholesterol in blood vessels (atherosclerotic Biochemistry
plaques) can develop, resulting in obstruction of blood vessels (atherosclerosis).
Atherosclerosis is linked to high levels of cholesterol in the blood, and
particularly to high levels of LDL-bound cholesterol; there is a negative
correlation between HDL levels and arterial disease. In familial
hypercholesterolemia, a human genetic disorder, blood levels of cholesterol are
extremely high and severe atherosclerosis develops in childhood.
The metabolism of LDL and HDL intersects in the production and control of fatty Notes
streaks and potential plaques in blood vessels. Damage to the endothelium may
be related to many factors, including normal turbulence of the blood, elevated
LDL, especially modified or oxidized LDL, free radicals from cigarette smoking,
homocystinemia, diabetes (glycation of LDL), and hypertension. The
atherosclerotic lesion represents an inflammatory response sharing several
characteristics with granuloma formation, and not simple deposition of cholesterol
in the blood vessel.
Local inflammation recruits monocytes and macrophages with subsequent
production of reactive oxygen species. LDL can become oxidized and then taken
up, along with other inflammatory debris, by macrophages, which can become
laden with cholesterol (foam cells). Initially the subendothelial accumulation of
cholesterol-laden macrophages produces fatty streaks. As the fatty streak
enlarges over time, necrotic tissue and free lipid accumulates, surrounded by
epithelioid cells and eventually smooth muscle cells, an advanced plaque with
a fibrous cap. The plaque eventually begins to occlude the blood vessel, causing
ischemia and infarction in the heart, brain, or extremities. Eventually the fibrous
cap may thin, and the plaque becomes unstable, leading to rupture and
thrombosis.
HDL may be protective by picking up accumulating cholesterol before the
advanced lesion forms. Apo-l activates LCAT, which in turn adds a fatty acid
to cholesterol to produce a cholesterol ester that dissolves in the core of the HDL.
The HDL may subsequently be picked up by the liver through the apoE receptor
or deliver cholesterol through the scavenger receptor SR-Bl (reverse cholesterol
transport from the periphery to the liver). The HDL may also transfer the
cholesterol to an IDL reforming a normal, unoxidized LDL particle.

INTEXT QUESTIONS 5.1

I. Choose the best answer
1. Lipid stored in the form of energy in the body adipose tissue is
(a) Steroid (b) Fatty acid
(c) Sphingolipid (d) Phospholipid

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Biochemistry 2. Combination of lipid and protein serving as the means of transporting

lipids in the blood is
(a) Glycoprotein (b) Phosphoprotein
(c) Lipoprotein (d) None
3. The enzyme which catalyzes the formation of cholesteryl esters from
lecithin and cholesterol.
Notes (a) Lecithin-cholesterol acyl transferase
(b) D(b)-3-hydroxybutyrate dehydrogenase
(c) Lipoprotein lipase
(d) Carnitine palmitoyltransferase
4. A human genetic disorder in which the extremely high levels of blood
cholesterol develops atherosclerosis is
(a) Familial hypercholesterolemia
(b) Obesity
(c) Apolipoproteinemia
(d) None
5. The molecule acting as both hydrophobic and hydrophilic is termed
as
(a) Amphipathic (b) Aliphatic
(c) Polar (d) Nonpolar
II. Fill in the blanks
1. .................... act as electrical insulators, allowing rapid propagation
of depolarization waves along myelinated nerves.
2. Vegetable oil contains high amount of .................... triglycerides.
3. Aggregation of bile salts into .................... facilitates the absorption
of lipids from the intestine.
4. A lipoprotein is a spherical molecule with water soluble ....................
on the exterior.
5. Triglyceride is hydrolysed to .................... and ....................
III. Match the following
1. Cholestrol (a) Phospholipid
2. Liposome (b) Diabetes mellitus
3. Linoleic (c) Steroid
4. Glycerophospholipid (d) Unsaturated fatty acid
5. Ketoacidosis (e) Amphipathic molecule

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Biochemistry

WHAT HAVE YOU LEARNT

z Lipids are a heterogeneous group of compounds, including fats, oils,
steroids, waxes, and related compounds and can be divided in two major
classes as nonsaponifiable and saponifiable lipids.
z Saturated fatty acids do not have any double bonds. Animal fats are a source
of saturated fatty acids. Unsaturated fatty acids can have one or more double Notes
bonds along its hydrocarbon chain. Plants are the source of unsaturated fatty
acids.
z Fatty acid that cannot be synthesized by the body is an essential fatty acid.
Linoleic and linolenic are the two essential fatty acids (both are unsaturated).
Nonessential fatty acids can be made by the human body and so do not need
to be obtained from diet alone.
z Animal fats (lard) contain a high amount of saturated triglycerides while
plant oils (vegetable oil) contain a high amount of unsaturated triglycerides.
While neither is healthy when consumed in excess, vegetable oils are far
healthier than lard.
z Phospholipids are amphipathic lipids seen as a bilayer and form the
structure in biologic membranes.
z Cholesterol is a sterol and an important component of cell membranes. It
is also the basis for the synthesis of other steroids, including the sex
hormones estradiol and testosterone, as well as other steroids such as
cortisone and vitamin D.
z Cholesterol is insoluble in the blood, so when it is released into the blood
stream it forms complexes with lipoproteins. Cholesterol can bind to two
types of lipoprotein, called high-density lipoprotein (HDL) and low-density
lipoprotein (LDL).
z High levels of blood cholesterol are associated with plaque formation in
the arteries, which can lead to heart disease and stroke.
z The major lipids in the diet are triacylglycerols and, to a lesser extent,
phospholipids. These are hydrophobic molecules and must be hydrolyzed
and emulsified to very small droplets (micelles) before they can be
absorbed.
z The fat-soluble vitamins – A, D, E, and K – and a variety of other lipids
(including cholesterol) are absorbed dissolved in the lipid micelles.
z Bile salts, formed in the liver and secreted in the bile, enable emulsification
of the products of lipid digestion into micelles and liposomes together with
phospholipids and cholesterol from the bile. Because the micelles are
soluble, they allow the products of digestion absorbed at the intestinal
region.
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Biochemistry z Low density lipoprotein (LDL), high density lipoprotein (HDL), very low
density lipoprotein (VLDL), and intermediate density lipoprotein (IDL) are
the types of lipoprotein.
z Ketogenesis occurs when there is a high rate of fatty acid oxidation in the
liver. Acetoacetate, β-hydroxybutyrate and acetone are the products of fatty
acid oxidation.
Notes z Atherosclerosis is linked to high levels of cholesterol in the blood, and
particularly to high levels of LDL-bound cholesterol; there is a negative
correlation between HDL levels and arterial disease. LDL-cholesterol is
called as bad cholesterol and HDL-cholesterol is called as good cholesterol.

TERMINAL QUESTIONS
1. Classify lipids with suitable examples.
2. What are essential and nonessential fatty acids.
3. Write short on importance of cholesterol.
4. Write short note on digestion and absorption of lipids.

ANSWERS TO INTEXT QUESTIONS

5.1
I. 1. (b) 2. (c) 3. (a) 4. (a) 5. (a)
II. 1. Nonpolar lipids
2. Unsaturated
3. Micelles
4. Proteins
5. Glycerol and free fatty acid
III. 11. (c) 12. (e) 13. (d) 14. (a) 15. (b)

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Nucleotides MODULE
Biochemistry

6
Notes
NUCLEOTIDES

6.1 INTRODUCTION
Nucleotides play major important roles in cellular metabolism. They are very
essential for chemical links in the response of cells to hormones and other
extracellular stimuli. They also act as the structural components of an array of
enzyme cofactors and metabolic intermediates. Most importantly they are the
constituents of nucleicacids: deoxyribonucleic acid (DNA) and ribonucleicacid
(RNA). Nucleic acids are the molecular repositories of genetic information. The
structure of every protein, and ultimately of every biomolecule and cellular
component are programmed in form of nucleotide sequence of a cell’s nucleic
acids. The ability of nucleic acids to store and transmit genetic information from
one generation to the next is a fundamental condition for life.

This chapter provides an overview of the structure of nucleic acids, chemistry

of nucleotides, nucleotide metabolism, function of nucleic acid and diseases of
error in nucleotide metabolism with special reference to gout.

OBJECTIVES
After reading this lesson, you will be able to:

z describe the chemistry of nucleotides

z describe the structure of nucleic acids
z explain the characteristics of nucleic acids
z describe the nucleotide metabolism
z enlist the functions of nucleic acid

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Biochemistry
6.2 STRUCTURE AND CHEMISTRY OF NUCLEIC ACIDS
In all living organisms the amino acid sequence of every protein and the
nucleotide sequence of every RNA, is specified by a nucleotide sequence in the
cell’s DNA. Segment of DNA molecule that encodes a protein or RNA, is
referred to as a gene.

Notes 6.2.1 Chemistry of nucleic acid

Nucleotides are organic compounds that are monomeric units of nucleic
acids,they are of different types and structures and have variety of physiological
functions. Nucleic acids are responsible for storage and transmission of genetic
information. Nucleic acids are chemically composed of polymers of nucleotides
joined together by phosphodiester linkages (bonds). Nucleic acids are broadly
divided into two major types; Ribonucleic acid (RNA) which is single stranded
containing Adenine (A), Uracil (U), Cytosine (C) and Guanine (G) ribonucleotides
and deoxyribonucleic acid which is double stranded containing Adenine,
Thymine (T), Cytosine and Guanine deoxyribonucleotides.

6.2.1.1 Nucleosides
The nitrogenous bases and pentose sugars associated structure gives a compound
called nucleoside. Based on the type of nitrogenous base and the types of sugar
it is liked to, different types of nucleotides are formed each having its own
characteristic and structure.

NHZ

N
N

N N
HO
O
H H
H H
OH OH

Fig. 6.1

Purines and pyrimidines are the components of nitrogenous bases. The purine
bases contains the purine ring (double ringsystem) while the pyrimidine base
contain pyrimidine ring (single ring structure). The purine bases include Adenine

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and Guanine. The unusual forms of purines are hypoxathine, 1 methylguanine, Biochemistry
1 methylhypoxanthine etc. While the pyrimidine includes cytosine, thymine
anduracil and its unusual forms are 5-methylcytosine, Thiouracil etc.
Purines Pyrimidines
NH2 O O O NH2

C N C N C C C
N 1 6 5C 7 HN 1 6 5C 7 HN 3 4 5 CH HN 3 4 5 CH3 N 3 4 5 CH
8 CH 8 CH 2 2 2
HC 2 3 4C 9 C 2 3 4C 9 C 1 6 CH C 1 6 CH C 1 6 CH Notes
N N H 2N N N O N O N O N
H H H H H
Adenine (A) Guanine (G) Uracil (U) Thymine (T) Cytosine (C)
Fig. 6.2

In nucleosides, nitrogenous bases are joined to pentose sugar through the

hemiacetal hydroxyl group on the C-1 (first carbon atom of the sugar). The
purines are attached to the sugar through the N-9 nitrogen atom while pyrimidine
are attached through the N-1 nitrogen atom.

6.2.1.2 Nucleotides
Nucleotides are phosphoric acid esters of nucleosides. Nucleotides contain
nitrogenous bases, sugars and phosphoric acids in ester linkage. The nitrogenous
base, presentin nucleotides are purines: Adenine and Guanine; pyrimidines:
Cytosine,Thymine and Uracil. The uracil can only be found in ribonucleotides
while thymine base can only be found in deoxyribonucleotides.
The sugar in nucleotides is the pentose sugar which could be ribose and
deoxyribose. Sugar are esterified to a phosphoric acid residue at positions (2,
3 or 5) in ribose and (3 or 5) in the deoxyribose where the ester bonds could
be formed. In addition, the nucleotides could be in form of mono,di and
triphosphates.
O

N
N

O N N NHZ

–O P O O
H H
O–
H H
OH OH
Fig. 6.3

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Biochemistry O
NHZ
N HN N
N
N H 2N N N
O– N O–
O –O P O CH2 O
–O P O CH2
O H H O H H
H H
H H
Notes
OH OH OH OH
Nucleotide: Deoxyadenylate Deoxyguanylate
(deoxyadenosine (deoxyguanosine
5¢-monophosphate) 5¢-monophosphate)
Symbols: A. dA. dAMP G. dG. dGMP

Nucleoside: Deoxyadenosine Deoxyadenosine

NHZ
O
CH3 N
HN
O N
O– O N O–
O –O P O CH2 O
–O P O CH2
O H H O H H
H H
H H
OH OH OH OH
Deoxythymidylate Deoxycytidylate
(deoxythymidine (deoxycytidine
5¢-monophosphate) 5¢-monophosphate)
T. dT. dTMP G. dG. dGMP

Deoxythymidine Deoxycytidine

(a) Deoxyribonucleotides

NHZ O

N HN N
N
N H2N N N
O– N O–
O –O P O CH2 O
–O P O CH2
O H H O H H
H H
H H
OH OH OH OH
Nucleotide: Adenylate (adenosine) Guanylate (guanosine
5¢-monophosphate 5¢-monophosphate)
Symbols: A. AMP G. dGMP
Nucleoside: Adenosine Deoxyadenosine

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Biochemistry
O NHZ

N
HN

O N O N
O– O–
O –O P O CH2 O
–O P O CH2
O H H O H H
H H Notes
H H
OH OH OH OH
Uridylate (uridine Cytidylate (cytidine
5¢-monophosphate) 5¢-monophosphate)
U. UMP C. CMP
Uridine Cytidine
(b) Ribonucleotides
Fig. 6.4

Compound that have their structures derived from nucleotide structures are
called as nucleotide derivatives. They share close structural features of
nucleotides. Nicotinamide Adenine dinucleotide (NAD), Nicotinamide Adenine
dinucleotide phosphate (NADP), flavine adeninedinucleotide (FAD) are some
of the examples for nucleotide derivatives.

INTEXT QUESTIONS 6.1

1. Segment of DNA molecule that encodes a protein is called as ................
(a) RNA (b) gene (c) interon (d) operon
2. Nucleotides are organic compounds that are monomeric units of ................
3. The nitrogenous bases and ................ associated structure gives a compound
called nucleoside
4. ................ are phosphoric acid esters of nucleosides

6.2.1.3 Structure of different types of DNA

The primary structure of a nucleicacid is based on nucleotide sequence. Any
regular, stable structure taken up by some or all of the nucleotides in a nucleic
acid can be referred to as secondary structure. The complex folding of large
chromosomes within eukaryotic chromatin and bacterial nucleoids is generally
considered tertiary structure. The structure of DNA was worked out by bringing
together a number of observations from various sources such as

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Biochemistry (a) That DNA from different sources have remarkable similarity in their Xray
diffraction patterns; suggesting that DNA molecules have uniform molecular
pattern and consists polynucleotide chains arranged in helical structure.
(b) The ratio of the bases (A: T and C: G) is very close to one. In base pairing
event A and T can be paired with a maximum of two hydrogen bonds
between them while C and G will have a maximum of three bonds.
Notes (c) The long polynucleotide chains were held together through bonds between
these residues.
Using these observations, Watson and Crick constructed a model of DNA
structures in 1953.

Fig. 6.5

The Watson-Crick Model consists of sugar molecule joined together by

phosphate diesters. Bases were observed to be projecting perpendicularly from
the chains into the central axis. For each adenine projecting inwards, there is a
corresponding thymine from the other chain and for each cytosine, there is a
guanine. A-T and C-G are held together by two and 3 hydrogen bonds
respectively. The two chains are however not identical because of base pairings.
The chains donot run in the same direction with respect to linkage between the
nucleotides, rather they are anti-parallel.
Structurally DNA exists as three different forms namely: A, B and Z form. The
A form of DNA posses’ right handed helix with a diameter of 26Å. The A form
of DNA contains 11 base pair per helix turn and base turns rise of helix is 2.6Å.
The B form of the DNA is considered as Watson-Crick DNA structure. In B form,
the DNA helix is arranged as left handed. The B form of DNA contains 0.34 nm

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between bp, 3.4 nm per turn, about 10 bp per turn1.9 nm (about 2.0 nm or 20 Biochemistry
Angstroms) in diameter. The Z form of DNA is a more radical departure from
B-DNA with left handed helical rotation. Ion Z form of DNA each helical turn
consist of 12 base pairs. Structures appear to be more slender and elongated.

Notes

Fig. 6.6

Comparison between different forms of DNA

Character A form B form Z form
helix sense Right handed Right handed Left handed
bp per turn 10 11 12
vertical rise per bp 3.4 Å 2.56 Å 3.7Å
rotation per bp +36° +33° +30°
helical diameter 19 Å 23 Å 18 Å

INTEXT QUESTIONS 6.2

1. The primary structure of a nucleic acid is based on ................. nucleotide
sequence
2. In base pairing event A and T can be paired with a maximum of two
hydrogen bonds
(a) 1 (b) 2 (c) 3 (d) 4
3. Watson and Crick constructed a model of DNA structures in the year
................. 1953
4. Structurally DNA exists as three different forms namely .................

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Biochemistry 6.2.2 Structure of different types of RNA

RNA in solution is dynamic molecules in solution. They undergo changes in
conformation during synthesis, processing and functioning. RNA (Ribo nucleic
acid) molecule exist as three different forms namely,
(a) Primary structure
(b) Secondary structure
Notes
(c) Tertiary structure
Unlike DNA, RNA molecules in primary structure are single stranded linear
polymer containing nucleotides joined by phosphodiester linkage. The linkage
of the ribonucleotides in RNA is 3’5’ phosphodiester link involve 3’-OH group
of ribose and 5’-phosphate group of another ribonucleotide. In eukaryotes the
length of RNA molecule ranges from 65 nucleotides to 6000 nucleotides.
In secondary structure the single stranded RNA molecule may have double
helical regions formed by hydrogen bonding between complimentary base
sequences within the RNA molecule. In tertiary structure the association of RNA
with proteins enables the RNA molecule to be stable and also fold into specific
conformations. The “L shaped” conformation of the tRNA conformations held
in position not only by the base pairing interactions but also other interactions.
RNA exist mainly as 3 major types namely:
(a) mRNA
(b) tRNA
(c) rRNA

6.2.2.1 Messenger RNA (mRNA)

The structure of messenger RNA (mRNA) especially the eukaryotic mRNA has
some unique. The 5’terminus of mRNA is “capped” with a methylated base of
Guanosine 5’ triphosphate. The methylation is on the 2’-hydroxyl group of the
ribose sugar. The methylated capping is followed by a non translated or “leader”
sequence. The leader sequence is followed by an initiation codon, most often
AUG. Coding region of mRNA are terminated by stop codon, usually UAA,
UAG, UGA. The stop codon are followed by a second non translated sequence
at the 3’ end. A series of adenylic acids called poly A tail which makes up 3’
terminus of the mRNA molecule.

6.2.2.2 Transfer RNA (tRNA)

The length of tRNAs ranges from 65-110 nucleotides with a corresponding
molecular weight of 22, 000-37500 Daltons. The tRNA is a single stranded. As
a result of intramolecular hydrogen bonding the 5’ to 3’ nucleotide stretch folded

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into a conformation and forms different loops. The various loops are: the D-loop, Biochemistry
anticodon loop, variable loop or arm, Tø C loop and the acceptor system, each
having its own function.

6.2.2.3 Ribosomal RNA (rRNA)

Ribosomal RNA are found associated with large number of proteins in an
ordered complex. Ribosomal RNA has a helical structure resulting from the Notes
folding back of single stranded polymer and constitute about 74-80% of total
RNA in a cell.

INTEXT QUESTIONS 6.3

1. RNA molecules in primary structure are single stranded linear polymer
containing nucleotides joined by .................... phosphodiester linkage
2. The three types of RNA are ....................
3. The 5’terminus of mRNA is “capped” with a methylated base of ....................
(a) Guanosine 5’ triphosphate (b) Adenosine 5’ triphosphate
(c) Cytocine 5’ triphosphate (d) Thyamine 5’ triphosphate
4. The length of tRNAs ranges from .................... to .................... nucleotides

6.2.3 Characteristics of nucleic acids

(a) The components of nucleic acids are nucleotides joined by phosphodiester
linkage to one another.
(b) Based on the type of sugar in nucleic acids, nucleic acids can either be
deoxyribonucleic acids in which case they contain deoxyribose sugar or
ribonucleic acids when the sugar is a ribose one.
(c) The double stranded forms of both DNA or RNA can be denatured.
(d) DNA is highly viscous at pH 7.0 and room temperature (25°C).
(e) At extreme pH or temperature above 80°C the viscosity of DNA decreases
sharply, which is an indicator of physical change.
(f) The denaturation of double helical DNA associated with the disruption of
hydrogen bonds between the paired bases is called melting of the.
(g) The temperature midpoint in the transition is called melting temperature
(tm).

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Biochemistry (h) For nucleic acids the marked absorption in the ultraviolet (UV) region of
the lights pectrum with Absorption maxima is near 260nm.

INTEXT QUESTIONS 6.4

1. The components of nucleic acids are nucleotides joined by ..................
Notes
2. .................. is highly viscous at pH 7.0
3. At temperature above .................. the viscosity of DNA decreases
4. The Absorption maxima of nucleic acid is near ..................

6.2.4 Metabolism of Nucleotides

Nucleotide metabolism involves several interconnected pathways. Nucleotides
can be synthesized de novo, or from components “salvaged” from the
degradation products of nucleic acids. When in excess, nucleotides are degraded
to products that can either be consumed by other pathways or excreted. Defects
in the pathways for de novo synthesis, salvage, and degradation of nucleotides
result in clinical disorders, and many drugs target these pathways.

DE NOVO SYNTHESIS

Nucleotide pool

RNA DNA Other

SALVAGE functions

DEGRADATION

Nucleosides
CATABOLISM Bases

Fig. 6.7

Nucleotide anabolism can be broadly characterized as purine and pyrimidine

biosynthesis.

6.2.4.1 Biosynthesis of purine nucleotides

Purine nucleotides are synthesized in cytoplasm of most of the tissues. The major
site for purine synthesis is liver. Since purine ring are synthesized from different

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small components, they are makorly denoted by de novo synthesis. Different Biochemistry
sources such as respiratory CO2, amino group from aspartate, formyl group,
amide group from glutamine and glycine etc., are required for formation of
purine ring. As a primary requirement of purine synthesis, purine ring are first
built upon a ribose-5-phosphate molecule. De novo synthesis of purine is a multi
enzyme reaction composed of 10 steps as follow as
H2O PP1 ATP ADP + P1 Notes
PRPP PRA GAR FGAR
1 2 3
ATP
glycine NM-
glutamine glutamate THE 4
formyl
-THF glutamine ADP + P1

fumarate ADP + P1 ATP glutamate FGAM

8 7 6 AIR 5
AICAR SAICAR CAIR
ATP
NM-formyl CO2 ADP + P1
aspartate
-THF
9 Enzyme names:
O
Step-1. glutamine phosphoribosy lpyrophosphate
THE N amidotransferase
H
N Step-2. glycinamide ribotide synthase
FAICAR
10 Step-3. glycinamide ribotide transformylase
N N Step-4. formy lgly cinamide synthase
Step-5. aminoimidazole ribotide synthase
Step-6. aminoimidazole ribotide carboxy lase
Step-7. succiny laminoimidazolecarboxamideribotide
synthase
Step-8. adenylosuccinate lyase
Step-9. aminoimidazole carboxamide ribotide transformy
lase
Step-10. IMP cyclohy drolase

Fig. 6.8
In salvage pathway purines were recycled from degraded nucleotide. Both
nucleosides and deoxy-nucleosides can be salvaged. Phospho ribosyl
phyrophosphate (PRPP) is the starting material for salvage pathway. In salvage
pathway purines are salvaged by adenine phosphor ribosyl transferase (APRTase)
and hypoxanthine guanine phosphoribosyl transferase (HGPRTase). Salvage
pathway are encountered in tissues such as RBC and brain, where there is
absence of de novo pathway.

6.2.4.2 Disorders of purine metabolism

The most common abnormality of purine metabolism is elevation of uric acid
level in blood. Elevated level of uric acid in blood is called as hyper-uricemia.
In hyperuricemia the serum uric acid level exceeds the level of 7 mg/dl for male

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Biochemistry and 6 mg/dl in female. This increased uric acid may or may not be excreated
via urine and the condition is called as uricosuria.

Gout
In gout the urate crystals accumulates in synovial fluid. The above condition
leads to inflammation mediated acute arthritis. The solubility of uric acid is
Notes lowered to 4.5 mg/dl at 30°C. Thus the uric acid is deposited in cooler zone such
as tophi. Deposition of uric acid crystrals are noticed with increase in excretion
of uric acid in urine. The deposition of uric acid crystals leads to calculi or stone
formation. The gout may be either primary or secondary in nature

Primary gout
Primary gout may show familial incidence and are about 1:500 in total
population. About 10 % of primary gout are idiopathic in nature. The main cause
of primary gout is because of error in synthesis of enzymes such as
(a) 5-phosphoribosyl amido transferase
The error in 5-phospho ribosyl amido transferase leads to over production
of purine nucleotides. Even though the abnormal nucleotide is active, they
are not sensitive to feedback inhibition by inhibitory nucleotides.
(b) Phospho ribosyl phyrophosphate (PRPP) synthetase
The abnormal leads to increase and accumulation of PRPP and are X-
linked recessive in inheritance.
(c) Glucose-6-phosphatase
Deficiency in glucose-6-phosphatase leads to a glycogen storage disease
called as Gierke’s disease. In this case more glucose is channeled to the
pentose-phosphate shunt pathway, leading increased availability of ribose5-
phosphate. As a result of this there will be a increase in PRPP formation.
(d) Glutathione reductase
Abnormality in glutathione reductase leads to increased production of
ribose-5-phosphate and thereby increases in PRPP.

Secondary gout
Secondary gout is characterized by increase in uric acid production and reduced
excretion rate. The increase in uric acid is due to increase in turnover rate of
nucleic acids. The increase in nucleic acid turnover rate are seen in
(a) Rapidly growing malignant tissues
(b) Increase in tissue breakdown after treatment of large malignant tumors

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(c) Increase in tissue damage due to trauma Biochemistry

(d) Increase in rate of catabolism as in starvation

Reduction in excretion rate as encountered in secondary gout during
(a) Renal failure
(b) Treatment with thiazide diuretics
(c) Interfrence of tubular secretion due to lactic acidosis and keto-acidosis. Notes

6.2.4.3 Pyrimidine synthesis

Purines are synthesized by building the ring system on the ribose. In contrast,
the pyrimidine ring is constructed first, followed by attachment of the pyrimidine
base to ribose using a phosphoribosyltransferase similar to those used for purine
salvage reactions. In both purine and pyrimidine synthesis, PRPP is used as the
ribose donor, but the stage of the pathway is different.
The first step of the pyrimidine synthesis pathway is the condensation of
bicarbonate with nitrogen derived from glutamine to form carbamoyl phosphate.
The enzyme involved is carbamoyl phosphate synthetase II and is different
from the enzyme catalyzing the equivalent step in the urea cycle.
Carbamoyl phosphate synthetase II has three major differences:
1. It uses nitrogen from glutamine rather than from ammonium
2. It is a cytosolic rather than a mitochondrial enzyme
3. Its regulation is completely different.
In animals two separate pools of carbamoyl phosphate were noticed, they are
1. Mitochondrial pool, used for the urea cycle
2. Cytosolic pool, used for pyrimidine synthesis.
While in bacteria a single pool of carbamoyl phosphate was used for both
purposes, and therefore their pathways are regulated slightly differently. The
pyrimidine ring skeleton comes from two molecules, the carbamoyl phosphate
from the first step, and the aspartate added in the second step.
The ribose ring is not added until the synthesis of the pyrimidine orotic acid is
complete. This orotic acid is then attached to PRPP with release of pyrophosphate.
UMP is the first “completed” product. UMP can then be phosphorylated to
produce UDP. UDP acts as a branch point; it can be converted to UTP and used
as a nucleotide, or it can serve as a substrate for the synthesis of the two other
major pyrimidine nucleotides. Both CTP and TTP were synthesized as described
in the following reaction.

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 O O

NH Uridine NH Uridine NH
Uridine diphosphate diphosphate
monophosphate (UDP) (UDP)
(UMP) N O N O N O
O O O O O O
O– P O CH2 O ATP ADP O P O P O CH2 O ATP ADP O P O P O P O CH2 O

O– O– O– O– O– O–

HO OH HO OH HO OH

Glutamine
+ATP
Deoxyuridine NADPH
monophosphate CTP synthetase
Ribonucleotide
(dUMP) reductase
NADP Glutamate
NH O +ADP + Pi
NH2
N NH Uridine
O O N
diphosphate
PPi dUTP N O (UDP)
O– P O CH2 O O O N
O O O O
O– Pi O P O P O CH2 O
O P O P O P O CH2 O
O– O– Deoxyuridine
HO diphosphate O– O– O–
(dUDP)

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HO
N5,N10. HO OH
Methylene Tetrahydrofolate

Thymidylate synthase

Dihydrofolate
O
CH2
NH

O N O

O– P O CH2 O

O–

HO
Thymidine
monophosphate
(TMP)
Nucleotides MODULE
Biochemistry

INTEXT QUESTIONS 6.5

1. Nucleotide anabolism can be broadly characterized as ...................
biosynthesis
2. Purine nucleotides are synthesized in ................... of most of the tissues and
major site is ...................
Notes
3. In salvage pathway purines both ................... and ................... can be
salvaged
4. The most common abnorbality of purine metabolism is ................... in blood
5. In gout the ................... accumulates in synovial fluid

6.2.5 Functions of nucleic acids

Both structurally and chemically, the nucleic acids differ from other
macromolecule found in living systems and poses their roles and functions.
Nucleic acid itself exist as two different forms namely DNA and RNA, both
differ chemically and structurally.

6.2.5.1 Functions of DNA

(a) DNA acts as a genetic material.
(b) It is worthwhile emphasizing that the main role of the DNA is storage and
transmission of genetic information from parents to offsprings or from one
generation to another.
(c) DNA is capable of undergoing replication (synthesis of another copy of
DNA) and being transcribed into RNA (transcription).
(d) These two processes enables the genetic information encoded in the DNA
found in the nucleus to be transformed into a functional biological material
e.g protein in the cytoplasm.

6.2.5.2 Functions of RNA

(a) RNA, like DNA has shown to be a general constituent of prokaryotic and
eukaryotic cells.
(b) RNA molecules are not as stable as DNA, they also serve as genetic
information carrier in some organisms e.g some viruses where RNA is their
genetic material.
(c) The main functions of tRNA include: Transportation of specific amino
acids to the ribosome’s decoding the genetic information in the messenger
RNA in terms of the proper amino acid to be inserted in the sequence of
protein/polypeptide synthesised.

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Biochemistry (d) tRNA carries one amino acid and also possess an anticodon by which it
recognizes the message on the mRNA template during protein synthesis.
(e) Transfer RNAs have two primary active sites, the 3’hydroxyl terminus to
which specific aminoacid are attached covalently and the anticodon triplet.
(f) Ribosomal RNAs (rRNA) serve as a structural framework for the ribosomes.
The hinging mechanism between the two ribosomal subunits enables
Notes translocation and mRNA movement.
(g) Messenger RNA (mRNA) are direct carriers of genetic information from
the nucleus to the cytoplasmic ribosomes.
(h) Eukaryotic mRNA contains information for only one polypeptide and is
therefore monocistronic whereas prokaryotic mRNA can contain information
for more than one polypeptide chain and therefore designated polycistronic.

INTEXT QUESTIONS 6.6

1. Nucleic acid itself exist as two different forms namely ................. and
.................
2. DNA acts as a .................
3. ................. are not as stable as DNA
4. ................. are direct carriers of genetic information from the nucleus to the
cytoplasmic ribosomes
(a) mRNA (b) rRNA (c) tRNA (d) DNA
5. Prokaryotic mRNA can contain information for more than one polypeptide
chain and therefore designated .................

WHAT HAVE YOU LEARNT

z There are different types of nucleic acids, each having its own characteristic
function.
z DNA has a structure different from RNA. Structurally there are 3 forms of
DNA. Structures of mRNA, rRNA and rRNA have been described. RNA
has primary, secondary and tertiary level structural organization.
z Nucleic acids are polymers of nucleotides. There are two types of nucleic
acids, DNA and RNA.
z The DNA is further subdivided into three (3) forms namely: A form, B form
and Z form.
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z DNA can be denatured and renatured. DNA is highly condensed in the Biochemistry
chromosome and its size can be determined under centrifugal field.
z RNA has a primary, secondary and tertiary structures. DNA and RNA have
differences and similarities. DNA serves in storage and transmission of
genetic material from one generation to another or from parents to
offsprings.
z Messenger RNA carries genetic information from nucleus to the cytoplasm. Notes
z Transfer RNA carries amino acids to the site of protein synthesis. Ribosomal
rRNA serves as the structural framework of the ribosome.

TERMINAL QUESTIONS
1. Describe the structure of DNA
2. Write short notes on the mRNA and tRNA
3. Elaborate in detail about nucleotide metabolism
4. Give details on Gout.

ANSWERS FOR INTEXT QUESTIONS

6.1
1. Gene
2. Nucleic acids
3. Pentose sugars
4. Nucleotides

6.2
1. Nucleotide sequence
2. 2
3. 1953
4. A, B and Z form

6.3
1. Phosphodiester linkage

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Biochemistry 2. mRNA, rRNA and tRNA

3. Guanosine 5’ triphosphate
4. 65 to 110

6.4
1. Phosphodiester linkage
Notes
2. DNA
3. 80°C
4. 260nm

6.5
1. Purine and pyrimidine
2. Cytoplasm and liver
3. Nucleosides and deoxy-nucleosides
4. Elevation of uric acid level
5. Urate crystals

6.6
1. DNA and RNA
2. Genetic material
3. RNA
4. Messenger RNA
5. Polycistronic

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Clinical Chemistry MODULE
Biochemistry

7
Notes
CLINICAL CHEMISTRY

7.1 INTRODUCTION
The clinical chemistry measures chemical changes in the body for diagnosis,
therapy and prognosis of disease. Primarily testing is performed using body fluids
such as serum, plasma, and urine to determine the chemical components.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the reglulation of blood sugar
z explain various blood glucose laboratory tests
z describe laboratory tests of Urea, Calcium and Phosphates
z describe Lipid Profile, Urine Creatinine

7.2 BLOOD SUGAR

The blood sugar concentration or blood glucose level is the amount of glucose
(sugar) present in the blood of a human or animal. The body naturally tightly
regulates blood glucose levels as a part of metabolic homeostasis. Glucose is the
primary source of energy for the body’s cells. Glucose is transported from the
intestines or liver to body cells via the bloodstream, and is made available for
cell absorption via the hormone insulin, produced by the body primarily in the
pancreas.
The mean normal blood glucose level in humans is about 100 mg/dL; however,
this level fluctuates throughout the day. Glucose levels are usually lowest in the
morning, before the first meal of the day (termed “the fasting level”), and rise
after meals for an hour or two by a few milligram. The normal blood glucose
level (tested while fasting) for non-diabetics, should be between 70 and 100

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Biochemistry milligrams per deciliter (mg/dL). Blood sugar levels for those without diabetes
and who are not fasting should be below 125 mg/dL. The blood glucose target
range for diabetics, according to the American Diabetes Association, should be
70–130 (mg/dL) before meal, and less than 180 mg/dL after meals (as measured
by a blood glucose monitor).
Blood sugar levels outside the normal range may be an indicator of a medical
condition. A persistently high level is referred to as hyperglycemia; low levels are
Notes
referred to as hypoglycemia. Diabetes mellitus is characterized by persistent
hyperglycemia from any of several causes, and is the most prominent disease
related to failure of blood sugar regulation. Intake of alcohol causes an initial
surge in blood sugar, and later tends to cause levels to fall. Also, certain drugs
can increase or decrease glucose levels.

7.2.1 Regulation
The body’s homeostatic mechanism keeps blood glucose levels within a narrow
range. It is composed of several interacting systems, of which hormone regulation
is the most important.
There are two types of mutually antagonistic metabolic hormones affecting blood
glucose levels:
z catabolic hormones (such as glucagon, cortisol and catecholamines) which
increase blood glucose
z anabolic hormone (insulin), which decreases blood glucose.

7.2.2 Abnormality in blood sugar levels

7.2.3 High blood sugar

If blood sugar levels remain too high the body suppresses appetite over the short
term. Long-term hyperglycemia causes many of the long-term health problems
including heart disease, eye, kidney, and nerve damage. The most common cause
of hyperglycemia is diabetes. When diabetes is the cause, physicians typically
recommend an anti-diabetic medication as treatment. From the perspective the
majority of patients, treatment with an old, well-understood diabetes drug such
as metformin will be the safest, most effective, least expensive, most comfortable
route to managing the condition. Diet changes and exercise implementation may
also be part of a treatment plan for diabetes.

7.2.4 Low blood sugar

If blood sugar levels drop too low, a potentially fatal condition called
hypoglycemia develops. Symptoms may include lethargy, impaired mental
functioning; irritability; shaking, twitching, weakness in arm and leg muscles; pale
complexion; sweating; paranoid or aggressive mentality and loss of consciousness.

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7.2.5 Glucose measurement Biochemistry

7.2.5.1 Sample type

Glucose is measured in whole blood, plasma or serum. Historically, blood glucose
values were given in terms of whole blood, but most laboratories now measure
and report plasma or serum glucose levels. Because red blood cells (erythrocytes)
have a higher concentration of protein (e.g., hemoglobin) than serum, serum has
a higher water content and consequently more dissolved glucose than does whole Notes
blood. Collection of blood in clot tubes for serum chemistry analysis permits the
metabolism of glucose in the sample by blood cells until separated by
centrifugation. Red blood cells, for instance, do not require insulin to intake
glucose from the blood. Higher than normal amounts of white or red blood cell
counts can lead to excessive glycolysis in the sample, with substantial reduction
of glucose level if the sample is not processed quickly. Ambient temperature at
which the blood sample is kept prior to centrifuging and separation of plasma/
serum also affects glucose levels. At refrigerator temperatures, glucose remains
relatively stable for several hours in a blood sample.
Loss of glucose can be prevented by using Fluoride tubes since fluoride inhibits
glycolysis. However, these should only be used when blood will be transported
from one hospital laboratory to another for glucose measurement. Red-top serum
separator tubes also preserve glucose in samples after being centrifuged isolating
the serum from cells. Arterial, capillary and venous blood has comparable glucose
levels in a fasting individual. Following meals, venous levels are somewhat lower
than those in capillary or arterial blood; a common estimate is about 10%.

7.2.6 Measurement techniques

Two major methods have been used to measure glucose. The first, still in use
in some places, is a chemical method exploiting the nonspecific reducing property
of glucose in a reaction with an indicator substance that changes color when
reduced. The more recent technique, using enzymes specific to glucose, is less
susceptible to this kind of error. The two most common employed enzymes are
glucose oxidase and hexokinase. In either case, the chemical system is commonly
contained on a test strip which is inserted into a meter, and then has a blood
sample applied. Test-strip shapes and their exact chemical composition vary
between meter systems and cannot be interchanged. More precise blood glucose
measurements are performed in a medical laboratory, using hexokinase, glucose
oxidase or glucose dehydrogenase enzymes.

7.2.7 Blood glucose laboratory tests

1. fasting blood sugar (i.e., glucose) test (FBS)
2. two-hr postprandial blood sugar test (2-h PPBS)
3. oral glucose tolerance test (OGTT)

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Biochemistry 4. intravenous glucose tolerance test (IVGTT)

5. glycosylated hemoglobin (HbA1C)
6. self-monitoring of glucose level via patient testing
7. Random blood sugar (RBS)
8. Average blood glucose may be estimated by measuring glycated hemoglobin
(HbA1c)
Notes
7.2.8 Clinical Correlation
The fasting blood glucose level, which is measured after a fast of 8 hours, is the
most commonly used indication of overall glucose homeostasis, largely because
disturbing events such as food intake are avoided. The metabolic response to a
carbohydrate challenge is conveniently assessed by a postprandial glucose level
drawn 2 hours after a meal or a glucose load. In addition, the glucose tolerance
test, consisting of several timed measurements after a standardized amount of
oral glucose intake, is used to aid in the diagnosis of diabetes.
Finally, there are several influences on blood glucose level aside from food intake.
Infection, for instance, tends to change blood glucose levels, as does stress either
physical or psychological. Exercise, especially if prolonged or long after the most
recent meal, will have an effect as well.

7.3 UREA
7.3.1 Blood urea nitrogen (BUN)
The liver produces urea in the urea cycle as a waste product of the digestion of
protein. Normal human adult blood should contain between 6 to 20 mg of urea
nitrogen per 100 ml. Individual laboratories may have different reference ranges,
and this is because the procedure may vary.

7.3.2 Interpretation
BUN is an indication of renal health. Normal ranges 8-20 mmol/L. If Glomerular
Filtration Rate (GFR) and blood volume decrease (hypovolemia) then BUN will
increase. Other factors responsible for its increment are fever, increased
catabolism, high protein diet and gastrointestinal bleeding.

7.4 CALCIUM-PHOSPHATES
Calcium metabolism or calcium homeostasis is the mechanism by which the body
maintains adequate calcium levels. Derangements of this mechanism lead to
hypercalcemia or hypocalcemia, both of which can have important consequences
for health.
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7.4.1 Calcium location and quantity Biochemistry

Calcium is the most abundant mineral in the human body. The average adult body
contains in total approximately 1 kg, 99% in the skeleton in the form of calcium
phosphate salts. The extracellular fluid (ECF) contains approximately 22.5 mmol,
of which about 9 mmol is in the serum. Approximately 500 mmol of calcium is
exchanged between bone and the ECF over a period of twenty-four hours.
Notes
7.4.2 Biological functions
z Structural function: Supporting material in bones. Present as calcium
phosphate.
z Signalling function: Intracellular calcium functions as a second messenger
for some hormones.
z Enzymatic function: Calcium acts as a coenzyme for clotting factors.
Calcium also causes the release of Acetylcholine from Pre-synaptic terminal in
the transmission of nerve impulse. Calcium causes the contraction of muscles.

7.4.3 Normal ranges

The serum level of calcium is closely regulated with normal total calcium of 2.2-
2.6 mmol/L (9-10.5 mg/dL) and normal ionized calcium of 1.1-1.4 mmol/L (4.5-
5.6 mg/dL). The amount of total calcium varies with the level of serum albumin,
a protein to which calcium is bound. The biologic effect of calcium is determined
by the amount of ionized calcium, rather than the total calcium. Ionized calcium
does not vary with the albumin level, and therefore it is useful to measure the
ionized calcium level when the serum albumin is not within normal ranges, or
when a calcium disorder is suspected despite a normal total calcium level.

7.4.4 Effector organs

7.4.4.1 Absorption
Calcium enters the body in a normal diet and is absorbed across the intestinal
brush border membrane. Calbindin is a vitamin D-dependent calcium-binding
protein inside intestinal epithelial cells actively transports calcium into the body.

7.4.4.2 Excretion
The kidney excretes 250 mmol a day in pro-urine, and resorbs 245 mmol, leading
to a net loss in the urine of 5 mmol/d. In addition to this, the kidney processes
Vitamin D into calcitriol, the active form that is most effective in assisting
intestinal absorption. Both processes are stimulated by parathyroid hormone.

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Biochemistry 7.4.4.3 The role of bone

Although calcium flow to and from the bone is neutral, about 5 mmol is turned
over a day. Bone serves as an important storage point for calcium, as it contains
99% of the total body calcium. Calcium release from bone is regulated by
parathyroid hormone. Calcitonin stimulates incorporation of calcium in bone,
although this process is largely independent of calcitonin. Low calcium intake
Notes may also be a risk factor in the development of osteoporosis.

7.4.5 Interaction with other chemicals

7.4.5.1 Potential positive interactions

z Vitamin D is an important co-factor in the intestinal absorption of calcium,
as it increases the number of calcium binding proteins, involved in calcium
absorption through the apical membrane of enterocytes in small intestine.
It also promotes re-absorption of calcium in the kidneys.
z Magnesium also plays an important role in calcium absorpsion by bones.
Release of calcium from the sarcoplasmic reticulum is inhibited by magnesium.
Thus hypomagnesemia results in an increased intracellular calcium level. This
inhibits the release of parathyroid hormone, which can result in
hypoparathyroidism and hypocalcemia. Furthermore, it makes skeletal and
muscle receptors less sensitive to parathyroid hormone.

7.4.5.2 Potential negative interactions

z Unesterified long-chain saturated fatty acids, i.e. palmitic acid, have a
melting point above body temperature and, with sufficient calcium in the
intestinal lumen, form insoluble calcium soaps.
z Sodium binding to calcium
z Phytic acid binding to calcium
z Oxalic acid binding to calcium
z Cortisol binding to calcium
z Low pH food and proteins (the latter promotes gastric acid)

7.5 LIPID PROFILE

Lipid profile or lipid panel, is a panel of blood tests that serves as an initial broad
medical screening tool for abnormalities in lipids, such as cholesterol and
triglycerides. The results of this test can identify certain genetic diseases and can
determine approximate risks for cardiovascular disease, certain forms of
pancreatitis, and other diseases.

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7.5.1 Components Biochemistry

The lipid profile typically includes:

z Low-density lipoprotein
z High-density lipoprotein
z Triglycerides
z Total cholesterol Notes
Using these values, a laboratory may also calculate:
z Very low-density lipoprotein

z Cholesterol:HDL ratio

7.5.2 Procedure
Traditionally, most laboratories have required patients to fast for 9–12 hours
before screening. However, recent studies have questioned the utility of fasting
before lipid panels, and some diagnostic labs now routinely accept non-fasting
samples.

7.5.3 Implications
This test is used to identify hyperlipidemia (various disturbances of cholesterol
and triglyceride levels), many forms of which are recognized risk factors for
cardiovascular disease and rarely pancreatitis. A total cholesterol reading can be
used to assess an individual’s risk for heart disease; however, it should not be
relied upon as the only indicator. The individual components that make up total
cholesterol reading LDL, HDL, and VLDL are also important in measuring risk.
The lipid profile includes total cholesterol, HDL-cholesterol (often called good
cholesterol), LDL-cholesterol (often called bad cholesterol), and triglycerides.
Sometimes the report will include additional calculated values such as the
Cholesterol/HDL ratio or a risk score based on lipid profile results, age, sex, and
other risk factors. The lipid profile is used to guide providers in deciding how
a person at risk should be treated. The results of the lipid profile are considered
along with other known risk factors of heart disease to develop a plan of
treatment and follow-up.

7.5.4 Normal range

LDL : 60–130 mg/dL
HDL : > 40 mg/dL
Total cholesterol : < 200 mg/dL
Triglycerides : 10–150 mg/dL
VLDL : 2–38 mg/dL

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Biochemistry
7.6 URINE LEVELS OF SUGAR
7.6.1 Glycosuria
Glycosuria or glucosuria is the excretion of glucose into the urine. Ordinarily,
urine contains no glucose because the kidneys are able to reclaim all of the filtered
glucose back into the bloodstream. Glycosuria is nearly always caused by
elevated blood glucose levels, most commonly due to untreated diabetes mellitus.
Notes
Rarely, glycosuria is due to an intrinsic problem with glucose reabsorption within
the kidneys themselves, a condition termed renal glycosuria. Glycosuria leads to
excessive water loss into the urine with resultant dehydration, a process called
osmotic diuresis.

7.6.2 Pathophysiology
Blood is filtered by millions of nephrons, the functional units that comprise the
kidneys. In each nephron, blood flows from the arteriole into the glomerulus, a
tuft of leaky capillaries. The Bowman’s capsule surrounds each glomerulus, and
collects the filtrate that the glomerulus forms. The filtrate contains waste
products (e.g. urea), electrolytes (e.g. sodium, potassium, chloride), amino acids,
and glucose. The filtrate passes into the renal tubules of the kidney. In the first
part of the renal tubule, the proximal tubule, glucose is reabsorbed from the
filtrate, across the tubular epithelium and into the bloodstream.

The proximal tubule can only reabsorb a limited amount of glucose. When the
blood glucose level exceeds about 160 – 180 mg/dl, the proximal tubule becomes
overwhelmed and begins to excrete glucose in the urine. This point is called the
renal threshold of glucose (RTG). Some people, especially children and pregnant
women, may have a low RTG (less than ~7 mmol/L glucose in blood to have
glucosuria). If the RTG is so low that even normal blood glucose levels produce
the condition, it is referred to as renal glycosuria. Glucose in urine can be
identified by Benedict’s qualitative test. A urine dipstick can show a false-positive
glucosuria if someone is taking Pyridium standard, medications that relieve
symptoms of urinary tract infection.

7.6.3 Diagnosis
A doctor normally can diagnose renal glycosuria when a routine urine test
(Urinalysis) detects glucose in the urine, while a blood test indicates that the
blood glucose level is normal. In most affected individuals, the condition causes
no apparent symptoms (asymptomatic) or serious effects. When renal glycosuria
occurs as an isolated finding with otherwise normal kidney function, the
condition is thought to be inherited as an autosomal recessive trait.

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Biochemistry
7.7 URINE LEVELS OF CREATININE
Creatinine is a breakdown product of creatine phosphate in muscle and is usually
produced at a fairly constant rate by the body (depending on muscle mass). Serum
creatinine (a blood measurement) is an important indicator of renal health
because it is an easily-measured by-product of muscle metabolism that is excreted
unchanged by the kidneys. Creatinine itself is produced via a biological system
involving creatine, phosphocreatine (also known as creatine phosphate), and
adenosine triphosphate (ATP, the body's immediate energy supply). Creatine is Notes
synthesized primarily in the liver from the methylation of glycocyamine by S-
adenosyl methionine. It is then transported through blood to the other organs,
muscle, and brain where, through phosphorylation, it becomes the high-energy
compound phosphocreatine. During the reaction, creatine and phosphocreatine
are catalyzed by creatine kinase, and a spontaneous conversion to creatinine may
occur.

7.7.1 Creatinine clearance

Creatinine is removed from the blood chiefly by the kidneys, primarily by
glomerular filtration but also via proximal tubular secretion. There is little or no
tubular reabsorption of creatinine. If the filtration in the kidney is deficient,
creatinine blood levels rise. Therefore, creatinine levels in blood and urine may
be used to calculate the creatinine clearance (CrCl), which correlates with the
glomerular filtration rate (GFR). Blood creatinine levels may also be used alone
to calulate the estimated GFR (eGFR).
The GFR is clinically important because it is a measurement of renal function.
However, in cases of severe renal dysfunction, the creatinine clearance rate will
overestimate the GFR because hypersecretion of creatinine by the proximal
tubules will account for a larger fraction of the total creatinine cleared. Ketoacids,
cimetidine, and trimethoprim reduce creatinine tubular secretion and, therefore,
increase the accuracy of the GFR estimate, in particular in severe renal
dysfunction. An alternate estimation of renal function can be made when
interpreting the blood (plasma) concentration of creatinine along with that of
urea. BUN-to-creatinine ratio (the ratio of blood urea nitrogen to creatinine) can
indicate other problems besides those intrinsic to the kidney; for example, a urea
level raised out of proportion to the creatinine may indicate a pre-renal problem
such as volume depletion. Each day, 1-2% of muscle creatine is converted to
creatinine. Men tend to have higher levels of creatinine than women because, in
general, they have a greater mass of skeletal muscle. Increased dietary intake of
creatine or eating a lot of meat can increase daily creatinine excretion.

7.7.2 Diagnostic use

7.7.2.1 Serum creatinine

Measuring serum creatinine is a simple test, and it is the most commonly used
indicator of renal function. A rise in blood creatinine level is observed only with

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Biochemistry marked damage to functioning nephrons. Therefore, this test is unsuitable for
detecting early-stage kidney disease. A better estimation of kidney function is
given by calculating the estimated glomerular filtration rate (eGFR). eGFR can
be accurately calculated using serum creatinine concentration and some or all of
the following variables: sex, age, weight, and race.

7.7.2.2 Urine creatinine

Notes Creatinine concentration is also checked during standard urine drug tests.
Normal creatinine levels indicate the test sample is undiluted, whereas low
amounts of creatinine in the urine indicate either a manipulated test or low
individual baseline creatinine levels. Random urine creatinine levels have no
standard reference ranges. They are usually used with other tests to reference
levels of other substances measured in the urine. Diuretics, such as coffee and
tea, cause more frequent urination, thus potently decreasing creatinine levels. A
decrease in muscle mass will also cause a lower reading of creatinine, as will
pregnancy.

7.7.3 Interpretation
The typical human reference ranges for serum creatinine are 0.5 to 1.0 mg/dl
(about 45-90 μmol/l) for women and 0.7 to 1.2 mg/dl (60-110 μmol/L) for men.
While a baseline serum creatinine of 2.0 mg/dl (150 μmol/l) may indicate normal
kidney function in a male body builder, a serum creatinine of 1.2 mg/dl (110
μmol/l) can indicate significant renal disease in an elderly female. Creatinine
levels may increase when ACE inhibitors (ACEI) or angiotensin II receptor
antagonists (or angiotensin receptor blockers, ARBs) are taken. Using both
ACEI and ARB concomitantly will increase creatinine levels to a greater degree
than either of the two drugs would individually. An increase of <30% is to be
expected with ACEI or ARB use.

7.7.4 Normal Results

Urine creatinine (24-hour sample) values can range from 500 to 2000 mg/day.
Results depend greatly on your age and amount of lean body mass.
Another way of expressing the normal range for these test results are:
z 14 to 26 mg per kg of body mass per day for men

z 11 to 20 mg per kg of body mass per day for women

7.7.5 Abnormal Results Mean

Abnormal results of urine creatinine are nonspecific, but may be due to any of
the following conditions such as, glomerulonephritis, high meat diet, kidney
infection (pyelonephritis), kidney failure, muscular dystrophy (late stage),
myasthenia gravis, prerenal azotemia, reduced kidney blood flow (as in shock
or congestive heart failure), urinary tract obstruction.

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Biochemistry
7.8 URINE LEVELS OF PROTEINS
7.8.1 Proteinuria
Proteinuria means the presence of an excess of serum proteins in the urine. The
excess protein in the urine often causes the urine to become foamy, although
foamy urine may also be caused by bilirubin in the urine (bilirubinuria), or drugs
such as pyridium. Up to 150 mg a day of protein may be excreted by a normal
person. Proteinuria can also be caused by certain biological agents, such as Notes
Avastin used in cancer treatment, or by excessive fluid intake (drinking in excess
of 4 litres of water per day).

7.8.2 Causes
There are three main mechanisms to cause proteinuria:
z Due to disease in glomerulus

z Because of increased quantity of proteins in serum (overflow proteinuria)

z Due to low reabsorption at proximal tubule (Fanconi syndrome)

7.8.3 Measurement
Conventionally, proteinuria is diagnosed by a simple dipstick test, although it is
possible for the test to give a false negative reading, if the protein in the urine
is composed mainly of globulins or Bence-Jones proteins. Alternatively the
concentration of protein in the urine may be compared to the creatinine level in
a spot urine sample. This is termed the protein/creatinine ratio (PCR). Proteinuria
is defined as a protein/creatinine ratio greater than 45 mg/mmol (which is
equivalent to albumin/creatinine ratio of greater than 30 mg/mmol or approximately
300 mg/g) with very high levels of proteinuria being for a PCR greater than
100 mg/mmol.

7.8.4 Associated conditions

Proteinuria may be a sign of renal (kidney) damage. Since serum proteins are
readily reabsorbed from urine, the presence of excess protein indicates either an
insufficiency of absorption or impaired filtration. Diabetics may suffer from
damaged nephrons and develop proteinuria.

7.8.5 Conditions with proteinuria as a sign

Proteinuria may be a feature of the following conditions such as, nephrotic
syndromes (i.e. intrinsic renal failure), toxic lesions of kidneys, amyloidosis,
collagen vascular diseases (e.g. systemic lupus erythematosus), dehydration,
glomerular diseases, strenuous exercise, stress, IgA nephropathy, IgM nephropathy,
diabetes mellitus (diabetic nephropathy), drugs (e.g. NSAIDs, nicotine,
penicillamine, lithium carbonate, antibiotics, or opiates (especially heroin),

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Biochemistry infections (e.g. HIV, syphilis, hepatitis, poststreptococcal infection), aminoaciduria,

hypertensive nephrosclerosis, sickle cell disease, hemoglobinuria, multiple
myeloma, myoglobinuria, organ rejection (Kidney transplant patients may have
gamma-globulins in their urine if the kidneys start to reject), systemic lupus
erythematosus, rheumatoid arthritis, glycogen storage disease type 1, and urinary
tract infection which has spread to the kidney(s). Conditions with proteinuria
consisting mainly of Bence-Jones proteins as a sign are waldenstrom’s
Notes macroglobulinemia, chronic lymphocytic leukemia, amyloidosis, malignancies
(e.g., lymphoma, other cancers), multiple myeloma.

7.8.6 Treatment
Treating proteinuria mainly needs proper diagnosis of the cause. The most
common cause is diabetic nephropathy; in this case, proper glycemic control may
slow the progression. Medical management consists of angiotensin converting
enzyme (ACE) inhibitors, which are typically first-line therapy for proteinuria.
In patients whose proteinuria is not controlled with ACE inhibitors, the addition
of an aldosterone antagonist or angiotensin receptor blocker may further reduce
protein loss. Caution must be used if these agents are added to ACE inhibitor
therapy due to the risk of hyperkalemia. Proteinuria secondary to autoimmune
disease should be treated with steroids or steroid-sparing agent plus the use of
ACE inhibitors.

INTEXT QUESTIONS 7.1

I. Choose the best answer
1. Measuring glucose levels before the first meal of the day is termed
as
(a) Post prandial blood glucose
(b) Fasting blood glucose
(c) Normal blood glucose
(d) None
2. The catabolic hormone which increases blood glucose level is
(a) Glucagon (b) Insulin
(c) Histamine (d) None
3. The coenzyme acts as blood clotting factor is
(a) Magnesium (b) Iron
(c) Calcium (d) Lead

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4. The vitamin D-dependent calcium-binding protein that actively Biochemistry
transports calcium into the body
(a) Calbindin (b) Calmodulin
(c) Transferrin (d) Globulin
5. Good cholesterol is termed for
(a) LDL-cholesterol (b) VLDL-cholesterol
(c) HDL-cholesterol (d) Triglycerides Notes
II. Fill in the blanks
6. Excretion of glucose into the urine is called as .................
7. ................. is the by-product of muscle metabolism
8. The protein in the urine is composed mainly of globulins is termed
medically as .................
9. The two most common employed enzymes in the blood glucose
measurement are .................
10. ................. increases if glomerular filtration rate and blood volume
decrease.
III. Match the following
11. Bone formation (a) LDL
12. Bad cholesterol (b) Calcium
13. Glycosuria (c) Diabetes mellitus
14. Bilirubin in the urine (d) Benedict’s qualitative test
15. Metformin (e) Bilirubinuria

WHAT HAVE YOU LEANRT

z Glucose is the primary source of energy for the body’s cells. Its level should
be between 70 and 100 mg/dL for normal person.
z Anabolic hormone (insulin) decreases blood glucose andcatabolic hormones
(such as glucagon, cortisol and catecholamines) increase blood glucose.
z More precise blood glucose measurements are performed in a medical
laboratory, using hexokinase, glucose oxidase or glucose dehydrogenase
enzymes.
z The liver produces urea in the urea cycle as a waste product of the digestion
of protein.
z Derangements of the calcium mechanism lead to hypercalcemia or
hypocalcemia, both of which can have important consequences for health.

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Biochemistry z Calbindin is a vitamin D-dependent calcium-binding protein inside intestinal

epithelial cells actively transports calcium into the body.
z Hyperlipidemia is recognized as a risk factor which leads to cardiovascular
disease.
z HDL-cholesterol is often called good cholesterol and LDL-cholesterol is
often called as bad cholesterol.
Notes z Glycosuria is nearly always caused by elevated blood glucose levels. Glucose
in urine can be identified by Benedict’s qualitative test.
z Serum creatinine is an important indicator of renal health because it is an
easily-measured by-product of muscle metabolism. Creatinine is removed
from the blood chiefly by the kidneys.
z Proteinuria means the presence of an excess of serum proteins in the urine.
z The three main mechanisms that cause proteinuria are disease in glomerulus,
increased quantity of proteins in serum, and low reabsorption at proximal
tubule (Fanconi syndrome).

TERMINAL QUESTIONS
1. Write short note on Laboratory tests of Blood glucose
2. Write short note on Lipid profile
3. Write short notes on urine levels of creatinine

ANSWERS TO INTEXT QUESTIONS

I. 1. (b) 2. (a) 3. (c) 4. (a) 5. (c)
II. 6. Glycosuria,
7. Creatinine,
8. Bence-Jones proteins,
9. Glucose oxidase and Hexokinase,
10. Blood urea nitrogen,
III. 11. (b) 12. (a) 13 (d) 14. (e) 15. (c)

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Enzymes MODULE
Biochemistry

8
Notes
ENZYMES

8.1 INTRODUCTION
The global life depends on a series of chemical reactions. Most of the chemical
reactions proceed too slowly on their own to sustain life. Hence catalysts are
required to greatly accelerate the rates of these chemical reactions. In nature
enzymes posses the catalytic power to facilitate life processes in essentially all
life-forms from viruses to man. Most of the enzymes retain their catalytic
potential even after extraction from the living organism. The above catalytic
power of enzyme leads to commercial usage of enzymes.

In ancient days enzymes are used in manufacture of cheeses, breads, and

alcoholic beverages, and for the tenderizing of meats. Today enzymes are also
of fundamental interest in the health sciences. Human disease processes can be
linked to the aberrant activities of one or a few enzymes. Because of their role
in maintaining life processes, the assay and pharmacological regulation of
enzymes have become key elements in clinical diagnosis and therapeutics.

The macromolecular components of almost all enzymes are composed of

protein, except for a class of RNA modifying catalysts known as ribozymes.
Ribozymes are molecules of ribonucleic acid that catalyze reactions on the
phosphodiester bond of other RNAs. Enzymes are found in all tissues and fluids
of the body. Intracellular enzymes catalyze the reactions of metabolic pathways.
Plasma membrane enzymes regulate catalysis within cells in response to
extracellular signals, and enzymes of the circulatory system are responsible for
regulating the clotting of blood.

Almost every significant life process is dependent on enzyme activity. So the

modern pharmaceutical research is based on the search for potent and specific
inhibitors of these enzymes. This chapter briefs you about basic nature,
classification and clinical significance of enzymes.

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Biochemistry

OBJECTIVES
After reading this lesson, you will be able to:
z define enzymes

z classify enzymes
Notes z explain co-enzymes
z explain the factors affecting enzyme activity
z describe isoenzymes
z explain the Clinical significance of enzymes

8.2 DEFINITION AND CHARACTERISTICS OF

ENZYMES
Enzymes are protein catalyst produced by a cell and responsible ‘for the high
rate’ and specificity of one or more intracellular or extracellular biochemical
reactions. Enzymes are biological catalysts responsible for supporting almost all
of the chemical reactions that maintain animal homeostasis. Enzyme reactions
are always reversible. The substance, upon which an enzyme acts, is called as
substrate. Enzymes are involved in conversion of substrate into product. Almost
all enzymes are globular proteins consisting either of a single polypeptide or of
two or more polypeptides held together (in quaternary structure) by non-covalent
bonds. Enzymes do nothing but speed up the rates at which the equilibrium
positions of reversible reactions are attained. In terms of thermodynamics,
enzymes reduce the activation energies of reactions, enabling them to occur
much more readily at low temperatures - essential for biological systems. The
basic characteristics of enzymes includes
(i) Almost all the enzymes are proteins and they follow the physical and
chemical reactions of proteins
(ii) Enzymes are sensitive and labile to heat
(iii) Enzymes are water soluble
(iv) Enzymes could be precipitated by protein precipitating agents such as
ammonium sulfate and trichloroacetic acid.

INTEXT QUESTIONS 8.1

1. Enzymes are ............... biocatalyst produced by cells.
2. Enzymes follow physical and chemical properties of ...............

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3. Enzymes are ............... soluble. Biochemistry

(a) Water (b) acid (c) organic acids

4. Precipitation of enzymes could be achieved by using .............. and ..............

8.3 CLASSIFICATION OF ENZYMES

Since earlier days to still date, fanciful names such as pepsin, chymotrypsin, etc
were used to name enzymes. Later the suffix “ase” to the substrate was used to Notes
name enzymes. For example the enzymes lactase acts upon the lactate and
produces glucose and galactose. The above method is known as “trivial naming”
of enzymes. Currently enzymes are grouped into six functional classes by the
International Union of Biochemists and Molecular Biology (IUBMB). As per the
IUBMB system, each enzyme name starts with EC (enzyme class) followed by
4 digits.
The first digit represents the class, the second digit strands for the subclass, the
third digit represents the sub-subclass or subgroup and the fourth digit provides
the particular enzyme (Table 8.1)

Table 8.1 Classification of enzymes

Sl.No. Classification Biochemical Properties
1. Oxidoreductases Act on many chemical groupings to add or
remove hydrogen atoms. e.g. Lactate
dehydrogenase
2. Transferases Transfer functional groups between donor and
acceptor molecules. Kinases are specialized
transferases that regulate metabolism by
transferring phosphate from ATP to other
molecules. e.g. Aminotransferase.
3. Hydrolases Add water across a bond, hydrolyzing it. E.g.
Acetyl choline esterase
4. Lyases Add water, ammonia or carbon dioxide across
double bonds, or remove these elements to
produce double bonds. e.g. Aldolase.
5. Isomerases Carry out many kinds of isomerization: L to D
isomerizations, mutase reactions (shifts of
chemical groups) and others. e.g. Triose
phosphate isomerase
6. Ligases Catalyze reactions in which two chemical
groups are joined (or ligated) with the use of
energy from ATP. e.g. Acetyl CoA carboxylase

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Biochemistry These rules give each enzyme a unique number and specifies a textual name for
each enzyme. Enzymes are also classified on the basis of their composition.
Enzymes composed wholly of protein are known as simple enzymes in contrast
to complex enzymes, which are composed of protein plus a relatively small
organic molecule. Complex enzymes are also known as holo-enzymes. The non-
protein component of an enzyme may be as simple as a metal ion or as complex
as a small non-protein organic molecule. Enzymes that require a metal in their
Notes composition are known as metalloenzymes. Metalloenzymes bind and retain
their metal atom(s) under all conditions with very high affinity. Enzymes with
lower affinity for metal ion, but still require the metal ion for activity, are known
as metal-activated enzymes. Based on requirement of ATP, enzymes are further
classified into two types namely synthetases and synthase. Synthetases are ATP-
dependent enzymes catalyzing biosynthetic reactions. Synthetases are enzyme
belong to the class 6 (Ligases). Enzymes such as Carbamoyl phosphate
synthetase, Arginino succinate synthetase and Glutamine synthetase are examples
of the synthetases group of enzymes. The enzyme class other than ligases
includes synthases. Synthases group of enzymes involves in catalyzing
biosynthetic reactions that do not require ATP directly. Enzymes such as
glycogen synthase and Alanine synthase are examples of synthase group.

INTEXT QUESTIONS 8.2

1. There are ................. main groups of enzymes.
2. Lactate dehydrogenase is example for ................. group of enzyme.
3. Enzymes that require a metal in their composition are known as .................
4. ................. are ATP dependent enzymes.
5. ................. and ................. are examples for synthase group of enzymes.

8.4 COENZYMES
Enzymes may be simple proteins, or complex enzymes. A complex enzyme
contains a non-protein part, called as prosthetic group (co-enzymes). Co-
enzymes are heat stable low molecular weight organic compound. The combined
form of protein and the co-enzyme are called as holo-enzyme. The heat labile
or unstable part of the holo-enzyme is called as apo-enzyme. The apo-enzyme
gives necessary three dimensional structures required for the enzymatic
chemical reaction. Co-enzymes are very essential for the biological activities of
the enzyme. Co-enzymes combine loosely with apo-enzyme and are released
easily by dialysis. Most of the co-enzymes are derivatives of vitamin B complex

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group of substance. One molecule of the co-enzyme with its enzyme is sufficient Biochemistry
to convert a large group of substrate.
Co-enzymes are further divided into two groups. The first groups of co-enzymes
are a part of reaction catalyzed by oxidoreductase by donating or accepting
hydrogen atoms or electrons. The first group of co-enzymes are also called as
co-substrates or secondary substrates. Because they are involved in counter-
balance in change occurring in the substrate. The second group of co-enzymes
Notes
involves in reactions transferring groups other than hydrogen.

8.4.1 Role of Coenzymes

The functional role of coenzymes is to act as transporters of chemical groups
from one reactant to another. The chemical groups carried can be as simple as
the hydride ion (H+ + 2e-) carried by NAD or the mole of hydrogen carried by
FAD; or they can be even more complex than the amine (-NH2) carried by
pyridoxal phosphate. Since coenzymes are chemically changed as a consequence
of enzyme action, it is often useful to consider coenzymes to be a special class
of substrates, or second substrates, which are common to many different
holoenzymes. In all cases, the coenzymes donate the carried chemical grouping
to an acceptor molecule and are thus regenerated to their original form. This
regeneration of coenzyme and holoenzyme fulfills the definition of an enzyme
as a chemical catalyst, since (unlike the usual substrates, which are used up
during the course of a reaction) coenzymes are generally regenerated.

INTEXT QUESTIONS 8.3

1. Non protein part of complex enzymes is called as .................
2. Combination of ................. and ................. are called as holo enzymes.
3. Co-enzymes are heat stable low molecular weight ................. compound.
4. There are ................. groups of co-enzymes.

8.5 FACTORS AFFECTING ENZYME ACTIVITY

Velocity or rate of enzymatic reaction is assessed by the rate of change in
concentration of substrate or product at a given time duration. Various factors
which affect the activity of enzymes include:
1. Substrate concentration
2. Enzyme concentration
3. Product concentration

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Biochemistry 4. Temperature
5. Hydrogen ion concentration (pH)
6. Presence of activators
7. Presence of inhibitor

8.5.1 Effect of substrate Concentration

Notes Reaction velocity of an enzymatic process increases with constant enzyme
concentration and increase in substrate concentration. The velocity (V) is
expressed in micromoles of substrate converted per minute. As the concentration
of substrate increases, the velocity of the reaction increases. Continued increase
in substrate concentration may lead to a reduction in rate of the reaction and leads
to flattened curve. The maximum velocity obtained from a enzymatic reaction
is called as Vmax. Vmax represents the maximum reaction rate possible in the
presence of excess substrate.

Vmax

½ Vmax

Km [S]
Fig. 8. 1: Effect of substrate concentration

8.5.2 Effect of enzyme Concentration

As there is optimal substrate concentration, rate of an enzymatic reaction or
velocity (V) is directly proportional to the enzyme concentration. Presence of
excess substrate and an increase in the enzyme concentration may result in some
limitations. It is worthy of note that the enzymes are rarely saturated with
substrates under physiological conditions.

[E]
Fig. 8.2: Effect of enzyme concentration

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Biochemistry
8.5.3 Effect of product concentration
In case of a reversible reaction catalyzed by a enzyme, as per the law of mass
action the rate of reaction is slowed down with equilibrium. So, rate of reaction
is slowed, stopped or even reversed with increase in product concentration. This
phenomena can be better explained by the equation
E1 E2 E3
A B C D
Notes
In the above equation, in case of absence of the enzyme E3, the product C will
accumulate. Enzymatic activity of E2 will be inhibited with accumulation of the
product C. In such inborn error of one enzyme will block the whole pathway.

8.5.4 Effect of temperature

Velocity of enzymatic reaction increases with temperature of the medium which
they are most efficient and the same is termed as optimum temperature. As
temperatures increases it leads to denaturation; a molecular arrangement which
causes a loss of the active sites of the enzyme surfaces and results in a loss of
efficiency.

Optimum temperature

Temperature in °C

Fig. 8.3: Effect of temperature

8.5.5 Effect of pH
Like temperature, all enzymes have a optimum pH, at which the enzymatic
activity will be at maximum. Many enzymes are most efficient in the region of
pH 6-7, which is the pH of the cell. Outside this range, enzyme activity drops
off very rapidly. Reduction in efficiency caused by changes in the pH is due to
changes in the degree of ionization of the substrate and enzyme. Highly acidic
or alkaline conditions bring about a denaturation and subsequent loss of
enzymatic activity. Some exceptions such as pepsin (with optimum pH 1-2),
alkaline phosphatase (with optimum pH 9-10) and acid phosphatase (with
optimum pH 4-5) are even noticed.

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Biochemistry Optimum pH

Notes pH range
Fig. 8.4: Effect of pH

8.5.6 Presence of activators

Presence of certain inorganic ions increases the activity of enzymes. The best
examples are chloride ions activated salivary amylase and calcium activated
lipases.

8.5.7 Effect of Inhibitors

The catalytic enzymatic reaction may be inhibited by substances which prevent
the formation of a normal enzyme-substrate complex. The level of inhibition
then depends entirely upon the relative concentrations of the true substrate and
the inhibitor. Such inhibition, which depends on competition with the substrate
for the active sites of the enzyme, is termed competitive inhibition. In other
cases, the inhibitor combines with the enzyme-substrate complex to give an
inactive enzyme-substrate-inhibitor complex, which cannot undergo further
reaction to give the usual product. This is termed uncompetitive inhibition. Non
competitive inhibition involves combination of the inhibitor with the enzyme
or the enzyme substrate complex, to give inactive complexes. In this case, the
inhibitor binds to sites, on the enzyme other than enzyme sites, resulting in
deformation of the enzyme molecule so that the formation of the enzyme-
substrate complex is slower than normal. Some enzymes undergo irreversible
inactivation; reaction of the inhibitor with a functional group of the enzyme,
resulting in a loss of its catalytic activity. Enzyme inhibitor plays a vital role in
clinical utility and is listed in table 8.2.
Table 8.2: Effect of Enzyme Inhibitors
Sl.No Enzymatic Enzyme inhibited Clinical use
inhibitor/drug
1. Allopurinol Xanthine oxidase gout
2. Dicoumarol Vitamin-K-epoxide-reductase Anti-coagulant
3. Penicillin Transpeptidase Anti-bacterial
4. Sulphonamide Pteroid synthetase Anti-bacterial
5. Pyrimethamine FH2-reductase Anti-malarial
6. 5-fluorouracil Thymidylate synthetase Anti-cancer

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INTEXT QUESTIONS 8.4

1. At optimal substrate concentration the rate of an enzymatic reaction or
velocity (V) is directly proportional to the ..................
2. Velocity of .................. increases with temperature of the medium.
3. The optimum pH of alkaline phosphatase are .................. Notes
4. Allopurinol are .................. inhibitor and used for treatment of ..................

8.6 ISO-ENZYMES
Iso-enzymes are physically distinct forms of the same enzyme activity. Higher
organisms have several physically distinct versions of a given enzyme, each of
which catalyzes the same reaction. Isozymes arise through gene duplication and
exhibit differences in properties such as sensitivity to particular regulatory
factors or substrate affinity that adapts them to specific tissues or circumstances.
Some isozymes enhance survival by providing a “backup” copy of an essential
enzyme. Isozymes are expressed only in specific cell types. The expression of
isozymes in specific cells occurs during certain periods of development, or in
response to specific physiologic or pathophysiologic changes. Thus analysis of
the presence and distribution of enzymes and isozymes are often useful in
diagnosis. Isoforms of Lactate dehydrogenase is useful in diagnosis of myocardial
infarction. While study of alkaline phosphatase isoforms are helpful in diagnosis
of various bone disorder and obstructive liver diseases.

INTEXT QUESTIONS 8.5

1. Physically distinct forms of the same enzyme are called as ................
2. Isozymes arise through ................ duplication.
3. Isoforms of ................ is useful in diagnosis of myocardial infarction.
4. Alkaline phosphatase isoforms are helpful in diagnosis of various ................
disorder.

8.7 CLINICAL SIGNIFICANCE OF ENZYMES

The measurement of enzymes level in serum is applied in diagnostic application.
Detection of certain enzymes in the serum indicates that tissue or cellular
damage has occurred resulting in the release of intracellular components into the
blood. Hence, when a physician indicates that he/she is going to assay for liver
enzymes, the purpose is to ascertain the potential for liver cell damage.

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Biochemistry Commonly assayed enzymes are the amino transferases: alanine transaminase,
ALT (sometimes still referred to as serum glutamate-pyruvate aminotransferase,
SGPT) and aspartate aminotransferase, AST (also referred to as serum glutamate-
oxaloacetate aminotransferase, SGOT); lactate dehydrogenase, LDH; creatine
kinase, CK (also called creatine phosphokinase, CPK); gamma-glutamyl
transpeptidase, GGT. Other enzymes are assayed under a variety of different
clinical situations but they will not be covered here.
Notes
8.7.1 Pancreatic Enzymes
Acute pancreatitis is an inflammatory process where auto digestion of gland was
noticed with activation of the certain pancreatic enzymes. Enzymes which
involves in pancreatic destruction includes α-amylase, lipase etc.,

8.7.1.1 α-amylase
α-amylase (AMYs) are calcium dependent hydrolyase class of metaloenzyme
that catalyzes the hydrolysis of 1, 4- α-glycosidic linkages in polysaccharides.
Molecular weights of AMYs are human plasma ranges from 54 to 62 kDa. Due
to its smaller size they could easily pass the glomeruli of the kidneys and AMY
is the only plasma enzyme physiologically found in urine. The normal values
of amylase is in range of 28-100 U/L. Marked increase of 5 to 10 times the upper
reference limit (URL) in AMYs activity indicates acute pancreatitis and severe
glomerular impairment. Pancreatic pseudocyst occurs if the plasma level of
amylase activity fails to fall after an attack of acute pancreatitis.

8.7.1.2 Lipase
Lipase is single chain glycoprotein of molecular weight 48 kDa. Bile salts and
a cofactor called colipase are required for full catalytic activity of lipase.
Colipase is secreted by pancreas. Lipase is small molecule filtered through the
glomerulus and totally reabsorbed by the renal tubules. Lipase is not normally
detected in urine samples. The normal value of lipase ranges from 40-200 U/
L. Increase in plasma lipase activity indicates acute pancreatitis and carcinoma
of the pancreas. So determination of both amylase and lipase together helps in
the diagnosis of acute pancreatitis.

8.7.2 Liver Enzymes

The assay of serum enzymes is very useful for the differential diagnosis and
monitoring of various liver disorders. Liver enzymes acts as marker of
hepatocellular damage, cholestasis and disturbances in the hepatocellular
synthesis.

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8.7.2.1 Markers of Hepatocellular Damage Biochemistry

In case of hepatocellular damage, the enzymes which are normally present inside
the hepatocytes are released into the blood. Aminotransaminases such as
aspartate transaminase (AST) and alanine transaminase (ALT) are routinely used
in diagnosis of hepatocellular damages. Transaminases are enzymes involved in
the transfer of an amino group from a 2-amino- to a 2-oxoacid. The 2-
oxoglutarate acts as amino group acceptor and the L-glutamate serves as donor
in all amino-transfer reactions. The specificity of the individual enzymes derives Notes
from the particular amino acid that serves as the other donor of an amino group.

8.7.2.1.1 Aspartate transaminase (AST)

Aspartate transaminase is present in high concentrations in cells of cardiac and
skeletal muscle, liver, kidney and erythrocytes. Damage to any of these tissues
may increase plasma AST levels. The normal value of AST for male is <35 U/
L and for female it is <31 U/L. Marked increase of AST activity in the range
of 10 to 100 times the upper adult reference limit indicates myocardial infarction
or acute viral or toxic hepatitis.

8.7.2.1.2 Alanine transaminase (ALT)

Alanine transaminase is present at high concentrations in liver and to a lesser
extent, in skeletal muscle, kidney and heart. Thus in case of liver damage
increase in both AST and ALT were noticed. While in myocardial infarction AST
is increased with little or no increase in ALT. The normal value of ALT is <45
U/L and <34 U/L for male and female respectively. In acute viral hepatitis there
is a 100-1000 times increase in both ALT and AST but ALT level is increased
more than that of AST.

8.7.2.2 Markers of cholestasis

Enzymatic markers of cholestasis are membrane bound enzymes. Markers of
cholestasis includes alkaline phosphatases, gamma-glutamyltransferase and
glutamate dehydrogenase.

8.7.2.2.1 Alkaline phosphatases

Alkaline phosphatases are a group of enzymes that hydrolyse organic phosphates
at high pH. They are present in osteoblasts of bone, the cells of the hepatobiliary
tract, intestinal wall, renal tubules and placenta.

8.7.2.2.2 Gamma-glutamyl-transferase (GGT)

Gamma-glutamyl-transferase catalyzes the transfere of the γ–glutamyl group
from peptides. The activity of GGT is higher in men than in women. In male

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Biochemistry the normal value of GGT activity is <55 U/L and for female it is <38 U/L. Rise
in plasma GGT activity is due to infectious hepatitis and induction of enzyme
synthesis, without cell damage, by drugs or alcohol.

8.7.2.2.3 Glutamate dehydrogenase (GLD)

Glutamate dehydrogenase is a mitochondrial enzyme found in liver, heart
Notes muscle and kidneys. Small amounts of GLD are even observed in brain, skeletal
muscle tissue and leukocytes. GLD is increased in serum of patients with
hepatocellular damage offering differential diagnostic potential in the investigation
of liver disease. GLD is released from necrotic cells and is of value in estimation
of the severity of liver cell damage. The GLD upper reference limits are 6U/L
(women) and 8U/L (men).

8.7.3 Muscle Enzymes

Clinically important muscle enzymes include creatine kinase and lactate
dehydrogenase.

8.7.3.1 Creatine Kinase

Creatine kinase (CK) is most abundant in cells of brain, cardiac and skeletal.
In addition to their abundance in above tissues, it also occurs in other tissues
such as smooth muscle. In normal physiological condition the CK activity is 46-
171 U/L (for male) and 34-145 U/L (for female). Serum CK level elevates in
all types of muscular dystrophy. Quite high values of CK are noted in viral
myositis, polymyositis and similar muscle disease. Under the circumstances of
neurogenic muscle disease such as: myasthenia gravis, multiple sclerosis and
Parkinsonism, the level of serum CK is normal. CK consist of two protein
subunits, M (for muscle) and B (for brain) and exist as three different isoforms
namely BB (CK-1), MB (CK-2) and MM (CK-3). CK-MM is the predominant
isoenzyme in skeletal and cardiac muscle and is detectable in the plasma of
normal subjects. CK-BB is present in high concentrations in the brain and in the
smooth muscle of the gastrointestinal and genital tracts.

8.7.3.2 Lactate Dehydrogenase

Lactate dehydrogenase (LD) catalyses the reversible interconversion of lactate
and pyruvate. LD has a molecular weight of 134 kDa and is widely distributed
in the body, with high concentrations in cells of cardiac and skeletal muscle,
liver, kidney, brain and erythrocytes. Five isoforms (LD-1 to LD-5) of LD are
existing. The normal physiological limit of LD is 180-360 U/L.
Other clinically important enzymes includes acid phosphatase, glucose -6-
phosphate dehydrogenase, cystathionine α-synthase and sphingomyelinase.

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Biochemistry

INTEXT QUESTIONS 8.6

1. .................. and .................. are enzymes which involves in pancreatic
destruction.
2. Molecular weights of amylases in human plasma ranges .................. to
.................. kDa.
Notes
3. Plasma level of amylase activity fails to fall after an attack of ..................
4. Alkaline phosphatases, gamma-glutamyltransferase and glutamate
dehydrogenase are markers of ..................
5. Clinically important muscle enzymes include .................. and ..................

WHAT HAVE YOU LEARNT

z Enzymes are protein catalyst produced by a cell and responsible ‘for the
high rate’ and specificity of one or more intracellular or extracellular
biochemical reactions.
z Enzymes posses the catalytic power to facilitates life processes in essentially
all life-forms from viruses to man.
z Enzymes are found in all tissues and fluids of the body.
z Intracellular enzymes catalyze the reactions of metabolic pathways.
z Plasma membrane enzymes regulate catalysis within cells in response to
extracellular signals.
z Enzymes are broadly classified into 6 major groups namely Oxidoreductases,
Transferases, Hydrolases, Lyases, Isomerases and Ligases.
z Enzymes may be simple proteins, or complex enzymes (presence of non-
protein part, called as prosthetic group).
z Coenzymes functions as transporters of chemical groups from one reactant
to another.
z Activity of a particular enzyme may be affected by many external factors
such as concentration of substrate, product, enzyme, hydrogen ion,
temperature, activators and inhibitors.
z Enzyme inhibitors plays a vital role in clinical utility and are useful as anti-
bacterial, anti-malarial, anti-cancer molecule and treatment of metabolic
disorders such as gout. Thus measurement of enzymes level in serum is
applied in diagnostic application.

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TERMINAL QUESTIONS
1. Define enzyme?
2. Write about classification of enzymes?
3. Give a brief discussion about various factors affecting enzyme activity?
Notes 4. What are metallo enzymes?
5. Define isozymes?
6. What are co-enzymes?
7. Differentiate synthase and synthatase?
8. Write a note on clinically important enzymes?
9. Describe in detail about the liver enzymes used in clinical diagnosis?
10. Give some examples of enzyme inhibitory drugs?

ANSWERS TO INTEXT QUESTIONS

8.1
1. Protein
2. Proteins
3. Water
4. Ammonium sulfate and Trichloroacetic acid

8.2
1. Six
2. Oxidoreductases
3. Metallo enzymes
4. Synthetases
5. Glycogen synthase and Alanine synthase

8.3
1. Co-enzymes
2. Protein and co-enzyme
3. Organic
4. Two

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8.4 Biochemistry

1. Enzyme concentration
2. Enzyme reaction
3. 9-10
4. Xanthine oxidase and gout

Notes
8.5
1. Isozymes
2. Gene
3. Lactate dehydrogenase
4. Bone

8.6
1. α-amylase and lipase
2. 54 to 62 kDa
3. Acute pancreatitis
4. Cholestasis
5. Creatine kinase and lactate dehydrogenase

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Biochemistry

9
Notes
BIOLOGICAL OXIDATION,
ELECTRON TRANSFER
CHAIN AND OXIDATIVE
PHOSPHORYLATION

9.1 INTRODUCTION
Chemically, oxidation is defined as the removal of electrons and reduction as
the gain of electrons. Thus, oxidation is always accompanied by reduction of an
electron acceptor. This principle of oxidation-reduction applies equally to
biochemical systems and is an important concept underlying understanding of
the nature of biologic oxidation. Many biologic oxidations can take place
without the participation of molecular oxygen, eg, dehydrogenations. The life
of higher animals is absolutely dependent upon a supply of oxygen for
respiration, the process by which cells derive energy in the form of ATP from
the controlled reaction of hydrogen with oxygen to form water. In addition,
molecular oxygen is incorporated into a variety of substrates by enzymes
designated as oxygenases; many drugs, pollutants, and chemical carcinogens
(xenobiotics) are metabolized by enzymes of this class, known as the cytochrome
P450 system. Administration of oxygen can be lifesaving in the treatment of
patients with respiratory or circulatory failure.

OBJECTIVES
After reading this lesson, you will be able to
z describe biological oxidation
z explain Electron transfer chain
z describe oxidation phosphorylation

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9.2 BIOLOGICAL OXIDATION-REDUCTION
9.2.1 Redox potential – free energy changes
In reactions involving oxidation and reduction, the free energy change is
proportionate to the tendency of reactants to donate or accept electrons. Free
energy change expressed as oxidation-reduction or redox potential. The redox
potential of a system is usually compared with the potential of the hydrogen
electrode (0.0 volts at pH 0.0). However, for biologic systems, the redox Notes
potential is normally expressed at pH 7.0, at which pH the electrode potential
of the hydrogen electrode is -0.42 volts. Enzymes involved in oxidation and
reduction are called oxidoreductases and are classified into four groups:
oxidases, dehydrogenases, hydroperoxidases, and oxygenases. Oxidases use
oxygen as a hydrogen acceptor. Oxidases catalyze the removal of hydrogen from
a substrate using oxygen as a hydrogen acceptor and form water or hydrogen
peroxide as a reaction product.

9.2.2 Some oxidases contain copper

Cytochrome oxidase is a hemoprotein widely distributed in many tissues, having
the typical heme prosthetic group present in myoglobin, hemoglobin, and other
cytochromes. It is the terminal component of the chain of respiratory carriers
found in mitochondria and transfers electrons resulting from the oxidation of
substrate molecules by dehydrogenases to their final acceptor, oxygen. The
enzyme is poisoned by carbon monoxide, cyanide, and hydrogen sulfide. It has
also been termed cytochrome a3. It is now known that cytochromes a and a3
are combined in a single protein, and the complex is known as cytochrome aa3.
It contains two molecules of heme, each having one Fe atom that oscillates
between Fe3+ and Fe2+ during oxidation and reduction. Furthermore, two atoms
of Cu are present, each associated with a heme unit.

9.2.3 Other oxidases are Flavoproteins

Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin adenine
dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed in the body
from the vitamin riboflavin. FMN and FAD are usually tightly – but not
covalently – bound to their respective apoenzyme proteins. Metalloflavoproteins
contain one or more metals as essential cofactors. Examples of flavoprotein
enzymes include L-amino acid oxidase, an FMN-linked enzyme found in kidney
with general specificity for the oxidative deamination of the naturally occurring
L-amino acids.

9.2.4 Dehydrogenases cannot use oxygen as a hydrogen acceptor

There are a large number of enzymes in this class. They perform two main
functions:

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Biochemistry 1. Transfer of hydrogen from one substrate to another in a coupled oxidation-

reduction reaction. These dehydrogenases are specific for their substrates
but often utilize common coenzymes or hydrogen carriers, eg, NAD+
(Figure 9.1). Since the reactions are reversible, these properties enable
reducing equivalents to be freely transferred within the cell. This type of
reaction, which enables one substrate to be oxidized at the expense of
another, is particularly useful in enabling oxidative processes to occur in
Notes the absence of oxygen, such as during the anaerobic phase of glycolysis.
NAD+ + AH2 ←⎯
→ NADH + H+ + A
Fig. 9.1: NAD acting as a hydrogen carrier in the dehydrogenase reaction.

2. As components in the respiratory chain of electron transport from substrate

to oxygen.

9.2.5 Many dehydrogenases depend on Nicotinamide Coenzymes

These dehydrogenases use nicotinamide adenine dinucleotide (NAD+) or
nicotinamide adenine dinucleotide phosphate (NADP+)—or both—and are
formed in the body from the vitamin niacin. These coenzymes are reduced by
the specific substrate of the dehydrogenase and reoxidized by a suitable electron
acceptor. They may freely and reversibly dissociate from their respective
apoenzymes. Generally, NAD-linked dehydrogenases catalyze oxidoreduction
reactions in the oxidative pathways of metabolism, particularly in glycolysis, in
the citric acid cycle, and in the respiratory chain of mitochondria. NADP-linked
dehydrogenases are found characteristically in reductive syntheses, as in the
extramitochondrial pathway of fatty acid synthesis and steroid synthesis— and
also in the pentose phosphate pathway.

9.2.6 Other dehydrogenases depend on Riboflavin

The flavin groups associated with these dehydrogenases are similar to FMN and
FAD occurring in oxidases. They are generally more tightly bound to their
apoenzymes than are the nicotinamide coenzymes. Most of the riboflavin-linked
dehydrogenases are concerned with electron transport in (or to) the respiratory
chain. NADH dehydrogenase acts as a carrier of electrons between NADH and
the components of higher redox potential. Other dehydrogenases such as
succinate dehydrogenase, acyl-CoA dehydrogenase, and mitochondrial glycerol-
3-phosphate dehydrogenase transfer reducing equivalents directly from the
substrate to the respiratory chain. Another role of the flavin-dependent
dehydrogenases is in the dehydrogenation of reduced lipoate, an intermediate in
the oxidative decarboxylation of pyruvate and α-ketoglutarate. The electron-

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transferring flavoprotein is an intermediary carrier of electrons between acyl-
CoA dehydrogenase and the respiratory chain.

9.2.7 Cytochromes may also be regarded as dehydrogenases

The cytochromes are iron-containing hemoproteins in which the iron atom
oscillates between Fe3+ and Fe2+ during oxidation and reduction. Except for
cytochrome oxidase, they are classified as dehydrogenases. In the respiratory Notes
chain, they are involved as carriers of electrons from flavoproteins on the one
hand to cytochrome oxidase on the other. Several identifiable cytochromes occur
in the respiratory chain, ie, cytochromes b, c1, c, a, and a3 (cytochrome oxidase).
Cytochromes are also found in other locations, eg, the endoplasmic reticulum
(cytochromes P450 and b5), and in plant cells, bacteria, and yeasts.

9.2.8 Hydroperoxidases use hydrogen peroxide or organic peroxide as

substrate
Two type of enzymes found both in animals and plants fall into this category:
peroxidases and catalase. Hydroperoxidases protect the body against harmful
peroxides. Accumulation of peroxides can lead to generation of free radicals,
which in turn can disrupt membranes and perhaps cause cancer and atherosclerosis.

9.2.9 Peroxidases reduce peroxides using various electron acceptors

Peroxidases are found in milk and in leukocytes, platelets, and other tissues
involved in eicosanoid metabolism. The prosthetic group is protoheme. In the
reaction catalyzed by peroxidase, hydrogen peroxide is reduced at the expense
of several substances that will act as electron acceptors, such as ascorbate,
quinones, and cytochrome c. The reaction catalyzed by peroxidase is complex,
but the overall reaction is as follows: In erythrocytes and other tissues, the
enzyme glutathione peroxidase, containing selenium as a prosthetic group,
catalyzes the destruction of H2O2 and lipid hydroperoxides by reduced
glutathione, protecting membrane lipids and hemoglobin against oxidation by
peroxides.

9.2.10 Catalase uses hydrogen peroxide as electron donor and electron

acceptor
Catalase is a hemoprotein containing four heme groups. In addition to possessing
peroxidase activity, it is able to use one molecule of H2O2 as a substrate electron
donor and another molecule of H2O2 as an oxidant or electron acceptor (Figure
9.2). Under most conditions in vivo, the peroxidase activity of catalase seems
to be favored. Catalase is found in blood, bone marrow, mucous membranes,

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Biochemistry kidney, and liver. Its function is assumed to be the destruction of hydrogen
peroxide formed by the action of oxidases. Peroxisomes are found in many
tissues, including liver. They are rich in oxidases and in catalase, Thus, the
enzymes that produce H2O2 are grouped with the enzyme that destroys it.
However, mitochondrial and microsomal electron transport systems as well as
xanthine oxidase must be considered as additional sources of H2O2.

Catalase
2H2O2 ⎯⎯⎯⎯
Notes → 2H2O + O2
Fig. 9.2: Catalase uses hydrogen peroxide as electron donor and electron acceptor.

9.2.11 Cytochromes P450 are monooxygenases important for the

detoxification of many drugs
Cytochromes P450 are an important superfamily of heme-containing
monooxgenases, and more than 1000 such enzymes are known. Both NADH and
NADPH donate reducing equivalents for the reduction of these cytochromes,
which in turn are oxidized by substrates in a series of enzymatic reactions
collectively known as the hydroxylase cycle (Figure 9.3). In liver microsomes,
cytochromes P450 are found together with cytochrome b5 and have an important
role in detoxification. Benzpyrene, aminopyrine, aniline, morphine, and
benzphetamine are hydroxylated, increasing their solubility and aiding their
excretion. Many drugs such as Phenobarbital have the ability to induce the
formation of microsomal enzymes and of cytochromes P450. Mitochondrial
cytochrome P450 systems are found in steroidogenic tissues such as adrenal
cortex, testis, ovary, and placenta and are concerned with the biosynthesis of
steroid hormones from cholesterol.
Reduced cytochrome P450 ⎯→ Oxidized cytochrome P450
RH + O2 ⎯→ R – OH + H2O
Fig. 9.3: Reduction and oxidation of cytochrome.

9.2.12 Superoxide dismutase protects aerobic organisms against oxygen

toxicity
Transfer of a single electron to O2 generates the potentially damaging superoxide
anion free radical (O2"-) (Figure 9.4), the destructive effects of which are
amplified by its giving rise to free radical chain reactions. The ease with which
superoxide can be formed from oxygen in tissues and the occurrence of
superoxide dismutase, the enzyme responsible for its removal in all aerobic
organisms (although not in obligate anaerobes) indicate that the potential
toxicity of oxygen is due to its conversion to superoxide. Superoxide is formed
when reduced flavins – present, for example, in xanthine oxidase – are
reoxidized univalently by molecular oxygen.
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Biochemistry
O2– + O2– + 2H+ ⎯⎯⎯⎯⎯⎯⎯⎯
Superoxide Dismutase
→ H2O + O2
Fig. 9.4: Removal of superoxide anion free radical by superoxide dismutase enzyme.

9.3 THE RESPIRATORY CHAIN AND OXIDATIVE

PHOSPHORYLATION
Aerobic organisms are able to capture a far greater proportion of the available Notes
free energy of respiratory substrates than anaerobic organisms. Most of this takes
place inside mitochondria, which have been termed the “powerhouses” of the
cell. Respiration is coupled to the generation of the high-energy intermediate,
ATP, by oxidative phosphorylation, and the chemiosmotic theory offers insight
into how this is accomplished. A number of drugs (eg, amobarbital) and poisons
(eg, cyanide, carbon monoxide) inhibit oxidative phosphorylation, usually with
fatal consequences. Several inherited defects of mitochondria involving
components of the respiratory chain and oxidative phosphorylation have been
reported. Patients present with myopathy and encephalopathy and often have
lactic acidosis.

9.3.1 Specific enzymes act as markers

Mitochondria have an outer membrane that is permeable to most metabolites,
an inner membrane that is selectively permeable, and a matrix within. The outer
membrane is characterized by the presence of various enzymes, including acyl-
CoA synthetase and glycerolphosphate acyltransferase. Adenylyl kinase and
creatine kinase are found in the intermembrane space. The phospholipid
cardiolipin is concentrated in the inner membrane together with the enzymes of
the respiratory chain.

9.3.2 The respiratory chain collects and oxidizes reducing equivalents

Most of the energy liberated during the oxidation of carbohydrate, fatty acids,
and amino acids is made available within mitochondria as reducing equivalents
(H+ or electrons). Mitochondria contain the respiratory chain, which collects and
transports reducing equivalents directing them to their final reaction with oxygen
to form water, the machinery for trapping the liberated free energy as high-energy
phosphate, and the enzymes of β-oxidation and of the citric acid cycle that
produce most of the reducing equivalents.

9.3.3 Components of the respiratory chain are arranged in order of

increasing redox potential
The respiratory chain consists of a number of redox carriers that proceed from
the NAD-linked dehydrogenase systems, through flavoproteins and cytochromes,

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Biochemistry to molecular oxygen. Not all substrates are linked to the respiratory chain
through NAD-specific dehydrogenases; some, because their redox potentials are
more positive (eg, fumarate/succinate; are linked directly to flavoprotein
dehydrogenases, which in turn are linked to the cytochromes of the respiratory
chain.

9.3.4 Ubiquinone or Q (coenzyme Q)

Notes
Coenzyme Q links the flavoproteins to cytochrome b, the member of the
cytochrome chain of lowest redox potential. Q exists in the oxidized quinone
or reduced quinol form under aerobic or anaerobic conditions, respectively. The
structure of Q is very similar to that of vitamin K and vitamin E and of
plastoquinone, found in chloroplasts. Q acts as a mobile component of the
respiratory chain that collects reducing equivalents from the more fixed
flavoprotein complexes and passes them on to the cytochromes. An additional
component is the iron-sulfur protein (FeS; nonheme iron). It is associated with
the flavoproteins (metalloflavoproteins) and with cytochrome b. The sulfur and
iron are thought to take part in the oxidoreduction mechanism between flavin
and Q, which involves only a single e-change, the iron atom undergoing
oxidoreduction between Fe2+ and Fe3+.

Fig. 9.5: In this simple representation of the chemiosmotic theory applied to mitochondria,
electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged
asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across
the membrane, producing both a chemical gradient (ΔpH) and an electrical gradient (Δψ). The
inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only
through proton-specific channels (Fo). The proton-motive force that drives protons back into
the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with
Fo.

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Pyruvate and α-ketoglutarate dehydrogenase have complex systems involving Biochemistry
lipoate and FAD prior to the passage of electrons to NAD, while electron trans
fers from other dehydrogenases, e.g., L(+)-3-hydroxyacyl- CoA dehydrogenase,
couple directly with NAD. The reduced NADH of the respiratory chain is in turn
oxidized by a metalloflavoprotein enzyme – NADH dehydrogenase. This
enzyme contains FeS and FMN, is tightly bound to the respiratory chain, and
passes reducing equivalents on to Q. Electrons flow from Q through the series
of cytochromes in order of increasing redox potential to molecular oxygen. The Notes
terminal cytochrome aa3 (cytochrome oxidase), responsible for the final
combination of reducing equivalents with molecular oxygen, has a very high
affinity for oxygen, allowing the respiratory chain to function at maximum rate
until the tissue has become depleted of O2. Since this is an irreversible reaction
(the only one in the chain), it gives direction to the movement of reducing
equivalents and to the production of ATP, to which it is coupled. Functionally
and structurally, the components of the respiratory chain are present in the inner
mitochondrial membrane as four protein-lipid respiratory chain complexes that
span the membrane. Cytochrome c is the only soluble cytochrome and, together
with Q, seems to be a more mobile component of the respiratory chain
connecting the fixed complexes. The overall reaction is given in the figure 9.5.

9.3.5 The respiratory chain provides most of the energy captured during
catabolism
ADP captures, in the form of high-energy phosphate, a significant proportion of
the free energy released by catabolic processes. The resulting ATP has been
called the energy “currency” of the cell because it passes on this free energy to
drive those processes requiring energy. There is a net direct capture of two high-
energy phosphate groups in the glycolytic reactions, equivalent to approximately
103.2 kJ/mol of glucose. (In vivo, ΔG for the synthesis of ATP from ADP has
been calculated as approximately 51.6 kJ/mol. (It is greater than ΔG0' for the
hydrolysis of ATP, which is obtained under standard concentrations of 1.0 mol/
L.) Since 1 mol of glucose yields approximately 2870 kJ on complete
combustion, the energy captured by phosphorylation in glycolysis is small. Two
more high-energy phosphates per mole of glucose are captured in the citric acid
cycle during the conversion of succinyl CoA to succinate.

All of these phosphorylations occur at the substrate level. When substrates are
oxidized via an NAD-linked dehydrogenase and the respiratory chain,
approximately 3 mol of inorganic phosphate are incorporated into 3 mol of ADP
to form 3 mol of ATP per half mol of O2 consumed; ie, the P:O ratio = 3. On
the other hand, when a substrate is oxidized via a flavoprotein- linked
dehydrogenase, only 2 mol of ATP are formed; ie, P:O = 2. These reactions are

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Biochemistry known as oxidative phosphorylation at the respiratory chain level. Such

dehydrogenations plus phosphorylations at the substrate level can now account
for 68% of the free energy resulting from the combustion of glucose, captured
in the form of high-energy phosphate. It is evident that the respiratory chain is
responsible for a large proportion of total ATP formation.

Notes 9.3.6 Respiratory control ensures a constant supply of ATP

The rate of respiration of mitochondria can be controlled by the availability of
ADP. This is because oxidation and phosphorylation are tightly coupled; ie,
oxidation cannot proceed via the respiratory chain without concomitant
phosphorylation of ADP. When work is performed, ATP is converted to ADP,
allowing more respiration to occur, which in turn replenishes the store of ATP.
Under certain conditions, the concentration of inorganic phosphate can also
affect the rate of functioning of the respiratory chain. There is also the possibility
that the ADP/ATP transporter, which facilitates entry of cytosolic ADP into and
ATP out of the mitochondrion, becomes rate limiting. Thus, the manner in which
biologic oxidative processes allow the free energy resulting from the oxidation
of foodstuffs to become available and to be captured is stepwise, efficient
(approximately 68%), and controlled – rather than explosive, inefficient, and
uncontrolled, as in many nonbiologic processes. The remaining free energy that
is not captured as high-energy phosphate is liberated as heat. This need not be
considered “wasted,” since it ensures that the respiratory system as a whole is
sufficiently exergonic to be removed from equilibrium, allowing continuous
unidirectional flow and constant provision of ATP. It also contributes to
maintenance of body temperature.

9.3.7 Many poisons inhibit the respiratory chain

Much information about the respiratory chain has been obtained by the use of
inhibitors, and, conversely, this has provided knowledge about the mechanism
of action of several poisons (Figure 9.6). They may be classified as inhibitors
of the respiratory chain, inhibitors of oxidative phosphorylation, and uncouplers
of oxidative phosphorylation. Barbiturates such as amobarbital inhibit NAD
linked dehydrogenases by blocking the transfer from FeS to Q. At sufficient
dosage, they are fatal in vivo. Antimycin A and dimercaprol inhibit the
respiratory chain between cytochrome b and cytochrome c. The classic poisons
H2S, carbon monoxide, and cyanide inhibit cytochrome oxidase and can
therefore totally arrest respiration. Malonate is a competitive inhibitor of
succinate dehydrogenase. Atractyloside inhibits oxidative phosphorylation by
inhibiting the transporter of ADP into and ATP out of the mitochondrion. The

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action of uncouplers is to dissociate oxidation in the respiratory chain from Biochemistry
phosphorylation. These compounds are toxic in vivo, causing respiration to
become uncontrolled, since the rate is no longer limited by the concentration of
ADP or Pi. The uncoupler that has been used most frequently is 2,4-
dinitrophenol, but other compounds act in a similar manner. The antibiotic
oligomycin completely blocks oxidation and phosphorylation by acting on a step
in phosphorylation.
Notes

Fig. 9.6: Proposed sites of inhibition (--) of the respiratory chain by specific drugs, chemicals,
and antibiotics. The sites that appear to support phosphorylation are indicated. BAL,
dimercaprol. TTFA, an Fe-chelating agent. Complex I, NADH:ubiquinone oxidoreductase;
complex II, succinate:ubiquinone oxidoreductase; complex III, ubiquinol:ferricytochrome c
oxidoreductase; complex IV, ferrocytochrome c:oxygen oxidoreductase.

9.3.8 The chemiosmotic theory explains the mechanism of oxidative

phosphorylation
Mitchell’s chemiosmotic theory postulates that the energy from oxidation of
components in the respiratory chain is coupled to the translocation of hydrogen
ions (protons, H+) from the inside to the outside of the inner mitochondrial
membrane. The electrochemical potential difference resulting from the asymmetric
distribution of the hydrogen ions is used to drive the mechanism responsible for
the formation of ATP (Figure 9.7).

9.3.9. The respiratory chain is a proton pump

Each of the respiratory chain complexes I, III, and IV act as a proton pump. The
inner membrane is impermeable to ions in general but particularly to protons,
which accumulate outside the membrane, creating an electrochemical potential
difference across the membrane. This consists of a chemical potential (difference
in pH) and an electrical potential.

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Notes

Fig. 9.7: Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton
circuit is created by the coupling of oxidation in the respiratory chain to proton translocation
from the inside to the outside of the membrane, driven by the respiratory chain complexes I,
III, and IV, each of which acts as a proton pump. Q, ubiquinone; C, cytochrome c; F1, F0,
protein subunits which utilize energy from the proton gradient to promote phosphorylation.
Uncoupling agents such as dinitrophenol allow leakage of H+ across the membrane, thus
collapsing the electrochemical proton gradient. Oligomycin specifically blocks conduction of
H+ through F0.

9.3.10 A membrane-located ATP synthase functions as a rotary motor to

form ATP
The electrochemical potential difference is used to drive a membrane-located
ATP synthase which in the presence of Pi + ADP forms ATP. Scattered over the
surface of the inner membrane are the phosphorylating complexes, ATP
synthase, responsible for the production of ATP. These consist of several protein
subunits, collectively known as F1, which project into the matrix and which
contain the phosphorylation mechanism. These subunits are attached to a
membrane protein complex known as F0, which also consists of several protein
subunits. F0 spans the membrane and forms the proton channel. The flow of
protons through F0 causes it to rotate, driving the production of ATP in the F1
complex. Estimates suggest that for each NADH oxidized, complex I translocates
four protons and complexes III and IV translocate 6 between them. As four
protons are taken into the mitochondrion for each ATP exported, the P:O ratio
would not necessarily be a complete integer, ie, 3, but possibly 2.5. However,
for simplicity, a value of 3 for the oxidation of NADH + H+ and 2 for the
oxidation of FADH2 will continue to be used throughout this text.

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9.3.11 The chemiosmotic theory can account for respiratory control and Biochemistry
the action of uncouplers
The electrochemical potential difference across the membrane, once established
as a result of proton translocation, inhibits further transport of reducing
equivalents through the respiratory chain unless discharged by back translocation
of protons across the membrane through the vectorial ATP synthase. This in turn
depends on availability of ADP and Pi. Uncouplers (eg, dinitrophenol) are Notes
amphipathic and increase the permeability of the lipoid inner mitochondrial
membrane to protons, thus reducing the electrochemical potential and short-
circuiting the ATP synthase. In this way, oxidation can proceed without
phosphorylation.

9.3.12 Impermeability of the inner mitochondrial membrane

The relative impermeability of the inner mitochondrial membrane necessitates
exchange transporters. Exchange diffusion systems are present in the membrane
for exchange of anions against OH- ions and cations against H+ ions. Such
systems are necessary for uptake and output of ionized metabolites while
preserving electrical and osmotic equilibrium. The inner bilipoid mitochondrial
membrane is freely permeable to uncharged small molecules, such as oxygen,
water, CO2, and NH3, and to monocarboxylic acids, such as 3-hydroxybutyric,
acetoacetic, and acetic. Long-chain fatty acids are transported into mitochondria
via the carnitine system, and there is also a special carrier for pyruvate involving
a symport that utilizes the H+ gradient from outside to inside the mitochondrion.
However, dicarboxylate and tri- carboxylate anions and amino acids require
specific transporter or carrier systems to facilitate their passage across the
membrane. Monocarboxylic acids penetrate more readily in their undissociated
and more lipid-soluble form. The transport of di- and tricarboxylate anions is
closely linked to that of inorganic phosphate, which penetrates readily as the
H2PO4– ion in exchange for OH–.

9.3.13. Ionophores permit specific cations to penetrate membranes

Ionophores are lipophilic molecules that complex specific cations and facilitate
their transport through biologic membranes, eg, valinomycin (K+). The classic
uncouplers such as dinitrophenol are, in fact, proton ionophores.

9.3.14 A proton-translocating transhydrogenase is a source of

intramitochon-drial NADPH
Energy-linked transhydrogenase, a protein in the inner mitochondrial membrane,
couples the passage of protons down the electrochemical gradient from outside

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Biochemistry to inside the mitochondrion with the transfer of H from intramitochondrial

NADH to NADPH for intramitochondrial enzymes such as glutamate
dehydrogenase and hydroxylases involved in steroid synthesis.

9.3.15 Oxidation of extramitochondrial NADH is mediated by substrate

shuttles
Notes NADH cannot penetrate the mitochondrial membrane, but it is produced
continuously in the cytosol by 3-phosphoglyceraldehyde dehydrogenase, an
enzyme in the glycolysis sequence. However, under aerobic conditions,
extramitochondrial NADH does not accumulate and is presumed to be oxidized
by the respiratory chain in mitochondria. The transfer of reducing equivalents
through the mitochondrial membrane requires substrate pairs, linked by suitable
dehydrogenases on each side of the mitochondrial membrane. The mechanism
of transfer uses glycerophosphate shuttle. Since the mitochondrial enzyme is
linked to the respiratory chain via a flavoprotein rather than NAD, only 2 mol
rather than 3 mol of ATP are formed per atom of oxygen consumed. Although
this shuttle is present in some tissues (eg, brain, white muscle), in others (eg,
heart muscle) it is deficient. It is therefore believed that the malate shuttle system
is of more universal utility. The complexity of this system is due to the
impermeability of the mitochondrial membrane to oxaloacetate, which must
react with glutamate and transaminate to aspartate and á-ketoglutarate before
transport through the mitochondrial membrane and reconstitution to oxaloacetate
in the cytosol.

9.3.16. Ion transport in mitochondria is energy-linked

Mitochondria maintain or accumulate cations such as K+, Na+, Ca2+, and
Mg2+, and Pi. It is assumed that a primary proton pump drives cation exchange.

9.3.17 The creatine phosphate shuttle facilitates transport of high-energy

phosphate from mitochondria
The creatine phosphate shuttle augments the functions of creatine phosphate as
an energy buffer by acting as a dynamic system for transfer of high-energy
phosphate from mitochondria in active tissues such as heart and skeletal muscle.
An isoenzyme of creatine kinase is found in the mitochondrial intermembrane
space, catalyzing the transfer of high-energy phosphate to creatine from ATP
emerging from the adenine nucleotide transporter. In turn, the creatine phosphate
is transported into the cytosol via protein pores in the outer mitochondrial
membrane, becoming available for generation of extramitochondrial ATP.
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Biochemistry

INTEXT QUESTIONS 9.1

I. Choose the best answer
1. Chemically, the removal and the gain of electrons is defined respectively
as
(a) Oxidation and reduction
Notes
(b) Reduction and oxidation
(c) Oxidation and dehydrogenase
(d) Reduction and dehydrogenase
2. Chemical carcinogens (xenobiotics) are metabolized by the enzymes
system known as
(a) Cytochrome P450 (b) Xanthine oxidase
(c) Succinate dehydrogenase (d) Hydroperoxides
3. Oxidative phosphorylation is inhibited usually with fatal consequences
by
(a) Quinalones (b) Cyanide
(c) Anacin (d) Amoxycillin
4. The action of uncouplers is to dissociate oxidation in the respiratory
chain from
(a) Gluconeogenesis (b) Glycolysis
(c) TCA cycle (d) Phosphorylation
5. This antibiotic completely blocks oxidation and phosphorylation by
acting on a step in phosphorylation.
(a) Dinitrophenol (b) Benzpyrene
(c) Oligomycin (d) Morphine
II. Fill in the blanks
6. Flavoprotein enzymes contain .................. or .................. as prosthetic
groups.
7. Generally, NAD-linked dehydrogenases catalyze .................. reactions
in the oxidative pathways of metabolism.
8. .................. protect the body against harmful peroxides.
9. The function of .................. is assumed to be the destruction of
hydrogen peroxide formed by the action of oxidases.
10. Cytochromes P450 are an important superfamily of heme-containing
..................

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Biochemistry III. Match the following

11. Mitochondria (a) Energy currency of the cell
12. Cardiolipin (b) Uncouplers
13. ATP (c) Powerhouses of the cell
14. Valinomycin (d) Phospholipid
15. Dinitrophenol (e) Ionophores
Notes

WHAT HAVE YOU LEARNT

z Oxidation is defined as the removal of electrons and reduction as the gain
of electrons.
z Many drugs, pollutants, and chemical carcinogens (xenobiotics) are
metabolized oxygenases known as cytochrome P450 system.
z Enzymes involved in oxidation and reduction are called oxidoreductases
and are classified into four groups: oxidases, dehydrogenases,
hydroperoxidases, and oxygenases.
z Flavoprotein enzymes contain flavin mononucleotide (FMN) or flavin
adenine dinucleotide (FAD) as prosthetic groups. FMN and FAD are formed
in the body from the vitamin riboflavin.
z Oxidation-reduction reactions carried out by dehydrogenases are specific
for their substrates but often utilize common coenzymes or hydrogen
carriers, eg, NAD+.
z The cytochromes are iron-containing hemoproteins in which the iron atom
oscillates between Fe3+ and Fe2+ during oxidation and reduction.
z Cytochromes are also found in the endoplasmic reticulum (cytochromes
P450 and b5), and in plant cells, bacteria, and yeasts.
z Two type of enzymes found both in animals and plants are peroxidases and
catalase. Hydroperoxidases protect the body against harmful peroxides.
z Accumulation of peroxides can lead to generation of free radicals, which
in turn can disrupt membranes and perhaps cause cancer and atherosclerosis.
z Mitochondrial cytochrome P450 systems are found in steroidogenic tissues
such as adrenal cortex, testis, ovary, and placenta and are concerned with
the biosynthesis of steroid hormones from cholesterol.
z The potential toxicity of oxygen is due to its conversion to superoxide in
tissues and the enzyme superoxide dismutase is responsible for its removal.
z Mitochondria are termed as the “powerhouses” of the cell. Respiration is
coupled to the generation of the high-energy intermediate, ATP, by oxidative

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phosphorylation, and the chemiosmotic theory offers insight into how this Biochemistry
is accomplished.
z A number of drugs (eg, amobarbital) and poisons (eg, cyanide, carbon
monoxide) inhibit oxidative phosphorylation, usually with fatal consequences.
z Mitochondria contain the respiratory chain, which collects and transports
reducing equivalents directing them to their final reaction with oxygen to
form water, the machinery for trapping the liberated free energy as high-
Notes
energy phosphate, and the enzymes of β-oxidation and of the citric acid
cycle that produce most of the reducing equivalents.
z Mitchell’s chemiosmotic theory postulates that the energy from oxidation
of components in the respiratory chain is coupled to the translocation of
hydrogen ions (protons, H+) from the inside to the outside of the inner
mitochondrial membrane. The electrochemical potential difference resulting
from the asymmetric distribution of the hydrogen ions is used to drive the
mechanism responsible for the formation of ATP.
z Uncouplers (eg, dinitrophenol) are amphipathic and increase the permeability
of the lipoid inner mitochondrial membrane to protons, thus reducing the
electrochemical potential and short-circuiting the ATP synthase. In this way,
oxidation can proceed without phosphorylation.
z Ionophores are lipophilic molecules that complex specific cations and
facilitate their transport through biologic membranes, eg, valinomycin (K+).

TERMINAL QUESTIONS
1. Write short note on electron transfer chain.
2. Write short note on oxidative phosphorylation.

ANSWERS TO INTEXT QUESTIONS

I. 1. (a) 2. (a) 3. (b) 4. (d) 5. (c)
II. 6. Flavin mononucleotide (FMN) or Flavin adenine dinucleotide (FAD)
7. Oxidoreduction
8. Hydroperoxidases
9. Catalase
10. Monooxgenases
III. 11. (c) 12. (d) 13. (a) 14. (e) 15. (b)

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 MODULE Vitamins

Biochemistry

10
Notes
VITAMINS

10.1 INTRODUCTION
The term “vitamin” is used to describe certain organic compounds that are
needed by the body but that cannot be manufactured by the body. They mainly
serve as catalysts for certain reactions in the body. The amounts of vitamins
required are very small, perhaps hundredths of grams. Vitamins are mainly
obtained from our foods.

OBJECTIVES
After reading this lesson, you will be able to
z classify vitamins
z describe Water soluble vitamins
z describe Fat soluble vitamins

10.2 CLASSIFICATION OF VITAMINS

Based on solubility Vitamins are classified as either fat-soluble (lipid soluble)
or water-soluble. Vitamins A, D, E and K are fat-soluble Vitamin C and B is
water soluble.

WATER-SOLUBLE VITAMINS

10.3 B-COMPLEX VITAMINS

Eight of the water-soluble vitamins are known as the vitamin B-complex group:
thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), vitamin B6
(pyridoxine), folate (folic acid), vitamin B12, biotin and pantothenic acid. The

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Vitamins MODULE
B vitamins are widely distributed in foods and their influence is felt in many parts Biochemistry
of the body. They function as coenzymes that help the body obtain energy from
food. The B vitamins are also important for normal appetite, good vision, and
healthy skin, nervous system, and red blood cell formation.

10.3.1 Thiamin: Vitamin B1

Thiamin, or vitamin B1, helps to release energy from foods, promotes normal
appetite, and is important in maintaining proper nervous system function. Notes

Food Sources for Thiamin

Sources include peas, pork, liver, and legumes. Most commonly, thiamin is
found in whole grains and fortified grain products such as cereal, and enriched
products like bread, pasta, rice, and tortillas. The process of enrichment adds
back nutrients that are lost when grains are processed. Among the nutrients
added during the enrichment process are thiamin (B1), niacin (B3), riboflavin
(B2), folate and iron.
RDA (Required Daily allowance)
Males: 1.2 mg/day; Females: 1.1 mg/day

Thiamin Deficiency
Under-consumption of thiamin is rare due to wide availability of enriched grain
products. However, low calorie diets as well as diets high in refined and
processed carbohydrates may place one at risk for thiamin deficiency. Alcoholics
are especially prone to thiamin deficiency because excess alcohol consumption
often replaces food or meals. Symptoms of thiamin deficiency include: mental
confusion, muscle weakness, wasting, water retention (edema), impaired
growth, and the disease known as beriberi. Thiamin deficiency is currently not
a problem in the United States.

Thiamin toxicity
No problem with overconsumption are known for thiamin.

10.3.2 Riboflavin: Vitamin B2

Riboflavin, or vitamin B2, helps to release energy from foods, promotes good
vision, and healthy skin. It also helps to convert the amino acid tryptophan
(which makes up protein) into niacin.

Food Sources
Sources include liver, eggs, dark green vegetables, legumes, whole and enriched
grain products, and milk. Ultraviolet light is known to destroy riboflavin, which
is why most milk is packaged in opaque containers instead of clear.

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Biochemistry RDA
Males: 1.3 mg/day; Females: 1.1 mg/day

Deficiency
Under consumption of riboflavin is rare. However, it has been known to occur
with alcoholism, malignancy, hyperthyroidism, and in the elderly. Symptoms of
deficiency include cracks at the corners of the mouth, dermatitis on nose and
Notes lips, light sensitivity, cataracts, and a sore, red tongue.

Riboflavin toxicity
No problems with overconsumption are known for riboflavin.

10.3.3 Niacin: Vitamin B3, Nicotinamide, Nicotinic Acid

Niacin, or vitamin B3, is involved in energy production, normal enzyme
function, digestion, promoting normal appetite, healthy skin, and nerves.

Food Sources for Niacin

Sources include liver, fish, poultry, meat, peanuts, whole and enriched grain
products.

RDA
Males: 16 mg/day; Females: 14 mg/day

Niacin Deficiency
Niacin deficiency is known to occur with alcoholism, protein malnourishment,
low calorie diets, and diets high in refined carbohydrates. Pellagra is the disease
state that occurs as a result of severe niacin deficiency. Symptoms include
cramps, nausea, mental confusion, and skin problems.

Niacin toxicity
Consuming large doses of niacin supplements may cause flushed skin, rashes,
or liver damage. Over consumption of niacin is not a problem if it is obtained
through food.

10.3.4 Vitamin B6: Pyridoxine, Pyridoxal, Pyridoxamine

Vitamin B6, otherwise known as pyridoxine, pyridoxal or pyridoxamine, aids
in protein metabolism and red blood cell formation. It is also involved in the
body’s production of chemicals such as insulin and hemoglobin.

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Vitamins MODULE
Food Sources for Vitamin B6 Biochemistry

Sources include pork, meats, whole grains and cereals, legumes, and green, leafy
vegetables. The RDA for vitamin B6 is 1.3 mg/day for adult males and females
through age fifty.

Vitamin B6 Deficiency
Deficiency symptoms include skin disorders, dermatitis, cracks at corners of
mouth, anemia, kidney stones, and nausea. A vitamin B6 deficiency in infants Notes
can cause mental confusion.

Too much Vitamin B6

Over consumption is rare, but excess doses of vitamin B6 over time have been
known to result in nerve damage.

10.3.5 Folate: Folic Acid, Folacin

Folate, also known as folic acid or folacin, aids in protein metabolism, promoting
red blood cell formation, and lowering the risk for neural tube birth defects.
Folate may also play a role in controlling homocysteine levels, thus reducing the
risk for coronary heart disease.

Food Sources for Folate

Sources of folate include liver, kidney, dark green leafy vegetables, meats, fish,
whole grains, fortified grains and cereals, legumes, and citrus fruits. Not all
whole grain products are fortified with folate..

RDA
The RDA for folate is 400 mcg/day for adult males and females. Pregnancy will
increase the RDA for folate to 600 mcg/day.

Folate Deficiency
Folate deficiency affects cell growth and protein production, which can lead to
overall impaired growth. Deficiency symptoms also include anemia and
diarrhea. A folate deficiency in women who are pregnant or of child bearing age
may result in the delivery of a baby with neural tube defects such as spina bifida.

Folate toxicity
Over consumption of folate offers no known benefits, and may mask B12
deficiency as well as interfere with some medications.

10.3.6 Vitamin B12: Cobalamin

Vitamin B12, also known as cobalamin, aids in the building of genetic material,
production of normal red blood cells, and maintenance of the nervous system.

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Biochemistry Food Sources for Vitamin B12

Vitamin B12 can only be found only in foods of animal origin such as meats,
liver, kidney, fish, eggs, milk and milk products, oysters, shellfish. Some
fortified foods may contain vitamin B12.

RDA
Notes The Recommended Dietary Allowance (RDA) for vitamin B12 is 2.4 mcg/day
for adult males and females

Vitamin B12 Deficiency

Vitamin B12 deficiency most commonly affects strict vegetarians (those who eat
no animal products), infants of vegan mothers, and the elderly. Symptoms of
deficiency include anemia, fatigue, neurological disorders, and degeneration of
nerves resulting in numbness and tingling. In order to prevent vitamin B12
deficiency, a dietary supplement should be taken. Some people develop a B12
deficiency because they cannot absorb the vitamin through their stomach lining.
This can be treated through vitamin B12 injections.

Vitamin B12 toxicity

No problems with overconsumption of vitamin B12 are known.

10.3.7 Biotin
Biotin helps release energy from carbohydrates and aids in the metabolism of
fats, proteins and carbohydrates from food.

Food Sources for Biotin

Sources of Biotin include liver, kidney, egg yolk, milk, most fresh vegetables,
yeast breads and cereals. Biotin is also made by intestinal bacteria.

RDA
The Adequate Intake (AI) for Biotin is 30 mcg/day for adult males and females

Biotin Deficiency
Biotin deficiency is uncommon under normal circumstances, but symptoms
include fatigue, loss of appetite, nausea, vomiting, depression, muscle pains,
heart abnormalities and anemia.
Biotin toxicity
No problems with overconsumption are known for Biotin.

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10.3.8 Pantothenic Acid Biochemistry

Pantothenic Acid is involved in energy production, and aids in the formation of

hormones and the metabolism of fats, proteins, and carbohydrates from food.

Food Sources for Pantothenic Acid

Sources include liver, kidney, meats, egg yolk, whole grains, and legumes.
Pantothenic Acid is also made by intestinal bacteria.
Notes
RDA
The Adequate Intake (AI) for Pantothenic Acid is 5 mg/day for both adult males
and females.

Pantothenic Acid Deficiency

Pantothenic Acid deficiency is uncommon due to its wide availability in most
foods.

Pantothenic Acid toxicity

No problems with overconsumption are known for Pantothenic Acid. Rarely,
diarrhea and water retention will occur with excessive amounts.

10.4 VITAMIN C: ASCORBIC ACID, ASCORBATE

The body needs vitamin C, also known as ascorbic acid or ascorbate. Vitamin
C benefits the body by holding cells together through collagen synthesis;
collagen is a connective tissue that holds muscles, bones, and other tissues
together. Vitamin C also aids in wound healing, bone and tooth formation,
strengthening blood vessel walls, improving immune system function, increasing
absorption and utilization of iron, and acting as an antioxidant.
Since our bodies cannot produce or store vitamin C, an adequate daily intake
of this nutrient is essential for optimum health. Vitamin C works with vitamin
E as an antioxidant, and plays a crucial role in neutralizing free radicals
throughout the body. An antioxidant can be a vitamin, mineral, or a carotenoid,
present in foods, that slows the oxidation process and acts to repair damage to
cells of the body. Studies suggest that vitamin C may reduce the risk of certain
cancers, heart disease, and cataracts. Research continues to document the degree
of these effects.

Food Sources for Vitamin C

Consuming vitamin C-rich foods is the best method to ensure an adequate intake
of this vitamin. While many common plant foods contain vitamin C, the best
sources are citrus fruits (orange, kiwi fruit, grape etc,)

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Biochemistry RDA
The Recommended Dietary Allowance (RDA) for Vitamin C is 90 mg/day for
adult males and 75 mg/day for adult females For those who smoke cigarettes,
the RDA for vitamin C increases by 35 mg/day, in order to counteract the
oxidative effects of nicotine.

Notes Vitamin C Deficiency

Severe vitamin C deficiency result in the disease known as scurvy, causing a loss
of collagen strength throughout the body. Loss of collagen results in loose teeth,
bleeding and swollen gums, and improper wound healing. More commonly,
vitamin C deficiency presents as a secondary deficiency in alcoholics, the
elderly, and in smokers.

Vitamin C toxicity
Despite being a water-soluble vitamin that the body excretes when in excess,
vitamin C overdoses have been shown to cause kidney stones, gout, diarrhea,
and rebound scurvy.

FAT-SOLUBLE VITAMINS
The fat-soluble vitamins, A, D, E, and K, are stored in the body for long periods
of time and generally pose a greater risk for toxicity when consumed in excess
than water-soluble vitamins. Eating a normal, well-balanced diet will not lead
to toxicity in otherwise healthy individuals. However, taking vitamin supplements
that contain megadoses of vitamins A, D, E and K may lead to toxicity. The body
only needs small amounts of any vitamin.
While diseases caused by a lack of fat soluble vitamins are rare symptoms of
mild deficiency can develop without adequate amounts of vitamins in the diet.
Additionally, some health problems may decrease the absorption of fat, and in
turn, decrease the absorption of vitamins A, D, E and K. Consult a medical
professional about any potential health problems that may interfere with vitamin
absorption.

10.5 VITAMIN A: RETINOL

Vitamin A, also called retinol, has many functions in the body. In addition to
helping the eyes adjust to light changes, vitamin A plays an important role in
bone growth, tooth development, reproduction, cell division, gene expression,
and regulation of the immune system. The skin, eyes, and mucous membranes
of the mouth, nose, throat and lungs depend on vitamin A to remain moist.

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Vitamin A is also an important antioxidant that may play a role in the prevention Biochemistry
of certain cancers.

Food Sources for Vitamin A

Eating a wide variety of foods is the best way to ensure that the body gets enough
vitamin A. The retinol, retinal, and retinoic acid forms of vitamin A are supplied
primarily by foods of animal origin such as dairy products, fish and liver. Some Notes
foods of plant origin contain the antioxidant, betacarotene, which the body
converts to vitamin A. Beta-carotene, comes from fruits and vegetables,
especially those that are orange or dark green in color. Vitamin A sources also
include carrots, pumpkin, winter squash, dark green leafy vegetables and
apricots, all of which are rich in beta-carotene.

How much Vitamin A

The recommendation for vitamin A intake is expressed as micrograms (mcg) of
retinol activity equivalents (RAE). Retinol activity equivalents account for the
fact that the body converts only a portion of betacarotene to retinol. One RAE
equals 1 mcg of retinol or 12 mcg of beta-carotene. The Recommended Dietary
Allowance (RDA) for vitamin A is 900 mcg/ day for adult males and 700 mcg/
day for adult females.
Compared to vitamin A, it takes twice the amount of carotene rich foods to meet
the body’s vitamin A requirements, so one may need to increase consumption
of carotene containing plant foods. Recent studies indicate that vitamin A
requirements may be increased due to hyperthyroidism, fever, infection, cold,
and exposure to excessive amounts of sunlight. Those that consume excess
alcohol or have renal disease should also increase intake of vitamin A.

Vitamin A Deficiency
Vitamin A deficiency is rare, but the disease that results is known as
xerophthalmia. It most commonly occurs in developing nations usually due to
malnutrition. Since vitamin A is stored in the liver, it may take up to 2 years for
signs of deficiency to appear. Night blindness and very dry, rough skin may
indicate a lack of vitamin A. Other signs of possible vitamin A deficiency include
decreased resistance to infections, faulty tooth development, and slower bone
growth.

Vitamin A toxicity
The Tolerable Upper Intake Level (UL) for adults is 3,000 mcg RAE. It would
be difficult to reach this level consuming food alone, but some multivitamin

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Biochemistry supplements contain high doses of vitamin A. If you take a multivitamin, check
the label to be sure the majority of vitamin A provided is in the form of beta-
carotene, which appears to be safe. Symptoms of vitamin A toxicity include dry,
itchy skin, headache, nausea, and loss of appetite. Signs of severe overuse over
a short period of time include dizziness, blurred vision and slowed growth.
Vitamin A toxicity also can cause severe birth defects and may increase the risk
for hip fractures.
Notes
10.6 VITAMIN D
Vitamin D plays a critical role in the body’s use of calcium and phosphorous.
It works by increasing the amount of calcium absorbed from the small intestine,
helping to form and maintain bones. Vitamin D benefits the body by playing a
role in immunity and controlling cell growth. Children especially need adequate
amounts of vitamin D to develop strong bones and healthy teeth.

Food Sources for Vitamin D

The primary food sources of vitamin D are milk and other dairy products fortified
with vitamin D. Vitamin D is also found in oily fish (e.g., herring, salmon and
sardines) as well as in cod liver oil. In addition to the vitamin D provided by
food, we obtain vitamin D through our skin which produces vitamin D in
response to sunlight.

RDA
The Recommended Dietary Allowance (RDA) for vitamin D appears as
micrograms (mcg) of cholecalciferol (vitamin D3). From 12 months to age fifty,
the RDA is set at 15 mcg. Twenty mcg of cholecalciferol equals 800 International
Units (IU), which is the recommendation for maintenance of healthy bone for
adults over fifty.
Exposure to ultraviolet light is necessary for the body to produce the active form
of vitamin D. Ten to fifteen minutes of sunlight without sunscreen on the hands,
arms and face, twice a week is sufficient to receive enough vitamin D. This can
easily be obtained in the time spent riding a bike to work or taking a short walk.
In order to reduce the risk for skin cancer one should apply sunscreen with an
SPF of 15 or more, if time in the sun exceeds 10 to 15 minutes.

Vitamin D Deficiency
Symptoms of vitamin D deficiency in growing children include rickets (long,
soft bowed legs) and flattening of the back of the skull. Vitamin D deficiency
in adults may result in osteomalacia (muscle and bone weakness), and
osteoporosis (loss of bone mass).

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Recently published data introduces a concern that some adults and children may Biochemistry
be more prone to developing vitamin D deficiency due to an increase in
sunscreen use. In addition, those that live in inner cities, wear clothing that
covers most of the skin, or live in northern climates where little sun is seen in
the winter are also prone to vitamin D deficiency. Since most foods have very
low vitamin D levels (unless they are enriched) a deficiency may be more likely
to develop without adequate exposure to sunlight. Adding fortified foods to the
diet such as milk, and for adults including a supplement, are effective at ensuring Notes
adequate vitamin D intake and preventing low vitamin D levels.
Vitamin D deficiency has been associated with increased risk of common
cancers, autoimmune diseases, hypertension, and infectious disease. In the
absence of adequate sun exposure, at least 800 to 1,000 IU of vitamin D3 may
be needed to reach the circulating level required to maximize vitamin D’s
benefits.

Vitamin D toxicity
The Tolerable Upper Intake Level (UL) for vitamin D is set at 100 mcg for people
9 years of age and older. High doses of vitamin D supplements coupled with large
amounts of fortified foods may cause accumulations in the liver and produce
signs of poisoning. Signs of vitamin D toxicity include excess calcium in the
blood, slowed mental and physical growth, decreased appetite, nausea and
vomiting.
It is especially important that infants and young children do not consume excess
amounts of vitamin D regularly, due to their small body size.

10.7 VITAMIN E: TOCOPHEROL

Vitamin E benefits the body by acting as an antioxidant, and protecting vitamins
A and C, red blood cells, and essential fatty acids from destruction. Research
from decades ago suggested that taking antioxidant supplements, vitamin E in
particular, might help prevent heart disease and cancer. However, newer findings
indicate that people who take antioxidant and vitamin E supplements are not
better protected against heart disease and cancer than non-supplement users.
Many studies show a link between regularly eating an antioxidant rich diet full
of fruits and vegetables, and a lower risk for heart disease, cancer, and several
other diseases. Essentially, recent research indicates that to receive the full
benefits of antioxidants and phytonutrients in the diet, one should consume these
compounds in the form of fruits and vegetables, not as supplements.

Food Sources for Vitamin E

About 60 percent of vitamin E in the diet comes from vegetable oil (soybean,
corn, cottonseed, and safflower). This also includes products made with

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Biochemistry vegetable oil (margarine and salad dressing). Vitamin E sources also include
fruits and vegetables, grains, nuts (almonds and hazelnuts), seeds (sunflower)
and fortified cereals.

RDA
The Recommended Dietary Allowance (RDA) for vitamin E is based on the most
active and usable form called alpha-tocopherol. Food and supplement labels list
Notes
alpha-tocopherol as the unit International units (IU) not in milligrams (mg). One
milligram of alpha-tocopherol equals to 1.5 International Units (IU). RDA
guidelines state that males and females over the age of 14 should receive 15 mcg
of alpha-tocopherol per day. Consuming vitamin E in excess of the RDA does
not result in any added benefits.

Vitamin E Deficiency
Vitamin E deficiency is rare. Cases of vitamin E deficiency usually only occur
in premature infants and in those unable to absorb fats. Since vegetable oils are
good sources of vitamin E, people who excessively reduce their total dietary fat
may not get enough vitamin E.

Vitamin E toxicity
Vitamin E obtained from food usually does not pose a risk for toxicity.
Supplemental vitamin E is not recommended due to lack of evidence supporting
any added health benefits. Megadoses of supplemental vitamin E may pose a
hazard to people taking blood-thinning medications such as Coumadin (also
known as warfarin) and those on statin drugs.

10.8 VITAMIN K
Vitamin K is naturally produced by the bacteria in the intestines, and plays an
essential role in normal blood clotting, promoting bone health, and helping to
produce proteins for blood, bones, and kidneys.

Food Sources for Vitamin K

Good food sources of vitamin K are green, leafy-vegetables such as turnip
greens, spinach, cauliflower, cabbage and broccoli, and certain vegetables oils
including soybean oil, cottonseed oil, canola oil and olive oil. Animal foods, in
general, contain limited amounts of vitamin K.
RDA
Males and females age 14 - 18: 75 mcg/day; Males and females age 19 and older:
90 mcg/day

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Vitamin K Deficiency Biochemistry

Hemorrhage can occur due to sufficient amounts of vitamin K. Vitamin K

deficiency may appear in infants or in people who take anticoagulants, such as
Coumadin (warfarin), or antibiotic drugs. Newborn babies lack the intestinal
bacteria to produce vitamin K and need a supplement for the first week. Those
on anticoagulant drugs (blood thinners) may become vitamin K deficient, but
should not change their vitamin K intake without consulting a physician. People
Notes
taking antibiotics may lack vitamin K temporarily because intestinal bacteria are
sometimes killed as a result of long-term use of antibiotics. Also, people with
chronic diarrhea may have problems absorbing sufficient amounts of vitamin K
through the intestine and should consult their physician to determine if
supplementation is necessary.

Vitamin K toxicity
Although no Tolerable Upper Intake Level (UL) has been established for vitamin
K, excessive amounts can cause the breakdown of red blood cells and liver
damage. People taking blood-thinning drugs or anticoagulants should moderate
their intake of foods with vitamin K, because excess vitamin K can alter blood
clotting times. Large doses of vitamin K are not advised.

INTEXT QUESTIONS 10.1

1. Fill in the balnks:
1. Vitamins are classified into ................... and ...................
2. Vitamin B complex comprises ................... vitamins in total.
3. Pellagra and Scurvy are caused by ................... and ...................
deficiency.
4. Water soluble vitamins are excreted through ...................
5. ................... can be synthesized by human body.

2. Match the following

1. Vitamin A deficiency (a) Vitamin D
2. Vitamin K (b) Vitamin E
3. Bone formation (c) Vitamin B12
4. Cobalamin (d) Night blindness
5. Tocopherol (e) Coagulation

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Biochemistry 3. True or false:

1. Vitamins are required in large amounts.
2. Vitamin B2 is otherwise known as Riboflavin.
3. All vitamins are synthesized in our body. (false)
4. Fat malabsorption leads to deficiency of fat soluble vitamins.
5. Water soluble vitamins are stored in our body.
Notes

WHAT HAVE YOU LEARNT

z The term vitamin is used to describe certain orgain compounds that are
needed by the body but they cannot be manufactured by the body
z Vitamins serve as catalysts for certain reactions in the body
z Based on solubility vitamins are classified as either fat soluble or vitamin
soluble
z Vitamins A, D, E and K are fat soluble and vitamins C and B is water soluble

TERMINAL QUESTIONS
1. Name the B complex vitamins.
2. Classification of vitamins.
3. Give the RDA of thiamine, folate, niacin, vitamin C.
4. Name the fat soluble vitamins and where they are stored?
5. What are the symptoms of Pellagra and Scurvy?

ANSWERS TO INTEXT QUESTIONS

1. 1. fat soluble and water soluble
2. Eight
3. Niacin and vitamin C
4. urine
5. vitamin D
2. 1. (d) 2. (e) 3. (a) 4. (c) 5. (b)
3. 1. false 2. true 3. false 4. true 5. false

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Minerals MODULE
Biochemistry

11
Notes
MINERALS

11.1 INTRODUCTION
Minerals are indispensable part of a complete diet of farm animals. In this unit,
dietary essential minerals functions, factors affecting the requirements and
sources will be discussed. Minerals are essential for the normal growth and
maintance of the body. If the daily requirement is more than 100mg/day they are
called major elements and if the daily requirements is less than 100mg/day they
are called minor elements. The roles of minerals and vitamins in the maintenance
of homeostatic balance and mediation of metabolic reactions in the skeleton,
tissues, body fluids, digestive juices, etc.

OBJECTIVES
After reading this lesson, you will be able to:
z classify minerals
z describe the functions, Daily requirements of minerals

11.2 CALCIUM
Total calcium in the human body is 1 to 1.5kg, out of which 99% is seen in bone
and 1% in extracellular fluid. The main source of calcium is milk. But in India
cereals is major source of calcium. The daily requirement of calcium for child
is 1200mg/day and for adult it is 500mg/day. During pregnancy /lactation the
calcium requirement is 1500mg/day.
The absorption of calcium takes place in 1st and 2nd part of deuodenum. Calcium
absorption requires carrier protein, helped by Ca2+ - dependent ATpase.
Factors responsible for increase in calcium absorption include Vitamin D,
Parathyroid hormone, acidity and amino acids. Factors such as phytic acid,oxalates,

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Biochemistry malabsorption syndromes and Phosphates decreases calcium absorption. The

normal calcium level in blood is 9-11mg/dl.

11.2.1 Function of Calcium

The major functions of calcium are
(a) Excitation and contraction of muscle fibres needs calcium. The active
Notes transport system utilizing calcium binding protein is called Calsequestrin.
Calcium decreases neuromuscular irritability.
(b) Calcium is necessary for transmission of nerve impulse from presynaptic
to postsynaptic region.
(c) Calcium is used as second messenger in system involving protein and
inositol triphosphate.
(d) Secretion of insulin, parathyroid hormone, calcium etc, from the cells
requires calcium.
(e) Calcium decrease the passage of serum through capillaries thus, calcium
is clinically used to reduce allergic exudates.
(f) Calcium is also required for coagulation factors such as prothrombin.
(g) Calcium prolongs systole.
(h) Bone and teeth contains bulk quantity of calcium.

11.2.2 Factors regulating blood calcium level

The factors regulating the blood calcium level includes

(i) Vitamin D
(a) Vitamin D and absorption of calcium:
Active form of calcium is calcitriol. Calcitriol enters intestinal wall and
binds to cytoplasmic receptor and then binds with DNA causes depression
and consequent transcription of gene code for calbindin. Due to increased
availability of calbindin, absorption of calcium increases leading to
increased blood calcium level.
(b) Vitamin D and Bone:
Vitamin D activates osteoblast, bone forming cells & also stimulates
secretion of alkaline phosphatase. Due to this enzyme, calcium and
phosphorus increase.
(c) Vitamin D and Kidney:
Calcitriol increase reabsorption of calcium and phosphorus by renal
tubules.

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(ii) Parathyroid hormone (PTH) Biochemistry

Normal PTH level in serum is 10-60ng/l.

(a) PTH and bones:
In bone, PTH causes demineralization. It also causes recreation of
collagenase from osteoclast leads to loss of matrix and bone resorption.
As a result, mucopolysacharides and hydroxyproline are excreted in urine.
Notes
(b) PTH and Kidney:
In kidney, PTH causes increased reabsorption of calcium but decreases
reabsorption of phosphorus from kidney tubules.

(iii) Calcitonin
Calcitonin decreases serum calcium level. It inhibits resorption of bone. It
decreases the activity of osteoclasts and increases osteoblasts.

11.2.3 Hyper Calcemia

When plasma Ca2+ level is more than 11mg/dl is called Hypercalcemia. It is due
to parathyroid adenoma or ectopic PTH secreting tumor. In this condition,
calcium excreted in urine decreases excretion of chloride causing hyperchloremic
acidosis. The main symptoms of hyperchloremic acidosis are:
(a) Anorexia, nausea,vomiting
(b) polyuria, polydypsia
(c) Confusion, depression, psychosis
(d) renal stones
(e) osteoporosis

11.2.4 Hypocalcemia
Plasma calcium level less than 8mg/dl is called hypocalcemia. Tetany due to
accidental surgical removal of parathyroid glands or by autoimmune disease. In
tetany, neuromuscular irritability is increased. Increased Q-7 internal in ECG is
seen. Main manifestation is carpopedal spasm. Laryngismus and stridor are also
observed. Laryngeal spasm may lead is death. The causes of laryngeal spasm
includes
(a) Deficiency of vitamin D
(b) Deficiency of parathyroid
(c) Increased calcitonin
(d) Deficiency of calcium intake

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Biochemistry

INTEXT QUESTION 11.1

1. The total calcium range in human is .................
2. Calcium absorption requires ................. dependent ATpase.
(a) Ca (b) Mn (c) Mg (d) fe
Notes 3. Factors responsible for increase in calcium absorption include .................
4. When plasma Ca2+ level is more than 11mg/dl is called .................
(a) Hypercalcemia (b) Anemia (c) Fluorosis (d) Selenosis

11.3 PHOSPHORUS
Total body content of phosphorus is 1 kg. Bone posses 80 % of total phosphorus
and muscle contains 10 % of total phosphorus. Human body requires 500 mg
of phosphorus per day. Milk is the good source of phosphorus and it contains
about 100 mg/dl of phosphrous. Apart from milk, cereals, nuts and meat are
moderate sources of phosprous.
Serum level of phosphate is 3-4 mg/dl for adults and 5-6 mg/dl in children.
Consumption of calcitriol increases phosphate absorption.

11.3.1 Functions of phosphorus

(a) Plays key role in formation of tooth and bone
(b) Production of high energy phosphate compounds such as ATP, CTP, GTP
etc.,
(c) Synthesis of nucleotide co-enzymes such as NAD and NADP
(d) Formation of phosphodiester backbone structure for DNA and RNA
synthesis
Hypophosphatemia is the condition which leads to decrease in absorption of
phosphorus. Further it leads to hypercalcamia, chronic alcoholism and increased
excretion of urinary phosphate. In case of hyperphosphatemia, increase in
absorption of phosphate was noticed. Hyperphosphatemia leads to cell lysis,
hypocalcemia and thyrotoxicosis.

INTEXT QUESTIONS 11.2

1. Bone posses ................. % of total phosphorus.
2. Serum level of phosphate is ................. mg/dl for adults.
(a) 4-8 (b) 3-4 (c) 8-9 (d) 1-2
3. ................. plays key role in formation of tooth and bone

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Biochemistry
11.4 MAGNESIUM
Optimal level of Magnesium for human consumption ranges 300-400 mg/day.
The main source of Magnesium includes cereals, beans, leafy vegetables and
fish. The normal serum level of Magnesium is 1.8 to 2.2. mg/dl.

11.4.1 Functions of Magnesium

(a) Irritability of neuromuscular tissues is lowered by Magnesium Notes
(b) Magnesium deficiency leads to decrease in Insulin dependent uptake of
glucose
(c) Magnesium supplementation improves glucose tolerance
Decrease in Magnesium content leads to the condition called as hypomagnesemia.
It causes increase in urinary loss. Causes such as liver cirrhosis, protein calorie
malnutrition and hypo para thyroidism leads to hypomagnesemia. Increase in the
level of Magnesium is called as hypermagnesemia. The main causes of
hypermagnesemia includes renal failure, hyper para thyroidism, rickets, oxalate
poisoning and multiple myeloma.

INTEXT QUESTIONS 11.3

1. Optimal level of ................... for human consumption ranges 300-400 mg/
day.
2. Irritability of neuromuscular tissues is lowered by ...................
3. Magnesium supplementation improves ................... tolerance
(a) lactose (b) glucose (c) toxin (d) microbial

11.5 IRON
Total body content of iron is 3 to 5 gm out of which 75 % is recorded in blood
and rest of them are recorded from liver, spleen, bone marrow and muscle. The
normal limit for iron consumption is 20 mg/day for adults, 20-30 mg/day for
children and 40 mg/day for pregnant women. The main source of iron is jaggery.
Other source of iron includes leafy vegetables and meat etc., Milk is considered
as a poor source of iron.

11.5.1 Factors influencing absorption of iron

Iron is absorbed by upper part of duodenum and is affected by various factors
(a) Only reduced form of iron (ferrous) is absorbed and ferric form are not
absorbed

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Biochemistry (b) Ascorbic acid (Vitamin C) increases the absorption of iron

(c) The interfering substances such as phytic acid and oxalic acid decreases
absorption of iron

11.5.2 Regulation of absorption of Iron

Absorption of iron is regulated by three main mechanisms, which includes
Notes
(a) Mucosal Regulation
(b) Storer regulation
(c) Erythropoietic regulation
In mucosal regulation absorption of iron requires DM-1 and ferroportin. Both
the proteins are down regulated by hepcidin secreted by liver. The above
regulation occurs when the body irons reserves are adequate. When the body iron
content gets felled, storer regulation takes place. In storer regulation the mucosal
is signaled for increase in iron absorption. The erythropoietic regulation occurs
in response to anemia. Here the erythroid cells will signal the mucosa to increase
the iron absorption.

11.5.3 Iron transport in blood

The transport form of iron in blood is transferin. Transferin are glycoprotein
secreted by liver. In blood, the ceruloplasmin is the ferroxidase which oxidizes
ferrous to ferric state.
Apo-Transferin + 2Fe2+ ⎯⎯→ Transferin combined with 2Fe3+
Storage form of iron is ferritin. Almost no iron is excreted through urine. Feces
contains iron as well as iron trapped in the intestine cells.

11.5.4 Anemia
Anemia is the most common nutritional deficiency disease. The microscopic
appearance of anemia is characterized by microcytic hypochromic anemia. Iron
toxicity or excess of iron is called as hemosiderosis. Iron toxicity occurs due to
repeated blood transfusion. The abnormal gene responsible for hemosiderosis
is located on the short arm of chromosome No.6. The main causes of iron
deficiency or anemia are
(a) Nutritional deficiency of iron
(b) Lack of iron absorption
(c) Hook worm infection
(d) Repeated pregnancy

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(e) Chronic blood loss Biochemistry

(f) Nephrosis
(g) Lead poisoning

INTEXT QUESTION 11.4

Notes
1. Match the following
Adults 20 - 30 mg/day
Children 40 mg/day
Pregnant women 20 mg/day
2. .................. is the most common nutritional deficiency disease
3. The transport form of iron in blood is ..................

11.6 COPPER
Total human body contains about 100 mg of copper and are encountered in
muscle, liver, bone marrow, brain, kidney, heart and in hairs. Enzymes such as
cytochrome oxidase, tyrosinase, lysyl oxidase, allanine synthase, monoamine
oxidase, superoxide dismutase and phenol oxidase contains copper. The normal
copper limit for human are 1.5 to 3 mg/day. The main sources of copper are
cereals, meat, liver, nuts and green leafy vegetables. From the total dietary
copper only 10% are absorbed. Copper are mainly excreated through bile. The
normal serum level of copper is 25 to 50 mg/dl.

11.6.1 Functions of copper

(a) Copper is necessary for iron absorption and incorporation of iron into
hemoglobin.
(b) It is very essential for tyrosinase activity
(c) It is the co-factor for vitamin C requiring hydroxylation
(d) Copper increases the level of high density lipo protein and protects the
heart

11.6.2 Abnormal metabolism of copper

The abnormal metabolism of copper leads to Wilson’s disease and Menke’s
kidney hair syndrome.

(a) Wilson’s disease

In case of Wilson’s disease ceruloplasmin level in blood is drastically reduced.
The incidence of Wilson’s disease is noticed for 1 in 50,000 populations. The

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Biochemistry defect in a gene encoding copper binding ATPase of liver cells leads to Wilson’s
disease. Wilson’s disease leads to
(i) Accumulation of copper in liver leads to hepatocellular degeneration and
cirrhosis
(ii) Deposition of copper in brain basal ganglia leads to leticular degeneration
(iii) Copper deposits as green pigmented ring around cornea and the condition
Notes is called as Kayser-Kleischer ring
Over accumulation of copper can be treated by consumption of diet containg low
copper and injection of D-penicillamine, which excretes copper through urine.

(b) Menke’s kidney hair syndrome

It is X-linked defect. In this condition copper is absorbed by GI tract, but cannot
be transported to blood. The defect in transport of copper to blood is due to
absence of an intracellular copper binding ATPase, which is due to mutation in
gene encoding them.

INTEXT QUESTIONS 11.5

1. Total human body conatins about ................. mg of copper
(a) 100 (b) 200 (c) 300 (d) 400
2. Menke’s kidney hair syndrome is ................. linked defect.
3. Deposition of copper in ................. leads to leticular degeneration

11.7 ZINC
The daily requirement of Zinc for human consumption ranges 10mg /day. The
major sources of Zinc includes grains, beans, nuts cheese, meat and shellfish.
The normal serum level of Zinc in human is 100mg/day. In the human total body
the content of Zinc is 2gm, out of which 60 % is encountered in skeletal muscle
and 30% in bones highest concentration is seen in hippocampus area of brain
and prostatic secretion.

More than 300 enzymes in human body are zinc-dependent; some of them are
carboxypeptidase, carbonic anhydrase, alkaline phosphatase, lactate
dehydrogenase, alcohol dehydragenase. The enzyme RNA polymerase, which
is required for transcription, contains zinc and it is essential for protein bio
synthesis.

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Deficiency in Zinc leads to poor wound healing, lesions of skin impaired Biochemistry
spermatogenesis, hyperkeratosis, dermatitis and alopecia. Consumption of more
than 1000 mg /day leads to zinc toxicity. Zinc toxicity leads to gastric ulcer,
pancreatitis, anemia, nausea and vomiting.

INTEXT QUESTIONS 11.6 Notes

1. ................. and ................. are major source of Zinc

2. ................. number of enzymes are zinc dependent
(a) 100 (b) 300 (c) 500 (d) 50
3. The normal serum level of Zinc in human is ................. mg/day

11.8 FLUORIDE
Fluoride is well known for their protective effect on caries. The safe limit of
fluorine is about 1PPM in water. But excess of fluoride causes Flourosis.
Flourosis is more dangerous than caries. When Fluoride content is more than
2 PPM, it will cause chronic intestinal upset, gastroenteritis, loss of weight,
osteosclerosis, stratification and discoloration of teeth. In India Flourosis is
widespread in Punjab, Rajasthan, Delhi and Tamilnadu. Fluoride rich source
includes sea fish, cheese, tea and jowar.

Flourosis could be prevented by providing and consumption of fluoride free

water, Further supplementation of vitamin c and usage of toothpaste containing
regulated level of Fluoride could prevent Fluorosis.

INTEXT QUESTIONS 11.7

1. .................. is well known for caries protection.
(a) calcium (b) fluoride (c) iron (d) Magnesium
2. Fluoride rich source includes ..................

11.9 SELENIUM
The normal limit of Selenium for human consumption is 50-100 mg/day and the
normal serum level is 50-100 mg/day. Selenium dependent enzymes include
glutathione Peroxidase and 5-de-iodinase. Selenium concentration in testis is the

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Biochemistry highest in adult. It is very necessary for normal development and maturation
of sperm.

Selenium toxicity could be termed as selenosis. The toxicity symptoms include

hair loss, falling of nails, diarrhea, weight loss and garlicky odor in breath.

Notes
INTEXT QUESTIONS 11.8
1. The normal limit of Selenium for human consumption is ............... mg/day
2. Glutathione peroxides is a .................. dependent enzyme

11.10 MANGANESE
The normal limit of Manganese for human is 5mg/day. The major source of
Manganese are nuts. In metabolism, its absorption is inhibited by iron. In blood,
it binds with transmanganin and excreted through bile.

INTEXT QUESTIONS 11.9

1. The major source of Manganese is ..................
2. Manganese are excreted through ..................

WHAT HAVE YOU LEARNT

z In this lesson you have learnt about the importance and types of clinically
significant mineral elements.
z Mineral elements are solid crystalline, chemical elements that cannot be
decomposed and synthesized by ordinary chemical reactions.
z They are present in both plants and animals to execute specific functions
and the amount to be required is largely determined by certain inherent
factors.
z Minerals although nutritionally required in smaller amounts are essential
for maintenance and production purposes. However the major and trace
minerals are needed by the body in large and small amount respectively.
z Generally, deficiencies of most minerals are shown by a reduced appetite
and production, slow growth and occasionally death.

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Biochemistry

TERMINAL QUESTIONS
1. Write a short note on minerals?
2. What are the elements of major and minor minerals?
3. Write a note on calcium and its finctions?
4. What are the functions of phosphorous? Notes
5. Narrate the importance of iron?

ANSWERS TO INTEXT QUESTIONS

11.1
1. 1 to 1.5 kg
2. Ca
3. Vitamin D
4. Hypercalcemia

11.2
1. 80 %
2. 3-4
3. Phosphorus

11.3
1. 300 to 400
2. Magnesium
3. Glucose

11.4
1. Adults 20 mg/day
Children 20 – 30 mg/day
Pregnant women 40 mg/day
2. Anemia
3. Transferin

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Biochemistry 11.5
1. 100
2. X-linked
3. Brain basal ganglia

11.6
Notes
1. Beans and nuts
2. 300
3. 10

11.7
1. Fluoride
2. Fish

11.8
1. 50-100
2. Selenium

11.9
1. Nuts
2. Bile

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Biochemistry

12
Notes
HORMONES

12.1 INTRODUCTION
A hormone is a chemical that acts as a messenger transmitting a signal from one
cell to another. When it binds to another cell which is the target of the message,
the hormone can alter several aspects of cell function, including cell growth,
metabolism, or other function. Hormones can be classified according to
chemical composition, solubility properties, location of receptors, and the nature
of the signal used to mediate hormonal action within the cell. Hormones that
bind to the surfaces of cells communicate with intracellular metabolic processes
through intermediary molecules called second messengers (the hormone itself
is the first messenger), which are generated as a consequence of the ligand-
receptor interaction. The second messenger concept arose from an observation
that epinephrine binds to the plasma membrane of certain cells and increases
intracellular cAMP. This was followed by a series of experiments in which
cAMP was found to mediate the effects of many hormones. To date, only one
hormone, atrial natriuretic factor (ANF), uses cGMP as its second messenger.

OBJECTIVES
After reading this lesson, you will be able to
z explain the characterizing hormone
z describe functional importance of hormones

12.2 CHARACTERIZING HORMONE

The first way of characterizing a hormone is by looking at the distance over
which the hormone acts. Hormones can be classified on three primary ways as
following:

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Biochemistry z Autocrine: An autocrine hormone is one that acts on the same cell that
released it.
z Paracrine: A paracrine hormone is one that acts on cells which are nearby
relative to the cell which released it. An example of paracrine hormones
includes growth factors, which are proteins that stimulate cellular proliferation
and differentiation. Specifically, consider the binding of white blood cells
to T cells. When the white blood cell binds to a T cell, it releases a protein
Notes growth factor called interleukin-1. This causes the T cell to proliferate and
differentiate.
z Endocrine: An endocrine hormone is one that is released into the bloodstream
by endocrine glands. The receptor cells are distant from the source. An
example of an endocrine hormone is insulin, which is released by the
pancreas into the bloodstream where it regulates glucose uptake by liver
and muscle cells.
There are three major classifications one should be aware of:
z Steroids: Steroid hormones are for the most part derivatives of cholesterol.
z Amino acid derivatives: Several hormones (and neurotransmitters) are
derived from amino acids.
z Polypeptides: Many hormones are chains of amino acids.

12.3 FUNCTIONAL IMPORTANCE OF HORMONES

12.3.1 Insulin
Insulin is a polypeptide hormone synthesized in the pancreas by β-cells, which
construct a single chain molecule called proinsulin. Enzymes excise a portion
of the proinsulin molecule called the C peptide, producing the actual insulin
molecule. When in demand, the β-cells will release insulin together with the c
peptide into the blood stream via exocytosis. The role of insulin in the body is
well known, with its primary role being to control the uptake of glucose by liver
and muscle cells and also the storage of glucose in the form of glycogen.
Diabetes results from a lack of insulin secretion by the pancreas. Without insulin,
cells take up glucose very slowly. The lack of insulin results in an inability to
use blood glucose for fuel. Insulin is a signal for high blood glucose levels and
increases glucose transport into cells. It stimulates synthesis of glycogen, fat, and
protein. It inhibits breakdown of glycogen, fat, and protein. Insulin, secreted by
the β-cells of the pancreas in response to rising blood glucose levels, is a signal
that glucose is abundant. Insulin binds to a specific receptor on the cell surface
and exerts its metabolic effect by a signaling pathway that involves a receptor
tyrosine kinase phosphorylation cascade. Note that insulin stimulates storage
processes and at the same time inhibits degradative pathways.

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12.3.1.1 The pancreas secretes insulin or glucagon in response to Biochemistry
changes in blood glucose
When glucose enters the bloodstream from the intestine after a carbohydrate-
rich meal, the resulting increase in blood glucose causes increased secretion of
insulin (and decreased secretion of glucagon). Insulin release by the pancreas
is largely regulated by the level of glucose in the blood supplied to the pancreas.
The peptide hormones insulin, glucagon, and somatostatin are produced by Notes
clusters of specialized pancreatic cells, the islets of Langerhans. Each cell type
of the islets produces a single hormone: α-cells produce glucagon; β-cells,
insulin; and δ-cells, somatostatin.

12.3.1.2 Insulin secretion

When blood glucose rises, GLUT2 transporters carry glucose into the b-cells,
where it is immediately converted to glucose 6-phosphate by hexokinase IV
(glucokinase) and enters glycolysis. The increased rate of glucose catabolism
raises [ATP], causing the closing of ATP-gated K+ channels in the plasma
membrane. Reduced efflux of K+ depolarizes the membrane, thereby opening
voltage-sensitive Ca2+ channels in the plasma membrane. The resulting influx
of Ca2+ triggers the release of insulin by exocytosis. Stimuli from the
parasympathetic and sympathetic nervous systems also stimulate and inhibit
insulin release, respectively. Insulin lowers blood glucose by stimulating glucose
uptake by the tissues; the reduced blood glucose is detected by the β-cell as a
diminished flux through the hexokinase reaction; this slows or stops the release
of insulin. This feedback regulation holds blood glucose concentration nearly
constant despite large fluctuations in dietary intake.

12.3.1.3 Insulin counters high blood glucose

Insulin stimulates glucose uptake by muscle and adipose tissue, where the
glucose is converted to glucose 6-phosphate. In the liver, insulin also activates
glycogen synthase and inactivates glycogen phosphorylase, so that much of the
glucose 6-phosphate is channelled into glycogen. Insulin also stimulates the
storage of excess fuel as fat. In the liver, insulin activates both the oxidation of
glucose 6-phosphate to pyruvate via glycolysis and the oxidation of pyruvate to
acetyl-CoA. If not oxidized further for energy production, this acetyl- CoA is
used for fatty acid synthesis in the liver, and the fatty acids are exported as the
triglyerides to the adipose tissue. These fatty acids are ultimately derived from
the excess glucose taken up from the blood by the liver. In summary, the effect
of insulin is to favor the conversion of excess blood glucose to two storage
forms: glycogen (in the liver and muscle) and triacylglycerols (in adipose tissue).

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Biochemistry 12.3.1.4 Diabetes Mellitus arises from defects in insulin production or

action
Diabetes mellitus, caused by a deficiency in the secretion or action of insulin,
is a relatively common disease. There are two major clinical classes of diabetes
mellitus: type I diabetes, or insulin-dependent diabetes mellitus (IDDM), and
type II diabetes, or non-insulin-dependent diabetes mellitus (NIDDM), also
called insulin-resistant diabetes. In type I diabetes, the disease begins early in
Notes
life and quickly becomes severe. This disease responds to insulin injection,
because the metabolic defect stems from the pancreatic β-cells and a consequent
inability to produce sufficient insulin. IDDM requires insulin therapy and
careful, lifelong control of the balance between dietary intake and insulin dose.
Characteristic symptoms of type I (and type II) diabetes are excessive thirst and
frequent urination (polyuria), leading to the intake of large volumes of water
(polydipsia) (“diabetes mellitus” means “excessive excretion of sweet urine”).
These symptoms are due to the excretion of large amounts of glucose in the urine,
a condition known as glucosuria. Type II diabetes is slow to develop (typically
in older, obese individuals), and the symptoms are milder and often go
unrecognized at first. This is really a group of diseases in which the regulatory
activity of insulin is defective: insulin is produced, but some feature of the
insulin-response system is defective. These individuals are insulin-resistant.
Individuals with either type of diabetes are unable to take up glucose efficiently
from the blood; recall that insulin triggers the movement of GLUT4 glucose
transporters to the plasma membrane of muscle and adipose tissue. Another
characteristic metabolic change in diabetes is excessive but incomplete oxidation
of fatty acids in the liver. Insulin secretion reflects both the size of fat reserves
(adiposity) and the current energy balance (blood glucose level). Insulin acts on
insulin receptors in the hypothalamus to inhibit eating. Insulin also signals
muscle, liver, and adipose tissues to increase catabolic reactions, including fat
oxidation, which results in weight loss.

12.3.2 Glucagon
Glucagon, a peptide hormone synthesized and secreted from the α-cells of the
islets of Langerhans of pancreas, raises blood glucose levels. Its effect is
opposite that of insulin, which lowers blood glucose levels. The pancreas
releases glucagon when blood sugar (glucose) levels fall too low. Glucagon
causes the liver to convert stored glycogen into glucose, which is released into
the bloodstream. High blood glucose levels stimulate the release of insulin.
Insulin allows glucose to be taken up and used by insulin-dependent tissues.
Thus, glucagon and insulin are part of a feedback system that keeps blood
glucose levels at a stable level.

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12.3.2.1 Regulation and function Biochemistry

Secretion of glucagon is stimulated by hypoglycemia, epinephrine, arginine,

alanine, acetylcholine, and cholecystokinin. Secretion of glucagon is inhibited
by somatostatin, insulin, increased free fatty acids and keto acids into the blood,
and increased urea production. Glucose is stored in the liver in the form of
glycogen, which is a starch-like polymer chain made up of glucose molecules.
Liver cells (hepatocytes) have glucagon receptors. When glucagon binds to the
Notes
glucagon receptors, the liver cells convert the glycogen polymer into individual
glucose molecules, and release them into the bloodstream, in a process known
as glycogenolysis. As these stores become depleted, glucagon then encourages
the liver and kidney to synthesize additional glucose by gluconeogenesis.
Glucagon turns off glycolysis in the liver, causing glycolytic intermediates to be
shuttled to gluconeogenesis.
Glucagon is a signal for low blood glucose levels. It stimulates breakdown of
glycogen, fat, and protein. It inhibits synthesis of glycogen, fat, and protein.
Several hours after the intake of dietary carbohydrate, blood glucose levels fall
slightly because of the ongoing oxidation of glucose by the brain and other
tissues. Although its primary target is the liver, glucagon (like epinephrine) also
affects adipose tissue, activating TAG breakdown by activating triacylglycerol
lipase and liberates free fatty acids, which are exported to the liver and other
tissues as fuel, sparing glucose for the brain. The net effect of glucagon is
therefore to stimulate glucose synthesis and release by the liver and to mobilize
fatty acids from adipose tissue, to be used instead of glucose as fuel for tissues
other than the brain.

12.3.2.2 During fasting and starvation, metabolism shifts to provide

fuel for the brain
The fuel reserves of a healthy adult human are of three types: glycogen stored
in the liver and, in relatively small quantities, in muscles; large quantities of
triacylglycerols in adipose tissues; and tissue proteins, which can be degraded
when necessary to provide fuel. In the first few hours after a meal, the blood
glucose level is diminished slightly, and tissues receive glucose released from
liver glycogen. There is little or no synthesis of lipids. By 24 hours after a meal,
blood glucose has fallen further, insulin secretion has slowed, and glucagon
secretion has increased.

12.3.3 Thyroid Hormones

Thyroid hormones (T4 and T3) are tyrosine-based hormones produced by the
follicular cells of the thyroid gland and are regulated by TSH made by the
thyrotropes of the anterior pituitary gland, are primarily responsible for

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Biochemistry regulation of metabolism. Iodine is necessary for the production of T3

(triiodothyronine) and T4 (thyroxine). A deficiency of iodine leads to decreased
production of T3 and T4, enlarges the thyroid tissue and will cause the disease
known as goitre. The thyronines act on nearly every cell in the body. They act
to increase the basal metabolic rate, affect protein synthesis, help regulate long
bone growth (synergy with growth hormone) and neural maturation, and increase
the body’s sensitivity to catecholamines (such as adrenaline). The thyroid
Notes hormones are essential to proper development and differentiation of all cells of
the human body. These hormones also regulate protein, fat, and carbohydrate
metabolism, affecting how human cells use energetic compounds. They also
stimulate vitamin metabolism. Numerous physiological and pathological stimuli
influence thyroid hormone synthesis. Thyroid hormone leads to heat generation
in humans. However, the thyronamines function via some unknown mechanism
to inhibit neuronal activity; this plays an important role in the hibernation cycles
of mammals and the moulting behaviour of birds.

12.3.3.1 Iodine is essential for thyroid hormone synthesis

Iodide is actively absorbed from the bloodstream by a process called iodide
trapping. In this process, sodium is cotransported with iodide from the
basolateral side of the membrane into the cell and then concentrated in the
thyroid follicles to about thirty times its concentration in the blood. Via a reaction
with the enzyme thyroperoxidase, iodine is bound to tyrosine residues in the
thyroglobulin molecules, forming monoiodotyrosine (MIT) and diiodotyrosine
(DIT). Linking two moieties of DIT produces thyroxine. Combining one particle
of MIT and one particle of DIT produces triiodothyronine. If there is a deficiency
of dietary iodine, the thyroid will not be able to make thyroid hormone. The lack
of thyroid hormone will lead to decreased negative feedback on the pituitary,
leading to increased production of thyroid-stimulating hormone, which causes
the thyroid to enlarge (the resulting medical condition is called endemic colloid
goiter).

12.3.3.2 Thyroid hormones are transported by Thyroid-Binding Globulin

Thyroxinebinding globulin (TBG), a glycoprotein binds T4 and T3 and has the
capacity to bind 20 μg/dL of plasma. Under normal circumstances, TBG binds
– noncovalently – nearly all of the T4 and T3 in plasma, and it binds T4 with
greater affinity than T3. The small, unbound (free) fraction is responsible for the
biologic activity. Thus, in spite of the great difference in total amount, the free
fraction of T3 approximates that of T4, and given that T3 is intrinsically more
active than T4, most biologic activity is attributed to T3. TBG does not bind any
other hormones.

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12.3.3.3 Circulation and transport Biochemistry

Most of the thyroid hormone circulating in the blood is bound to transport

proteins. Only a very small fraction of the circulating hormone is free (unbound)
and biologically active, hence measuring concentrations of free thyroid hormones
is of great diagnostic value.
When thyroid hormone is bound, it is not active, so the amount of free T3/T4
is what is important. For this reason, measuring total thyroxine in the blood can Notes
be misleading.

12.3.3.4 Related diseases

Both excess and deficiency of thyroxine can cause disorders.
z Hyperthyroidism (an example is Graves Disease) is the clinical syndrome
caused by an excess of circulating free thyroxine, free triiodothyronine, or
both. It is a common disorder that affects approximately 2% of women and
0.2% of men.
z Hypothyroidism (an example is Hashimoto’s thyroiditis) is the case where
there is a deficiency of thyroxine, triiodiothyronine, or both.

12.3.4 Parathyroid Hormone

Parathyroid hormone (PTH), parathormone or parathyrin, is secreted by the chief
cells of the parathyroid glands. It acts to increase the concentration of calcium
(Ca2+) in the blood, whereas calcitonin (a hormone produced by the parafollicular
cells of the thyroid gland) acts to decrease calcium concentration. PTH acts to
increase the concentration of calcium in the blood by acting upon the parathyroid
hormone 1 receptor (high levels in bone and kidney) and the parathyroid
hormone 2 receptor (high levels in the central nervous system, pancreas, testis,
and placenta). PTH was one of the first hormones to be shown to use the G-
protein, adenylyl cyclase second messenger system. Parathyroid hormone
regulates serum calcium through its effects (Table 12.1).
Table 12.1: Effect of parathyroid hormone in regulation of serum calcium.

Region Effect
Bone PTH enhances the release of calcium from the large
reservoir contained in the bones. Bone resorption is the
normal destruction of bone by osteoclasts, which are
indirectly stimulated by PTH forming new osteoclasts,
which ultimately enhances bone resorption.

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Biochemistry
Kidney PTH enhances active reabsorption of calcium and magnesium
from distal tubules of kidney. As bone is degraded, both
calcium and phosphate are released. It also decreases the
reabsorption of phosphate, with a net loss in plasma
phosphate concentration. When the calcium:phosphate ratio
increases, more calcium is free in the circulation.

Notes Intestine PTH enhances the absorption of calcium in the intestine by

increasing the production of activated vitamin D. Vitamin
D activation occurs in the kidney. PTH converts vitamin D
to its active form (1,25-dihydroxy vitamin D). This activated
form of vitamin D increases the absorption of calcium (as
Ca2+ ions) by the intestine via calbindin.

12.3.4.1 Regulation of PTH secretion

Secretion of parathyroid hormone is controlled chiefly by serum [Ca2+] through
negative feedback. Calcium-sensing receptors located on parathyroid cells are
activated when [Ca2+] is low. Hypomagnesemia inhibits PTH secretion and also
causes resistance to PTH, leading to a form of hypoparathyroidism that is
reversible. Hypermagnesemia also results in inhibition of PTH secretion.
Stimulators of PTH includes decreased serum [Ca2+], mild decreases in serum
[Mg2+], and an increase in serum phosphate. Inhibitors include increased serum
[Ca2+], severe decreases in serum [Mg2+], which also produces symptoms of
hypoparathyroidism (such as hypocalcemia), and calcitriol.

12.3.4.2 Clinical significance

z Hyperparathyroidism, the presence in the blood of excessive amounts of
parathyroid hormone, occurs in two very distinct sets of circumstances.
Primary hyperparathyroidism is due to autonomous, abnormal hypersecretion
of PTH in the parathyroid gland while secondary hyperparathyroidism is
an appropriately high PTH level seen as a physiological response to
hypocalcemia
z A low level of PTH in the blood is known as hypoparathyroidism and is
most commonly due to damage to or removal of parathyroid glands during
thyroid surgery.
z There are a number of rare but well-described genetic conditions affecting
parathyroid hormone metabolism, including pseudohypoparathyroidism,
familial hypocalciuric hypercalcaemia, and autosomal dominant
hypercalciuric hypocalcaemia.

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12.3.5 Growth hormone Biochemistry

Growth hormone (GH or HGH), also known as somatotropin or somatropin, is

a peptide hormone that stimulates growth, cell reproduction and regeneration in
humans. It is a type of mitogen which is specific only to certain kinds of cells.
Growth hormone is a single-chain polypeptide that is synthesized, stored, and
secreted by somatotropic cells within the lateral wings of the anterior pituitary
gland. The anterior pituitary gland secretes hormones that tend to elevate the Notes
blood glucose and therefore antagonize the action of insulin. Growth hormone
secretion is stimulated by hypoglycemia; it decreases glucose uptake in muscle.
Some of this effect may not be direct, since it stimulates mobilization of free
fatty acids from adipose tissue, which themselves inhibit glucose utilization.
Growth hormone increases amino acid transport in all cells, and estrogens do
this in the uterus. A deficiency of this hormone produces dwarfism, and an excess
leads to gigantism.

12.3.5.1 Regulation of growth hormone secretion

Secretion of growth hormone (GH) in the pituitary is regulated by the
neurosecretory nuclei of the hypothalamus. These cells release the peptides
Growth hormone-releasing hormone (GHRH or somatocrinin) and Growth
hormone-inhibiting hormone (GHIH or somatostatin) into the hypophyseal
portal venous blood surrounding the pituitary. GH release in the pituitary is
primarily determined by the balance of these two peptides, which in turn is
affected by many physiological stimulators (e.g., exercise, nutrition, sleep) and
inhibitors (e.g., free fatty acids) of GH secretion. A number of factors are known
to affect GH secretion, such as age, sex, diet, exercise, stress, and other
hormones.

12.3.5.2 Regulation
Stimulators of growth hormone (GH) secretion include peptide hormones,
ghrelin, sex hormones, hypoglycemia, deep sleep, niacin, fasting, and vigorous
exercise. Inhibitors of GH secretion include somatostatin, circulating
concentrations of GH and IGF-1 (negative feedback on the pituitary and
hypothalamus), hyperglycemia, glucocorticoids, and dihydrotestosterone.

12.3.5.3 Effects of growth hormone

Increased height during childhood is the most widely known effect of GH. In
addition to increasing height in children and adolescents, growth hormone has
many other effects on the body such as:

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Biochemistry
z Increases calcium retention, and strengthens and increases the mineralization
of bone
z Increases muscle mass through sarcomere hypertrophy
z Promotes lipolysis
z Increases protein synthesis
z Stimulates the growth of all internal organs excluding the brain
Notes
z Plays a role in homeostasis
z Reduces liver uptake of glucose
z Promotes gluconeogenesis in the liver
z Contributes to the maintenance and function of pancreatic islets
z Stimulates the immune system

12.3.5.4 Clinical significance

12.3.5.4.1 Excess
The most common disease of GH excess is a pituitary tumor composed of
somatotroph cells of the anterior pituitary. These somatotroph adenomas are
benign and grow slowly, gradually producing more and more GH. For years, the
principal clinical problems are those of GH excess. Eventually, the adenoma may
become large enough to cause headaches, impair vision by pressure on the optic
nerves, or cause deficiency of other pituitary hormones by displacement.
Prolonged GH excess thickens the bones of the jaw, fingers and toes. Resulting
heaviness of the jaw and increased size of digits is referred to as acromegaly.
Accompanying problems can include sweating, pressure on nerves (e.g., carpal
tunnel syndrome), muscle weakness, excess sex hormone-binding globulin
(SHBG), insulin resistance or even a rare form of type 2 diabetes, and reduced
sexual function.
GH-secreting tumors are typically recognized in the fifth decade of life. It is
extremely rare for such a tumor to occur in childhood, but, when it does, the
excessive GH can cause excessive growth, traditionally referred to as pituitary
gigantism.

12.3.5.4.2 Deficiency
The effects of growth hormone deficiency vary depending on the age at which
they occur. In children, growth failure and short stature are the major
manifestations of GH deficiency, with common causes including genetic
conditions and congenital malformations. It can also cause delayed sexual

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maturity. In adults, deficiency is rare, with the most common cause a pituitary Biochemistry
adenoma, and others including a continuation of a childhood problem, other
structural lesions or trauma, and very rarely idiopathic GHD. Adults with GHD
“tend to have a relative increase in fat mass and a relative decrease in muscle
mass and, in many instances, decreased energy and quality of life”.

Notes
INTEXT QUESTIONS 12.1
I. Choose the best answer
1. The hormone that is released into the bloodstream having the receptor
cells at distant from the source is
(a) Exocrine (b) Paracrine
(c) Endocrine (d) None
2. This hormone is a chain of amino acids
(a) Thyroxine (b) Insulin
(c) Vitamin D3 (d) Epinephrin
3. Glucose transporter to the plasma membrane of muscle and adipose
tissue is
(a) GLUT1 (b) GLUT2
(c) GLUT3 (d) GLUT4
4. The storage form of lipid in adipose tissue is
(a) Triacylglycerol (b) Free fatty acid
(c) Low density lipoprotein (d) Phospholipid
5. Excess of somatotropin leads to
(a) Dwarfism (b) Gigantism
(c) Goitre (d) Diabetes Mellitus
II. Fill in the blanks
6. Hormones communicate with intracellular metabolic processes through
intermediary molecules called .................
7. ................. transporters carry glucose into the b-cells of pancreas
8. ................. stimulates breakdown of glycogen, fat, and protein.
9. Thyroid hormones are transported by .................
10. Vitamin D3 increases the absorption of calcium (as Ca2+ ions) by the
intestine via .................

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Biochemistry III. Match the following

11. Frequent urination (a) Insulin
12. Endocrine hormone (b) Glucokinase
13. Second messenger (c) Hypothyroidism
14. Hashimoto’s thyroiditis (d) cAMP
Notes 15. Hexokinase IV (e) Polyuria

WHAT YOU HAVE LEARNT

z A hormone acts as a messenger by transmitting signal from one cell to
another.
z Hormones are derived from steroids, amino acids and polypeptides.
z Insulin secreted from β-cells of pancreas controls the uptake of glucose by
liver and muscle cells and also the storage of glucose in the form of
glycogen.
z Diabetes Mellitus results from the lack of insulin secretion by the pancreas.
z Glucagon, a peptide hormone synthesized and secreted from the α-cells of
pancreas, raises blood glucose levels.
z Secretion of glucagon is stimulated by hypoglycemia, epinephrine, arginine,
alanine, acetylcholine, and cholecystokinin. Inhibited by somatostatin,
insulin, increased free fatty acids and keto acids into the blood, and
increased urea production.
z Thyroid hormones (T4 and T3) are tyrosine-based hormones produced by
the follicular cells of the thyroid gland.
z The thyroid hormones are essential to proper development and differentiation
of all cells of the human body. These hormones also regulate protein, fat,
and carbohydrate metabolism.
z The free thyroid hormones in the circulation are the active form, and is of
great diagnostic value.
z The diseases associated with the excess and deficiency of thyroid hormones
are Graves disease and Hashimoto’s thyroiditis, respectively.
z Parathyroid hormone (PTH), parathormone or parathyrin, is secreted by the
chief cells of the parathyroid glands.
z PTH acts to increase the concentration of calcium (Ca2+) in the blood.

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Biochemistry
z Growth hormone (GH or HGH), also known as somatotropin or somatropin,
is secreted from anterior pituitary gland, is a peptide hormone that
stimulates growth, cell reproduction and regeneration in humans.
z A deficiency of this hormone produces dwarfism, and an excess leads to
gigantism.

Notes
TERMINAL QUESTIONS
1. Classify Hormones
2. Write short notes on
Insulin
Thyroid Hormone
Growth Hormone

ANSWERS TO INTEXT QUESTIONS

I. 1. (c) 2. (b) 3. (d) 4. (a) 5. (b)
II. 6. second messengers
7. GLUT2
8. Glucagon
9. Thyroxine binding globulin
10. calbindin,
III. 11. (e) 12. (a) 13. (d) 14. (c) 15. (b)

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Biochemistry

13
Notes
CLINICAL BIOCHEMISTRY

13.1 INTRODUCTION
Clinical Biochemistry deals with the study of biochemical events or parameters
in the body.

OBJECTIVES
After reading this lesson, you will be able to:
z describe clinically important enzymes
z explain liver and renal functional test
z explain specimen collection & other pre-analytical variables.
z describe general laboratory techniques, procedures & safety measure

13.2 CLINICALLY IMPORTANT ENZYMES

Certain enzymes, proenzymes, and their substrates are present at all times in the
circulation of normal individuals and perform a physiologic function in the
blood. Examples of these functional plasma enzymes include lipoprotein
lipase, pseudocholinesterase, and the proenzymes of blood coagulation and
blood clot dissolution. The majority of these enzymes are synthesized in and
secreted by the liver.

Enzymes Principle Sources Clinical Applications

of Enzymes in Blood
Alanine Liver Hepatic parenchymal
aminotransferase diseases

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Biochemistry
Alkaline Liver, bone, intestinal Bone diseases,
phosphatase mucosa, placenta hepatobiliary diseases
Amylase Salivary glands, Pancreatic diseases
pancreas
Aspartate Liver, skeletal muscle, Hepatic parenchymal
aminotransferase heart erythrocytes disease, muscle disease
Notes
Cholinesterase Liver Organophosphorus
insecticide, hepatic
parenchymaldisease
Creatine kinase Skeletal muscle, heart Muscle diseases
γ- glutamyl Liver Hepatobiliary diseases,
transferase marker of alcohol
abuse.
Lactate Heart, liver, skeletal Hemolysis, hepatic
dehydrogenase muscle, erythrocytes, parenchymal diseases,
platelets, lymph nodes tumor marker
Lipase Pancreas Pancreatic disesases
5’- nucleotidase Liver Hepatobiliary diseases
Trypsin (Serine Pancreas Pancreatic diseases
protease)

Alanine aminotransferase (ALT)

Clinical applications of ALT assays are mainly used for the evaluation of hepatic
disorders. Higher elevations are found in hepatocellular disorders than in
extrahepatic or intrahepatic obstructive disorders. In acute inflammatory
conditions of the liver, ALT elevations are frequently higher than those of AST
and tend to remain longer half-life of ALT in serum (16 hours and 24 hours,
respectively). ALT levels have historically been compared with levels of AST
to help determine the source of an elevated AST level and to detect liver
involvement with myocardial injury.

Normal values of ALT:

Male: <45 U/L = <0.77 μkat/L

Female: <34 U/L = <0.58 μkat/L

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Biochemistry Aspartate aminotransferase (AST)

The clinical use of AST is limited mainly to the evaluation of hepatocellular
disorders and skeletal muscle involvement. In Acute myocardial infarction
(AMI), AST levels begin to rise within 6-8 hours, peak at 24 hours, and generally
return to normal within 5 days. However, because of the wide tissue distribution,
AST levels are not useful in the diagnosis of AMI.
Notes AST elevations are frequently seen in pulmonary embolism. Following congestive
heart failure, AST levels also may be increased, probably reflecting liver
involvement as a result of inadequate blood supply to that organ. AST levels are
highest in acute hepatocellular disorders. Skeletal muscle disorders, such as the
muscular dystrophies, and inflammatory conditions also cause increases in AST
levels.
Normal values of AST:
Male: <35U/L = <0.60 μkat/L
Female: <31 U/L = <0.53 μkat/L

α-Amylase
It is an enzyme of the hydrolyase class that catalyzes the hydrolysis of 1,4-α-
glycosidic linkages in polysaccharides. AMYs are calcium metaloenzymes, with
the calcium absolutely required for functional integrity. AMYs normally
occurring in human plasma are small molecules with molecular weights varying
from 54 to 62 kDa. The enzyme is thus small enough to pass the glumeruli of
the kidneys and AMY is the only plasma enzyme physiologically found in urine.
The AMY activity present in normal serum and urine is of pancreatic (P-AMY)
and salivary gland (S-AMY) origin. If the plasma amylase activity fails to fall
after an attack of acute pancreatitis there may be leakage of pancreatic fluid into
the lesser sac (a pancreatic pseudocyst). Urinary amylase levels are high,
differentiating it from macroamylasaemia. This is one of the few indications for
estimating urinary amylase activity, which is inappropriately low relative to the
plasma activity if there is glomerular impairment or macroamylasaemia.
Normal values of amylase: 28-100 U/L = 0.48-1.7 μkat/L

Lipase
It is a single–chain glycoprotein with molecular weight of 48 kDa. For full
catalytic activity and greatest specificity the presence of bile salts and a cofactor
called colipase, which is secreted by the pancreas, is required. LPS is a small
molecule and is filtered through the glomerulus. It is totally reabsorbed by the

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renal tubules, and it is not normally detected in urine. Plasma lipase levels are Biochemistry
elevated in acute pancreatitis and carcinoma of the pancreas.
Normal values: 40-200 U/L

Gamma-glutamyl-transferase
It catalyzes the transfer of the γ–glutamyl group from peptides and compounds
that contain it to an acceptor Gamma-glutamyl transferase occurs mainly in the Notes
cells of liver, kidneys, pancreas and prostate. Plasma GGT activity is higher in
men than in women.

Causes of raised plasma GGT activity

z Induction of enzyme synthesis, without cell damage, by drugs or alcohol.
z Hepatocellular damage, such as that due to infectious hepatitis: A patient
should never be labeled an alcoholic because of a high plasma GGT activity
alone.
Normal values for GGT

Male: <55 U/L = <0.94 μkat/L

Female: <38 U/L = <0.65 μkat/L

Lactate Dehydrogenase (LD)

It catalyses the reversible interconversion of lactate and pyruvate. The enzyme
is widely distributed in the body, with high concentrations in cells of cardiac and
skeletal muscle, liver, kidney, brain and erythrocytes: measurement of plasma
total LD activity is therefore a non-specific marker of cell damage.

It is increased in plasma in Myocardial Injury, acute leukemias, generalized

carcinomatosis and in acute hepatitis. Estimation of isoenzymes in more useful
in clinical diagnosis between hepatic disease and M.I.

Normal range of total LDH: 180-360 U/L= 3.1-6.1 μkat/L.

13.3 LIVER FUNCTION TESTS

Definition
Liver function tests, or LFTs, include tests that are routinely measured in all
clinical laboratories. LFTs include bilirubin, a compound formed by the
breakdown of hemoglobin; ammonia, a breakdown product of protein that is

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Biochemistry normally converted into urea by the liver before being excreted by the kidneys;
proteins that are made by the liver including total protein, albumin, prothrombin,
and fibrinogen; cholesterol and triglycerides, which are made and excreted via
the liver; and the enzymes alanine aminotransferase (ALT), aspartate
aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl
transferase (GGT), and lactate dehydrogenase (LDH).

Notes Other liver function tests include serological tests (to demonstrate antibodies)
and DNA tests for hepatitis and other viruses; and tests for antimitochondrial
and smooth muscle antibodies, transthyretin (prealbumin), protein electrophoresis,
bile acids, alpha-fetoprotein, and a constellation of other enzymes that help
differentiate necrotic (characterized by death of tissues) versus obstructive liver
disease.
The hepatic function panel evaluates:
Alanine aminotransferase (ALT)
Alkaline phosphatase (ALP)
Aspartate aminotransferase (AST)
Total bilirubin and direct bilirubin. Bilirubin is a byproduct of the normal
breakdown of red blood cells. It usually passes through the liver and is excreted
from the body. But if that doesn’t happen due to a liver disease, bilirubin levels
in the blood can rise and the skin can take on the yellow discoloration known
as jaundice. Tests for bilirubin may be total (measuring the level of all of the
bilirubin in the blood) or direct (measuring only bilirubin that has been processed
by the liver and attached to other chemicals).
Albumin and total protein. Protein is needed to build and maintain muscles,
bones, blood, and organ tissue. Sometimes when there’s a problem with the liver,
it can’t make proteins as well, so protein levels decrease. Liver function tests
measure albumin specifically (the major blood protein produced by the liver),
as well as the total amount of all proteins in the blood.

Normal results
Reference ranges vary from laboratory to laboratory and also depend upon the
method used. However, normal values are generally framed by the ranges shown
below. Values for enzymes are based upon measurement at 37°C.
ALT: 5–35 IU/L. (Values for the elderly may be slightly higher, and values also
may be higher in men and in African-Americans).
AST: 0–35 IU/L

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ALP: 30–120 IU/LALP is higher in children, older adults and pregnant females Biochemistry

GGT: males 2–30 U/L; females 1–24 U/L

LDH: 12–60 years: 100–190 U/L
Bilirubin: (Adult, elderly, and child)
Total bilirubin: 0.1–1.0 mg/dL
Notes
Indirect bilirubin: 0.2–0.8 mg/dL
Direct bilirubin: 0.0–0.3 mg/dL. (Newborn)
Note: Critical values for adult: greater than 1.2 mg/dL
Critical values for newborn (requiring immediate treatment): greater than 15 mg/
dL
Ammonia: 10–70 micrograms per dL (heparinized plasma). Normal values for
this test vary widely, depending upon the age of the patient and the type of
specimen.
Albumin: 3.2–5.4 g/L

13.4 RENAL FUNCTION TEST

Definition
Kidney function tests are a collective term for a variety of individual tests and
procedures that can be done to evaluate how well the kidneys are functioning.
A doctor who orders kidney function tests and uses the results to assess the
functioning of the kidneys is called a nephrologist.

Laboratory tests
There are a number of urine tests that can be used to assess kidney function. A
simple, inexpensive screening test a routine urinalysis is often the first test
conducted if kidney problems are suspected. A small, randomly collected urine
sample is examined physically for things like color, odor, appearance, and
concentration (specific gravity); chemically, for substances such a protein,
glucose, and pH (acidity/alkalinity); and microscopically for the presence of
cellular elements (red blood cells [RBCs], white blood cells [WBCs], and
epithelial cells), bacteria, crystals, and casts (structures formed by the deposit
of protein, cells, and other substances in the kidneys’s tubules). If results indicate
a possibility of disease or impaired kidney function, one or more of the following
additional tests is usually performed to pinpoint the cause and the level of decline
in kidney function.

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Biochemistry Creatinine clearance test

This test evaluates how efficiently the kidneys clear a substance called creatinine
from the blood. Creatinine, a waste product of muscle energy metabolism, is
produced at a constant rate that is proportional to the individual’s muscle mass.
Because the body does not recycle it, all creatinine filtered by the kidneys in a
given amount of time is excreted in the urine, making creatinine clearance a very
specific measurement of kidney function. The test is performed on a timed urine
Notes specimen – a cumulative sample collected over a two to 24-hour period.
Determination of the blood creatinine level is also required to calculate the urine
clearance.

Urea clearance test

Urea is a waste product that is created by protein metabolism and excreted in
the urine. The urea clearance test requires a blood sample to measure the amount
of urea in the bloodstream and two urine specimens, collected one hour apart,
to determine the amount of urea that is filtered, or cleared, by the kidneys into
the urine.

Urine osmolality test

Urine osmolality is a measurement of the number of dissolved particles in urine.
It is a more precise measurement than specific gravity for evaluating the ability
of the kidneys to concentrate or dilute the urine. Kidneys that are functioning
normally will excrete more water into the urine as fluid intake is increased,
diluting the urine. If fluid intake is decreased, the kidneys excrete less water and
the urine becomes more concentrated. The test may be done on a urine sample
collected first thing in the morning, on multiple timed samples, or on a
cumulative sample collected over a 24-hour period. The patient will typically
be prescribed a high-protein diet for several days before the test and be asked
to drink no fluids the night before the test.

Urine protein test

Healthy kidneys filter all proteins from the bloodstream and then reabsorb them,
allowing no protein, or only slight amounts of protein, into the urine. The
persistent presence of significant amounts of protein in the urine, then, is an
important indicator of kidney disease. A positive screening test for protein
(included in a routine urinalysis ) on a random urine sample is usually followed
up with a test on a 24-hour urine sample that more precisely measures the
quantity of protein.
There are also several blood tests that can aid in evaluating kidney function.
These include:

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Blood urea nitrogen test (BUN) Biochemistry

Urea is a byproduct of protein metabolism. Formed in the liver, this waste

product is then filtered from the blood and excreted in the urine by the kidneys.
The BUN test measures the amount of nitrogen contained in the urea. High BUN
levels can indicate kidney dysfunction, but because BUN is also affected by
protein intake and liver function, the test is usually done together with a blood
creatinine, a more specific indicator of kidney function.
Notes

Creatinine test
This test measures blood levels of creatinine, a by-product of muscle energy
metabolism that, similar to urea, is filtered from the blood by the kidneys and
excreted into the urine. Production of creatinine depends on an person’s muscle
mass, which usually fluctuates very little. With normal kidney function, then,
the amount of creatinine in the blood remains relatively constant and normal.
For this reason, and because creatinine is affected very little by liver function,
an elevated blood creatinine level is a more sensitive indicator of impaired
kidney function than the BUN.

Other blood tests

Measurement of the blood levels of other elements regulated in part by the
kidneys can also be useful in evaluating kidney function. These include sodium,
potassium, chloride, bicarbonate, calcium, magnesium, phosphorus, protein,
uric acid, and glucose.

Results
Normal values for many tests are determined by the patient’s age and gender.
Reference values can also vary by laboratory, but are generally within the
following ranges:

Urine tests
Creatinine clearance. For a 24-hour urine collection, normal results are 90 mL/
min–139 mL/min for adult males younger than 40, and 80–125 mL/min for adult
females younger than 40. For people over 40, values decrease by 6.5 mL/min
for each decade of life.
Urine osmolality. With restricted fluid intake (concentration testing), osmolality
should be greater than 800 mOsm/kg of water. With increased fluid intake
(dilution testing), osmolality should be less than 100 mOsm/kg in at least one
of the specimens collected. A 24-hour urine osmolality should average 300–900
mosm/kg. A random urine osmolality should average 500–800 mOsm/kg.

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Biochemistry Urine protein. A 24-hour urine collection should contain not more than 150 mg
of protein.
Urine sodium. A 24-hour urine sodium should be within 75–200 mmol/day.

Blood tests
Blood urea nitrogen (BUN) should average 8–20 mg/dL.

Notes Creatinine should be 0.8–1.2 mg/dL for males, and 0.6–0.9 mg/dL for females.
Uric acid levels for males should be 3.5–7.2 mg/dL and for females 2.6–6.0 mg/
dL.
Low clearance values for creatinine indicate a diminished ability of the kidneys
to filter waste products from the blood and excrete them in the urine. As
clearance levels decrease, blood levels of creatinine, urea, and uric acid increase.
Because it can be affected by other factors, an elevated BUN, alone, is
suggestive, but not diagnostic for kidney dysfunction.
The inability of the kidneys to concentrate the urine in response to restricted fluid
intake, or to dilute the urine in response to increased fluid intake during
osmolality testing, may indicate decreased kidney function. Because the kidneys
normally excrete almost no protein in the urine, its persistent presence, in
amounts that exceed the normal 24-hour urine value, usually indicates some type
of kidney disease.

13.5 COLLECTION OF SPECIMENS

Collection of Blood sample
Blood and separated serum are the most common specimens taken to investigate
outbreaks of communicable disease. Venous blood can be used for isolation and
identification of the pathogen in culture.

Venous blood samples

Materials for collection

z Skin disinfection: 70% alcohol (isopropyl alcohol, ethanol) or 10% povidone
iodine, swabs, gauze pads, band aid
z Disposable latex or vinyl gloves
z Tourniquet, Vacutainer, Monovette, or similar vacuum blood collection
devices, or disposable syringes and needles
z Vacutainer or sterile screw-cap tubes (or cryotubes if indicated), blood
culture bottles (50ml for adults, 25ml for children) with appropriate media
z Labels and indelible marker pen.

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Method of collection Biochemistry

z Place a tourniquet above the venepuncture site.

z Palpate and locate the vein. It is critical to disinfect the venepuncture site
meticulously with 10% povidone iodine or 70% isopropyl alcohol by
swabbing the skin concentrically from the centre of the venepuncture site
outwards. Let the disinfectant evaporate. Do not repalpate the vein again.
Perform venepuncture.
Notes
z If withdrawing with conventional disposable syringes, withdraw 5-10 ml
of whole blood from adults, 2-5ml from children and 0.5-2ml for infants.
z If withdrawing with vacuum systems, withdraw the desired amount of blood
directly into each transport tube and culture bottle.
z Remove the tourniquet. Apply pressure to site until bleeding stops, and
apply sticking plaster (if desired).
z Using aseptic technique, transfer the specimen to relevant cap transport
tubes and culture bottles. Secure caps tightly.
z Label the tube, including the unique patient identification number, using
indelible marker pen.
z Discard the sharps into disposal container without recaping.
z Complete the case investigation and the laboratory request forms using the
same identification number.

Handling and transport

z Blood culture bottles and blood sample tubes should be transported upright
and secured in a screw cap container or in a rack in a transport box.
z Cushion or suspend bottles during transport over rough terrain to prevent
lysis of red cells. They should have enough absorbent paper around them
to soak up all the liquid in case of a spill.
z If the specimen will reach the laboratory within 24 hours, most bacterial
pathogens can be recovered from blood cultures transported at ambient
temperature.

Urine specimen collection

Materials for collection
z Sterile plastic cup with lid (50 ml or more)
z Clean, screw-top specimen transport containers (“universal” containers are
often used)
z Gauze pads
z Soap and clean water (or normal saline) if possible.

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Biochemistry Method of collection

z Give the patient clear instructions to pass urine for a few seconds, and then
to hold the cup in the urine stream for a few seconds to catch a midstream
urine sample. This should decrease the risk of contamination from organisms
living in the urethra.
z To decrease the risk of contamination from skin organisms, the patient
Notes should be directed to avoid touching the inside or rim of the plastic cup
with the skin of the hands, legs or external genitalia. Tighten the cap firmly
when finished.
z For hospitalized or debilitated patients, it may be necessary to wash the
external genitalia with soapy water to reduce the risk of contamination.
z Urine collection bags may be necessary for infants. If used, transfer urine
from the urine bag to specimen containers as soon as possible to prevent
contamination with skin bacteria. Use a disposable transfer pipette to
transfer the urine and label it.

Handling and transport

z Transport to the laboratory within 2–3 hours of collection. If this is not
possible, do not freeze but keep the specimen refrigerated at 4-8°C. Keeping
the specimen refrigerated will decrease the risk of overgrowth of
contaminating organisms.
z Ensure that transport containers are leak-proof and tightly sealed.

13.6 GENERAL LABORATORY TECHNIQUES

Laboratory services are an integral part of disease diagnosis, treatment,
monitoring response to treatment, disease surveillance programmes and clinical
research. Essential Health Technology as an important ingredient of Essential
Clinical Services. Use of diagnostic techniques aid early diagnosis enabling
appropriate and prompt intervention thereby reducing overall disease burden and
promoting health. All laboratories are not equipped with facilities for carrying
out complex investigations. The structure and function of a clinical laboratory
varies according to the level of health care facility. Peripheral laboratories carry
out simple tests like urine analysis and haemoglobin estimation whereas higher
centers are equipped with sophisticated technology and trained manpower to
carry out complex investigations. Establishing a network between peripheral and
higher laboratories allows collection of specimen at periphery and their storage
and transport for testing at higher centers and communicating report to the
peripheral center efficiently without actually having to transfer the patient. In
the event of patient transfer, the higher centers do not need to repeat

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investigations carried out at the peripheral health center, thereby saving crucial Biochemistry
time as well as cost and providing continuity in patient care. Networking
between laboratories is also essential in disease surveillance programmes and
outbreak investigations in order to obtain quick and reliable results.

Scope
Good Clinical Laboratory Practices should be used by all laboratories where Notes
tests are done on biological specimens for diagnosis, patient care, disease control
and research such as:

z Microbiology & Serology

z Hematology & Blood Banking
z Molecular Biology and Molecular Pathology
z Clinical Pathology
z Clinical Biochemistry
z Immunology (Immunohematology and Immunobiochemistry)
z Histopathology/Pathology and Cytology

Equipment
z Each laboratory should prepare an exhaustive list of equipment and
consumables required and available for general functioning of the laboratory
and specialized equipment for special tests.
z Laboratory equipment should be of adequate capacity to meet work load
requirement.
z Equipment should be suitably located in the laboratory so as to allow
accessibility and sequential utilization thus minimizing the need for
frequent movement of specimens or reagents.
z All equipment should be in good working condition at all times. Periodic
inspection, cleaning, maintenance of equipment should be done. An
equipment log book should be maintained for all major equipment.
Laboratories should maintain necessary instructions for operation and
maintenance of equipment in the form of Standard Operating Procedures
(SOPs). A copy of SOP should be readily available.
z Maintenance contracts including warranty cards, telephone numbers of staff
to be contacted in case of equipment malfunction should be kept safely. User
manual should be available readily for reference. The staff should be aware
of trouble shooting measures to be adopted for preventing equipment

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Biochemistry malfunction. A format of the equipment log book provided in Annexure 1

can be used.
z New equipment should be calibrated and validated before routine use. AMR
(Analytical Measurement range) should be verified, manufacturer can be
consulted for verification and selection of range.
z Periodic performance check/calibration check for all equipment should be
Notes done using reference standard/reference material. The frequency of
performance check should be based on the day-to-day performance of the
equipment.
z Equipment performance should be verified from Internal Quality Control
results and External Quality Assessment results. Outlier parameter trend
analysis record should be maintained in respect of its effect on the
equipment.
z All analytical equipment should be calibrated and calibration certificate
provided by equipment company. Non-analytical equipment such as pipette,
thermometer, weighing balance and centrifuge should be calibrated by
accredited calibration laboratory or done in-house with traceability to
National Physical Laboratory (NPL). For in-house calibration, laboratories
should use :
z Calibrated tachometer - for centrifuge
z Calibrated digital temperature sensor - for checking temperature of
refrigerator, incubator etc.
z Calibrated glass thermometer- for temperature checking of oven,
water bath etc.
z Calibrated weights - for balance
z National Institute of Science and Technology (NIST) buffer – for pH
meter.Standard buffer solutions bought from reputed manufacturers
with certifiable traceability can be used as alternative.

Standard Operating Procedure (SOP)

z SOP is a document, which contains detailed, written instructions describing
the stepwise process and technique of performing a test or procedure in the
laboratory.
z SOP helps to ensure uniformity, consistency and control over the processes
carried out. It ensures that the procedures are done in exactly the same way
each time irrespective of the operator.
z SOP should contain information on who can perform the test, their
qualification and training, how to carry out the test including pre-analytical,
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analytical and post-analytical stages of test/procedure, laboratory conditions Biochemistry
required for the test/ procedure, routine care and maintenance of equipment,
precautions and safety instructions, trouble shooting measures, waste
disposal and linkage with reference laboratories.
z SOP should be simple and written in an easy to understand language.
z The procedure described in the SOP must be followed exactly by all staff
members to ensure high quality results. Notes

z It should be titled along with version number, dated and signed by an

authorized person and updated regularly.
z It is important for the SOP document to be readily available in the working
area and is therefore also referred to as ‘laboratory bench work manual’.
z SOPs are controlled documents and can be changed only with approval
of the laboratory quality manager and/or Head of the laboratory.

Safety in Laboratories
Personnel working in laboratories may be exposed to risks from various
chemicals, infectious materials, fire hazard, gas leak etc. The environment is also
at risk of being contaminated by hazardous materials used and wastes generated
in the laboratory. Safety in laboratories therefore includes protection of both
the staff and the environment from hazardous materials.

General Safety Measures

z Documentation of Laboratory Safety Policies and Procedures.
z All laboratory personnel should be aware about the laboratory safety
policies and procedures and follow these at all times.
z List of hazardous materials used in the laboratory should be prepared. All
hazardous materials should be accounted for on a continuous basis.
z Laboratory personnel should follow safe hygienic practices which include
hand washing, wearing protective clothing, gloves, eye protection etc.
z Eye wash facility should be available as “stand-alone” facility or attached
to sink. Portable, sealed, refillable bottles should also be available.
z Biohazard symbol should be used on all container/equipment containing
biohazardous material.
z Laboratories should ensure proper preservation and security of specimens.

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Biochemistry z Destruction/disposal of hazardous material should be authorized, supervised

and handled according to standard procedures.
z Laboratory personnel should be thoroughly trained in managing fire, and
nonfire emergencies such as large spillage, gas leakage etc.
z Adequate fire extinguishers should be readily available in the laboratory
z Periodic checking of all safety equipment and accessories should be
Notes ensured.
z Accident/incident/injuries record of laboratory personnel should be
maintained and reported to the designated authority. The report should
include description of the event, factors contributing to the event and
information on first aid or other health care provided. This information can
be analyzed periodically towards effectively controlling and preventing
future events. The records should be checked periodically by the laboratory
safety officer even in the absence of fresh entries.

INTEXT QUESTIONS 13.1

1. Fill in the blanks:
1. Clinical applications of ALT assays are mainly used for the evaluation
of .................. disorders.
2. The normal value of a-amylase in blood is ..................
3. Bilirubin is a byproduct of the normal breakdown of ..................
4. Urea is a waste product that is created by .................. metabolism.
5. .................. is a by-product of muscle energy metabolism that is
similar to urea and is filtered from the blood by the kidneys.

TERMINAL QUESTIONS
1. Name some clinically important enzymes?
2. What is the normal value of ALT and AST?
3. What is Bilirubin?
4. What is Urea clearance test?
5. What is the normal range of creatinine in blood?

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ANSWERS TO INTEXT QUESTIONS

13.1
1. hepatic
2. 28-100U/L
3. red blood cells Notes

4. protein
5. creatinine

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14
Notes
BODY WATER, OSMOLARITY
AND IONIC COMPOSITION OF
BODY FLUIDS

14.1 INTRODUCTION
Water is the solvent of life. It bathes our cells, dissolves and transports
compounds in the blood, provides a medium for movement of molecules into
and throughout cellular compartments, separates charged molecules, dissipates
heat, and participates in chemical reactions. In spite of the variation in the
amount of water we ingest each day and produce from metabolism, our body
maintains a nearly constant amount of water that is approximately 60% of our
body weight.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the fluid component in the body
z explain ionic composition of body fluids
z explain osmolarity and osmolality
z describe the buffer systems of human body

14.2 FLUID COMPARTMENTS IN THE BODY

Total body water is roughly 50 to 60% of body weight in adults and 75% of body
weight in children. Because fat has relatively little water associated with it, obese

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people tend to have a lower percentage of body water than thin people, women Biochemistry
tend to have a lower percentage than men, and older people have a lower
percentage than younger people.

Total body water is approximately around 42L. Approximately 40% (1/3rd) of

the total body water is intracellular fluid (ICF) and 60% (2/3rd) is extracellular
fluid (ECF).
Notes
ECF : The extracellular fluid includes

(i) Plasma: fluid part of blood after the blood cells have been removed. It
constitutes about 25% of ECF.

(ii) Interstitial water: the fluid in the tissue spaces, lying between cells and
also includes lymph. It constitutes about 75 % of ECF..

(iii) Transcellular water : a small, specialized portion of extracellular water that

includes gastrointestinal secretions, urine, sweat.

42 L

Extracellular Fluid Volume (ECF) Intracellular Fluid (ICF)

1/3 of TBW 2/3 of TBW
14 L 28 L

Interstitial Fluid Plasma Transcellular Fluid

3/4 of ECF 1/4 of ECF 0.5 L
10.5 L 3. L

Fig. 14.1: Distribution of body fluid compartments

14.3 IONIC COMPOSITIN OF BODY FLUIDS

The ions are unequally distributed between the ECF and ICF compartments.

ECF
Major cation: Na+
Major anion: Cl- and HCO3-

ICF
Major cation: K+
Major anion: Inorganic Phosphates

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Biochemistry Table 14.1: Concentration of ions in body fluids

Ions mmol/L ECF ICF
Cations
Na+ 145 12
K+ 4 150
Notes Anions
Cl– 105 5
HCO3– 25 12
Inorganic 2 100
Phosphates

14.4 OSMOLARITY AND OSMOLALITY

The number of molecules of a particular substance per unit volume of a particular
body fluid is expressed in moles or osmoles.

A mole is the gram molecular weight of a substance i.e. the molecular weight
of the substance in grams.

Osmolarity: The number of osmoles per litre of solution is called osmolarity.

Osmolality: The number of osmoles per kilogram of solvent is called osmolality

14.4.1 Body Fluid Osmolality

Normally, the osmolality of the ECF and ICF are at equilibrium in spite of
difference in composition of electrolytes. The osmotic equilibrium between ECF
and ICF is maintained by easy transfer of water between the compartments.
Plasma osmolality can be easily measured which reflects the osmolality of ECF
and ICF.

Normal Plasma osmolality ranges between 280 to 295 mosm/kg H2O. Sodium
and Chloride contributes to 90% of plasma osmolality and the rest is by glucose,
urea, proteins and other ions. Chloride movement from one compartment to
other always follows the movement of sodium. Therefore, it is mainly sodium
that maintains the osmolality.

Hence Plasma osmolality is calculated as,

Plasma osmolality (mmol/kg) = 2 × Plasma Na+ (mmol/L)

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Biochemistry
14.5 TONICITY OF FLUID
Isotonic fluid
Isotonic fluid is fluid having the same osmolality as that of plasma. Isotonic
fluid do not change the volume of cells. Examples: 0.9% NaCl, 5% glucose, 10%
mannitol, 20% urea.
Notes
Hypotonic fluid
Hypotonic fluid is fluid having osmolality less than that of plasma. The cells
swell when placed in hypotonic solution.

Hypertonic fluid
Hypertonic fluid is fluid having osmolality more than that of plasma. The cells
shink when placed in hypertonic solution.

14.6 DISORDERS OF FLUID VOLUME

Iso-osmotic dehydration
This occurs during loss of isotonic fluid from the body. Examples are diarrhea,
vomiting, hemorrhage

Hypo-osmotic dehydration
This occurs during loss of salt in excess of water from the body. It’s seen in
Adrenocortical insufficiency.

Hyperosmotic dehydration
This occurs during loss of water in excess of solutes from the body. It’s seen in
Diabetes insipidus, excessive sweating.

Iso-osmotic volume expansion

This occurs during excessive infusion of isotonic fluid into the body.

Hypo-oosmotic volume expansion

This occurs during excessive water gain into the body. It’s seen during ingestion
of large volume of water.

Hyperosmotic volume expansion

This occurs during excessive infusion of hypertonic saline.

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INTEXT QUESTIONS 14.1

1. Short notes
1. Classify the body fluid comparments
2. Write down the ionic composition of different fluid compartment in
Notes the body.
3. Define osmolality and give normal value of plasma osmolality. Give
examples of isotonic, hypotonic and hypertonic fluid.
2. Fill in the blanks.
1. Plasma constitutes …………… percentage of ECF.
2. Major cation present in the ECF is…………………..
3. Major cation present in TCF is ………………
4. Plasma osmolality is determined by …………… ion.
5. 20% urea is an example of ………………… fluid.
3. State if the following statements are true or false.
1. 0.9% saline is hypotonic fluid.
2. Cells swell when placed in hypotonic solution.
3. Diarrhoea causes hypotonic dehydration.
4. K+ ion is the major ion contributing to plasma osmolality.
5. 60% of body fluid is comprised by ECF.

14.7 BUFFER SYSTEM IN BLOOD, ROLE OF LUNGS AND

KIDNEYS IN ACID-BASE BALANCE
Acid – Base homeostasis is essential to the body. Many enzymatic activities and
metabolic processes need an optimal pH. Hence, any disturbance in the acid-
base balance leads to derangement in the vital functions of the body.

14.7.1 Definitions
Acid: chemical substance that can donate H+
Base: chemical substance that can accept H+
Examples : Strong acids (e.g. HCl, H2SO4)
Strong bases (e.g. NaOH, KOH)
Weak acids (e.g. H2CO3, CH3COOH)
Weak bases (e.g. NH3)

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pH: The pH of the solution is defined as the negative logarithm of the hydrogen Biochemistry
ion concentration in the solution.
pK and Ka : The pK is defined as the negative logarithm of the ionization
constant of an acid (Ka) in a solution. pK is the pH at which concentration of
acid and its conjugate base are in equal proportion.
Buffer: An aqueous solution of weak acid & its conjugate base or a weak base
& its conjugate acid, that resist changes in pH to addition or removal of small Notes
amounts of acids/ bases.
HB ↔ H+ + B-
The efficiency of a buffer is given by Henderson-Hasselbach equation:
[Anion]
pH = pKa + log
[Acid]

14.7.2 Concept of Acid–Base Balance

Normal pH of the body is maintained over a very narrow range of 7.35 to 7.45.
Acids are continuously produced in the body. But there are mechanisms that
buffer the acids.
Acidosis : decreased pH.
Alkalosis: increased pH.
Volatile acid: Carbonic acid (H2CO3) à dissociates into CO2: excreted by lungs
Non-volatile/ fixed acids: H2SO4, H3PO4 & organic acids; excreted exclusively
by kidneys
Acid load in the body :
z volatile acids
z non volatile acids ingested in diet
z non volatile acids produced from metabolism
z Bicarbonate excreted in feces also contributes to acid load
This acid load is excreted by both lungs and kidneys to maintain the homeostasis.
The reabsorption of the bicarbonate which is filtered by the kidneys also
corresponds to the acid – base balance, apart from the excretion of the acid load.

14.7.3 Buffer Systems in the Body

There are four buffer systems in the body:

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Biochemistry I. Bicarbonate-carbonic acid buffer system

II. Plasma protein buffer system
III. Phosphate buffer system
IV. Hemoglobin buffer system

Bicarbonate-carbonic acid buffer system

Notes
z Most important buffer system in plasma
z In lungs it is involved in removal of acid in the form of CO2
z In kidneys it is involved in reabsorption of bicarbonates.

Plasma protein buffer system

z Accounts for 95 % of non bicarbonate buffer in plasma.
z Mainly is contributed by abumin.

Phosphate buffer system

z Is an important intracellular buffer
z Has organic and inorganic components
z Organic phosphate : ATP, AMP, ADP
z Inorganic phosphate : Disodium hydrogen phosphate, sodium dihydrogen
phosphate

Hemoglobin buffer system

z Major buffer in RBCs, around 85%.
z The rest 15 % is by 2,3- Diphosphoglycerate.
z Histidine residue in Hb is responsible for buffering action.

14.7.4 Role of Lungs in Acid – Base Balance

The respiratory mechanism gets mainly operated by changing the rate and depth
of respiration. The bicarbonate buffer system is the major regulator.

The carbonic acid in the body reacts with H+ and produces CO2..

HCO3- + H+ ⎯⎯→ H2O + CO2

The level of CO2 in the body in turn stimulates the chemoreceptors and increases
ventilation. The increased ventilation causes removal of CO2.

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14.7.5 Role of Kidneys in Acid – Base Balance Biochemistry

Three major mechanisms operate in kidneys:

HCO3– reabsorption
Excretion of H+ as titratable acid (with PO43- buffer)
Excretion of H+ as NH4+
Notes
14.7.5.1 HCO3– reabsorption
z Almost all the filtered of HCO3- reabsorbed (4320 mEq per day)
„ 80% in Proximal Convoluted Tubule
„ 10% in Thick Ascending Limb
„ 6% in Distal Convoluted Tubule
„ 4% in Collecting Duct
z Mechanisms: different in different sites

14.7.5.1.1 HCO3– reabsorption In PCT and TAL

1. H+ secretion in the apical membrane using:
(a) Na+ - H+ exchanger/ antiporter (NHE3):
„ predominant pathway
„ a form of secondary active transport
(b) H+-ATPase: a form of active transport
–
2. HCO3 exit at the basolateral membrane using:
(a) Na+-HCO3– cotransporter (NBC1)
(b) Cl–-HCO3– antiporter

Fig. 14.2: Bicarconate reabsorption in PCT

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Biochemistry 14.7.5.1.2 HCO3– reabsorption In DCT and CD

1. H+ secretion in the apical membrane:
(a) H+-ATPase: a form of active transport
(b) H+/K+-ATPase: similar to that found in gastric parietal cell
2. HCO3– exit at the basolateral membrane:
(a) by Cl– - HCO3– antiporter (AE-1)
Notes

Fig. 14.3: Bicarconate reabsorption in DCT and CD

14.7.5.2 Role of phosphate buffer

H+ excreted in urine binds with phosphate buffer (HPO4-)and gets excreted as
H2PO4-. There is formation of new bicarbonate.

Fig. 14.4: Role of phosphate buffer

*There is no new bicarbonate formation in bicarbonate buffer system.

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Titratable acid Biochemistry

z amount of base to be added to a sample of urine to bring its pH to that of

plasma.
z reflects the amount of H+ excreted by phosphate buffer.

14.7.5.3 Role of ammonium buffer

z NH3 is synthesized in PCT by glutamine metabolism (Ammoniagenesis) Notes

z This NH3 gets excreted in collecting duct as NH4+ by combining with

H+ excreted in urine.
z There is also new bicarbonate formation during the process.

14.7.6 Acid-Base Disorders

14.7.6.1 Metabolic acidosis:

z Decreased pH
z Decreased ECF bicarbonate
z Increased metabolic acid production or reduced acid elimination
z Eg. diabetes mellitus

14.7.6.2 Metabolic alkalosis

z Increased pH
z Increased ECF bicarbonate
z Increased bicarbonate or increased acid elimination
z Eg. Excess vomiting

14.7.6.3 Respiratory acidosis

z Decreased pH
z Decreased CO2 elimination
z Eg. Respiratory diseases

14.7.6.4 Respiratory alkalosis

z Increased pH
z Increased CO2 elimination
z Eg. Hyperventilation

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INTEXT QUESTIONS 14.2

1. Enumerate the role of kidneys in acid- base homeostasis.
2. Write briefly on the buffer systems of the body.
3. Give a brief description on various acid – base disorders.
Notes 4. Role of lungs in acid-base balance.
5. Fill in the blanks
1. The important ECF buffer is ....................
2. Phosphate is an important .................... buffer.
3. New bicarbonate formation occurs in .................... & ....................
buffer.
4. Normal pH of the body is ....................
5. Titrable acid is contributed by .................... buffer.

WHAT HAVE YOU LEARNT

z Water is solvent of life. It bathes our cells, dissolves and transposrts
compounds in the blood, provides a medium for movement of molecules
into and throughout cellular components, separates charged molecules,
dissipates heat
z Total body weight is roughly 50 to 60% of body weight in adults and 75%
of body weights in children
z Total body water is present as Extracellular and Intracellular fluids
z Extracellular fluids are plasma, Interstitial water and Transcellular water
z Osmolarity is the number of osmoles per litre of solution
z Osmolality is the number of osmoles per kilogram of solvent
z Normality, the osmolality of the ECF and ICF are at equilibrium in spite
of difference in composition of electrolytes
z Based on the Osmolality fluids are classified as Isotonic, Hypotonic and
Hypertonic
z Acid-Base homeostasis is essential to the body. Many enzymatic activities
and metabolic processes need an optimal pH.

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z pH of a solution is the negative logarithm of the hydrogen ion concentration Biochemistry
in the solution
z pK is the negative logarithm of the ionization constant of an acid in a
solution
z Buffer is an aqueous solution of weak acid and its conjugate base or a weak
base and its conjugate acid, that resist changes in pH to addition or removal
of small amounts of acids/bases Notes
z Bicarbonate-carbonate acid buffer system, Plasma protein buffer system,
Phosphte byffer system and Hemoglobin buffer system are the buffer sytems
in the body

ANSWERS TO INTEXT QUESTIONS

14.1
2. 1. 25%
2. Sodium (Na+)
3. Potassium (K+)
4. Sodium
5. Isotonic Fluid
3. 1. False
2. True
3. False
4. False
5. True

14.2
5. 1. Bicarbonate carbonic acid buffer
2. Intracellular
3. Phosphate and Ammonium
4. 7.35-7.45
5. Phosphate

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Biochemistry

15
Notes
NUTRITION

15.1 INTRODUCTION
Nutrition is the requirement of the body from external sources to sustain life. The
requirement comprises of foods that provide energy i.e. carbohydrates, fats,
proteins, provide the building blocks such as proteins and amino acids or act as
the functional molecules such as micro nutrients and vitamins acting as co
enzymes and carrier molecules.

OBJECTIVES
After reading this lesson, you will be able to:
z define nutrition. basal metabolic rate
z explain factors affecting basal metabolic rate
z describe the nutritional aspects of carbohydrates proteins and fats
z enumerate the various essential amino acids
z explain the composition of food and what constitutes a balanced diet
z describe the causes, symptoms and treatment of protein energy malnutrition
disease such as kwashiorkor and marasmus.

15.2 NUTRITION
As explained earlier nutrition is the requirement of the body from external sources
to sustain life. Nutrition consists of intake of food that provides both macro as
well as micro nutrients in the correct amount.
A reduced, unbalanced or excessive intake of nutrients can result in various health
related problems. A reduced intake can cause deficiency disorders such as vitamin
deficiency disorders

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Biochemistry
i.e scurvy, beri beri etc, or protein energy malnutrition disorders such as
kwashiorkor, marasmus. Similarly over nutrition in general or of a certain
nutrient can cause various diseases. Excessive intake of Vitamins can cause
hypervitaminosis. Excessive intake of calories by any mean can cause obesity and
associated disorders such as Diabetes mellitus, cardio vascular disease etc.
Hence we should have a complete knowledge of the nutritional requirement of
the human body to prevent these disorders of over and under nutrition. Notes

15.3 BASAL METABOLIC RATE (BMR)

Basal metabolic rate is the energy requirement of the body when the body as well
as the brain is at rest i.e. Basal condition. It is the energy requirement of the body
for its life sustaining processes such as respiration, circulation, peristalsis,
maintenance of body temperature etc. It is expressed in K cal of energy consumed
per hour by the person for ever square meter of the body’s surface area. Its unit
hence is Kcal/ hour/m2.
BMR is calculated by measuring the consumption of oxygen by a person who
is awake but at complete rest and at a comfortable room temperature. The oxygen
consumption gives a good approximation of energy metabolism in our body. The
oxygen consumption for 10 minutes is measured and then multiplied by 6 to get
the oxygen use for an hour. This is done for 24 hours. For each litre of oxygen
consumed it is calculated that and approximate amount of 4.825 Kcal of energy
is used.
The body surface area can be calculated by the Du Bois formulae:

Area (m 2 ) =
{Height (cm) 0.725 × Weight (kg) 0.425 × 71.
10,000

Average BMR of an adult male is 40 Kcal / Hr/ sq m.

Average BMR of an Adult female is 36 Kcal/ Hr/ sq m.
BMR is not a static property of the body. It is dependent on various physiological
and pathological states.
BMR is higher in ‘hypermetabolic’ states such as in pregnancy, fever etc. It is
also higher in growing age groups such as children and adolescents. Athletes and
individuals with higher muscle mass also have significantly higher BMR. So BMR
is affected by age, sex, activity level, body mass and various pathological and
physiological states.

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15.4 THERMOGENESIS
It is the process of heat production in the body to maintain the body temperature
in warm blooded animals. It is done through various methods. Thermogenesis
can be done by muscle movements. A proportion of the energy produced to
support muscle action is leaked from the electron transport chain and results in
thermogenesis. Similarly shivering also results in thermogenesis by this mechanism.
Notes Brown adipose tissue also plays an important role in thermogensis. It has a unique
protein that uncouples the electron transport chain resulting in leaking of
electrons from the chain. In this case the energy is lost ion form of heat instead
of being stored as ATP.

15.5 NUTRITIONAL ASPECTS OF CARBOHYDRATES

Carbohydrates are the most important sources of energy. Even though their
calorific value at 4 Kcal/g is less than half of fats they form the chief source of
energy in our body because they are easiest to digest also the most abundant and
cheap source of energy.
Brain uses glucose exclusively as a source of energy. Almost 20 to 25% of our
total energy intake is used by the brain and almost 50% of our carbohydrate
intake.
The main forms of carbohydrate that we consume are:
1. Starch: It is the form of carbohydrate we consume the most and hence forms
the most important source of energy in our body. It is present in all food
grains, pulses, tuberous vegetables etc.
2. Sugars: Sugar is a term generally used for disaccharides and monosaccharides.
They are present in fruits and vegetables in form of glucose, fructose,
sucrose. It is also found in milk in form of lactose. Glucose and fructose
are examples of naturally occurring monosaccharides. Lactose, Sucrose,
Maltose are naturally occurring disaccharides.
3. Cellulose: Cellulose is a polysaccharide which is present in the structural
elements of plants. It cannot be digested by humans but it plays a very
important role as roughage. Roughage provides bulk to our food. The bulk
helps in the movement of food through our digestive system and also has
a role to play in digestion. It traps water in the large intestine and helps in
its absorption.
Carbohydrates have a protein sparing effect which means that when adequate
carbohydrates are taken in diet the amino acids in diet are spared from energy
metabolism and used for synthesis of proteins in the body.

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All carbohydrates required by our body from other sources hence there are no Biochemistry
essential amino acids.

15.6 NUTRITIONAL ASPECTS OF LIPIDS

Lipids play many roles in our body. They act as a storage of energy, structural
elements of cells and tissues. They act as hormones and vitamins. As a source
of energy it has very high calorific value 9kcal/g. Notes
Lipids as a group is composed of hydrophobic non polar organic compounds that
are insoluble in water and soluble in organic solvents. They are composed of
trigycerides (fats and oils), cholesterol, cholesterol esters.
Lipids can be divided in to saturated, mono unsaturated and poly unsaturated
lipids based on the number of double bonds present in the fatty acid. All poly
unsaturated fatty acids cannot be synthesized in our body hence certain lipids that
have essential fatty acids need to be present in our diet to prevent deficiency
disease. Essential fatty acids are alpha-linolenic acid, linoleic acid and
arachidonic acid. These essential fatty acids should make up for at least 3% of
the energy requirement in adults and 5% to 6% of energy requirements in
children. These essential fatty acids are present in plant oils rich in poly
unsaturated fatty acids. Fish oil is also a good source of poly unsaturated fatty
acids.
Our diet should be rich in poly unsaturated fatty acids and low in saturated fatty
acids as saturated fatty acids are linked to atherosclerosis and cardiac disorders.
Lipids also act as carriers of fat soluble vitamins. Certain fats also act as hormones
such as steroid hormones.
Cholesterol is also an important component of our diet. It is responsible for the
structural integrity of the cell membrane and hence some amount needs to be
taken exogenously. The yolk of an egg is a rich source of cholesterol. But
excessive cholesterol intake has been linked to atherosclerosis and cardiovascular
disorders. The RDA for cholesterol intake in adults and children above four years
of age is 300 mg / day.

15.7 NUTRITIONAL ASPECTS OF PROTEINS

Proteins form the main building blocks of our body both functionally and
structurally. Almost all of our body is composed of proteins. Our muscles, bones,
cartilages, blood vessels, connective tissues, skin, hair, nails etc are all mainly
composed of proteins. Apart from these almost all functional elements of our
body are also composed of proteins such as the actin myosin involved in

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Biochemistry movement, the enzymes responsible for all metabolic reactions in our body, the
hormones, the cellular channels, the receptors for various signaling molecules
etc. In cases of energy deprivation proteins can also be broken down as a source
of energy. They have a calorific value similar to carbohydrates, i.e. 4 Kcal /g, but
their metabolism is much less efficient than that of carbohydrates.
Proteins are composed of amino acids. They are polymers of the 20 amino acids
Notes that exist in our body. Of these 11 amino acids can be synthesized in the body
from non protein sources and hence are deemed non essential in our diet. The
rest 9 amino acids cannot be synthesized in the body and hence need to be taken
in food to prevent any deficieny disorder from occurring. They are histidine,
isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and
valine.
Not all proteins are nutritionally equivalent when it comes to food. Certain
proteins such as keratin undergoes almost nil digestion while proteins like
albumin present in the egg undergo almost complete digestion. Hence the
nutritional quality of proteins has to be decided depending upon the balanced
presence of all essential amino acids, their digestibility (% of protein ingested
digested known as digestibility coefficient, DC), and the biological value, BV
(which is the percentage of protein retained by the body after digestion and
absorption).
Net protein utilization (NPU) is a measure of the utilization of ingested protein
for protein synthesis inside the body. It is calculated by:

(DC × BV)
NPU =
100

Animal proteins are superior biologically as they contain all essential amino acids
in the needed amount and are better digested and absorbed. They have a high
biological value and are termed as first class proteins. While vegetable proteins
have a low biological value and are termed as second class proteins as they are
generally deficient in one amino acid or the other and they are also more difficult
to digest and absorb. Whole egg protein is taken as a reference protein due to
its excellent bioavailability and completeness as far as amino acids are concerned.
This short coming of plant proteins can be overcome are mutual supplementation
i.e. taking two kinds of plant proteins together. A perfect example is rice and
pulses. This results in a complete protein diet being taken by using two proteins
that are deficient in two different amino acids.
Protein requirement is higher in growing years as well as in hyper metabolic states
such as pregnancy, fever, infection etc. Protein requirement is expressed in

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relation to body weight. In infants, upto 3 months of age, the protein requirement Biochemistry
is about 2.5 gm per kg body weight. Then post this the protein requirement is
between 2 to 1.5 gm/ kg body weight uptill adolescence. In adulthood, after 18
yrs of age, the protein requirement in about 1gm/ kg body weight. During
pregnancy and lactation the requirement is higher nearly 2.5 to 2 gm / kg body
weight.

Notes
15.8 BALANCED DIET
A balanced diet is a diet that provides all the nutrients in their correct proportion
so as to fulfill the biological requirement of an individual. It neither creates a
deficiency nor an excess of any of the nutrients. Balanced diet is different for
different individuals and is dependent on the biological need of an individual. For
example the biological demand for carbohydrates fats proteins as well of the
vitamins required for their metabolism will be much higher in an athlete than in
a person with a sedentary desk job.

A balanced diet should contain

(a) Cereals
(b) Pulses
(c) Vegetables and Fruits
(d) Milk and milk products
(e) Oils and fats
(f) Sugars
(g) In cases of non vegetarians it should also contain animal proteins and eggs
on a regular bases.

A balanced diet should contain all the above said food groups in our daily diet
in a balanced manner so that all the nutritional requirements of the body are
fulfilled.

The cereals are energy rich and form a major source of carbohydrates in our diet.
Their energy yield is about 350 kcal/100g.

They consist of staples such as wheat, rice, maize, sorghum, jowar, bajra etc. The
cereals are deficient in proteins and the proteins present in them are generally
deficient in one amino acid or another.

They contain adequate minerals but the absorption of minerals from them is
inefficient.

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Biochemistry Their outer covering is rich in vitamin B complex. But in most cases the outer
covering is lost due to milling.
Pulses are a rich source of proteins among vegetarian food. But similar to other
vegetable proteins they are deficient in certain amino acids but these deficiencies
can be overcome by supplementing them with cereals. They have a energy density
of about 350Kcal/100 gm and 20% to 25% of their mass is protein. Germinating
pulses are an important source of vitamin C and vitamin B complex.
Notes
Vegetables and fruits are the most important source of water soluble and and
certain fat soluble vitamins in our diet. Apart from that they are also an important
source of fiber. They provide minerals and carbohydrates. Their contribution to
proteins in our diet is negligible.
Dairy and its products form almost a complete diet. It contains almost all
nutrients other than vitamin C and Iron. It is a specially good source of Calcium.
Milk is rich in saturated fats.
The protein content of milk can be increased by converting to curd. Also curd
can be eaten by those who are lactose intolerant as during the fermentation
process most of the lactose is fermented.
Fats and oils form an essential part of our diet. They are obtained from the fats
and oils we add to our food during cooking. Other than these lipids they are
obtained also from lipids contained in food items such as dairy and its products,
lipids present in cereals and pulses and fats present in meats and eggs. We should
try to avoid saturated fats in our diet and include unsaturated lipids in form of
oils in our diet. Refined sugars are used as flavoring agents in our diet but excess
of these sugars should be avoided as they provide empty calories.

Fig. 15.1: Food pyramid showing the servings of various

food items in a balanced diet

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Notes

Fig. 15.2: This pie graph shows the percentage of energy that should be
provided by the various macro nutrients in a balanced diet.

Eggs are an excellent source of protein and cholesterol in our diet. An average
egg is about 100 k cal in energy value. Similarly non vegetarian diet such as meat,
poultry and fish are an excellent source of high quality proteins. They are also
a good source of fat soluble vitamins as well as vitamins of the B complex except
for vitamin C.

A balanced diet should take care of the energy and nutrient requirement of the
body and these should be provided by a mix of food items.

The energy requirement of an individual has to provide for the energy

requirement for the various activities as well as growth and repair of wear and
tear of the body. Hence the energy requirement is dependent on the sex, built
as well as the life style of an individual. The following are the average energy
requirements for various population groups

Group Particulars Body Wt.kg Net EnergyKcal

Man Sedentary 60 2320
Moderate ” 2730
Heavy work ” 3490
Woman Sedentary 55 1900
Moderate ” 2230
Heavy work ” 2850
Pregnancy 55kg + GWG +350
Lactation (0-6 m) 55kg + WG +600
Lactation (6-12 m) ” +520

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Biochemistry Daily energy requirement for an average Indian man and woman as per ICMR
guideline (2010)
Energy requirement of infants and children according to ICMR (2010)
Group Age Weight Energy requirement (Kcal)
Infants 0-6 months 5.4 500
Notes
6-12 months 8.4 670
Children 1-3 y 12.9 1060
4-6 y 18.1 1350
7-9 y 25.1 1690
Boys 10-12 y 34.3 2190
Girls 10-12 y 35.0 2010
Boys 13-15 y 47.6 2750
Girls 13-15 y 46.6 2330
Boys 16-17 y 55.4 3020
Girls 16-17 y 52.1 2440

15.9 PROTEIN ENERGY MALNUTRITION

Protein energy malnutrition is generally seen in children being weaned off
mother’s milk. It presents as specific syndromes depending on whether the
protein deficiency is more severe or carbohydrate deficiency.

(a) Kwashiorkor: It is a disorder seen in children weaned off milk onto a diet
that is deficient in proteins but adequate in calories. It results in the child
developing a protein deficiency. This results in muscle wasting and retarded
growth. Patchy areas of hyper and hypo pigmented skin are seen and also
brittle hypo pigmented hair with flagging sign.
A classical feature of kwashiorkor is a child with a distended belly and
edema. The distended belly and edema are due to loss of plasma oncotic
pressure and may completely mask muscle wasting. This loss is seen due
to loss of plasma albumin. Fatty liver caused by deficiency of apo-
lipoproteins also causes distention of abdomen. Also there is anaemia
caused by decreased globin synthesis.

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Notes

Fig. 15.3: A classical picture of kwashiorkor

Kwashiorkor can be easily prevented by adding protein rich food to the

diet of the child being weaned off mother’s milk. This can be done by add
undiluted animal milk, eggs, pulses, groundnuts, bengal gram, cotton seed
flour etc.
(b) Marasmus is a disorder of both protein as well as calorie intake. Because
of deficiency in calorie intake most of the protein in diet is used for energy.
There is gross muscle wasting with loss of subcutaneous fat. The child
shows growth retardation.
There are frequent infections that put a further stress on the metabolism.
Diarrhea results in loss of further nutrients.

Fig. 15.4: A marasmic child

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Biochemistry A marasmic child

Marasmus can be corrected by :
1. Increasing protein and energy intake.
2. Supplementing Vitamins and minerals.
3. Correcting dehydration.
Notes 4. Correcting the infections.

TERMINAL QUESTIONS
1. What is nutrition?
2. What is basal metabolic rate (BMR)?
3. How is Basal metabolic rate calculated?
4. What are the various factors that influence BMR?
5. Define thermogenesis. How does it happen?
6. What are nutritional aspects of carbohydrates?
7. What is the importance of roughage in diet?
8. What is the importance of protein in diet?
9. What are the essential amino acids? Enumerate the essential amino acids
10. What does the Bioavailability of a protein mean. How is it sued to calculate
net protein utilization?
11. What is the nutritional importance of lipids?
12. What are essential fatty acids?
13. Define balanced diet. What are the factors that determine the balanced diet
for an individual.
14. What is Kwashioko? What are its symptoms and how can it be prevented.
15. What is marasmus? What are its symptoms and how can it be corrected.

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Biochemistry

16
Notes
KIDNEY FUNCTION TEST

16.1 INTRODUCTION
The main function of the kidney is excretion of water soluble waste products
from our body. The kidney has various filtration, excretion and secretary
functions. Derangement of any of these function would result in either decreased
excretion of waste products and hence their accumulation in the body or loss
of some vital nutrient from the body. Based on the level of these excretory
products and nutrients in the urine as well as in blood we can make an accurate
calculation to decipher the efficieny of the kidney to undertake its various
functions.

OBJECTIVES
After reading this lesson, you will be able to:
z explain the importance of kidney function test.
z describe the types of lesions detected by the renal function tests.
z describe the various components of the kidney function test.
z explain the importance of various components of the kidney function test.

16.2 THE FUNCTIONAL COMPONENTS OF A KIDNEY

The functional unit of the kidney is called a nephron. It consists of two main
parts, the glomerulus and the tubular system.
The glomerulus is composed of a bowman’s capsule and a tuft of leaky blood
vessels encapsulated by the bowman’s capsule. The primary purpose of the
glomerulus is filtrations. The leaky vessels filter into the glomerulus almost all
the water, electrolytes, small proteins, nutrients such as sugar etc and excretory

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Biochemistry products such as urea etc. The filtrations is dependent on the size and charge
of the particles. The average pore size is 8 nm hence particles of only smaller
size will pass through. Also the basement membrane carries a negative charge
hence preventing negatively charged particles from passing through.
The Tubular system is responsible for re absorption of most of the water,
electrolytes, nutrients as well as excretion of the remaining nutrients by means
of secretion into the tubules. These tubules are responsible for the concentration
Notes of urine.

Fig. 16.1: A nephron showing the glomerulus as well as the tubules

16.3 COMPONENTS OF KIDNEY FUNCTION TEST

The components of the Kidney function test can be broadly divided into two
categories.

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Notes

Fig. 16.2
The tests that are part of the Kidney Function test panel are:
(a) Urine examination
(b) Serum Urea
(c) Serum creatinine
(d) Blood urea nitrogen (BUN)
(e) Calcium
(f) Phosphorus
(g) Protein
(h) Albumin
(i) Creatinine clearance
(j) Urea clearance
(k) Inulin clearance
(l) Dilution and Concentration test
(l) Serum electrolyte levels

16.4 URINE EXAMINATION

Before we do a quantitative examination of urine a qualitative examination is
necessary as it can provide excellent clues to the nature and location of the lesion
in the renal system.
This examination consists of a physical examination where the colour, odour,
quantity, specifc gravity etc of the urine is noted. Microscopic examination of
urine is done to rule out any pus cells, Rbc casts, Crystals.

16.5 SERUM UREA

Urea is the end product of protein catabolism. The urea is produced from the
amino group of the amino acids and is produced in the liver by means of the
Urea cycle.

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Biochemistry Urea undergoes filtrations at the glomerulus as well as secretion and re

absorption at the tubular level. The rise in the level of serum urea is generally
seen as a marker of renal dysfunction specially glomerular dysfunction. Urea
level only rises when the glomerular function is reduced below 50%.
The normal serum urea level is between 20-45 mg/dl. But the level may also
be affected by diet as well as certain non kidney related disorders. A high protein
Notes diet may increase the blood urea level. Similarly a low protein diet may decrease
blood urea level. Other causes of protein catabolism such as any hyper metabolic
conditions, starvation etc also cause increased blood urea levels. Similarly the
level of urea may also be decreased in case of hepatic injury.
So even though blood urea is not an excellent marker of renal dysfunction as
it rises quite late in the dysfunction and its rise is also not exclusive to kidney
dysfunction, but for practical purposes serum urea level is still one of the most
ordered test and forms an important part of the kidney function test.
Urea is measured in diagnostic labs either by UV kinetic method using á keto
glutarate as an NH3+ acceptor in presence of enzyme glutamate dehydrogenase.
It is also measured calorimetrically by Berthelot’s end point method and is read
in visible range using a calorimeter.

16.6 BLOOD UREA NITROGEN (BUN)

Sometimes the Serum urea level is expressed as blood urea nitrogen. BUN can
be easily calculated from the serum urea level. The molecular weight of urea is
60 and it contains two nitrogen atoms of combined atomic weight of 28. Hence
the contribution of nitrogen to the total weight of urea in serum is 28/60 that
is equal to 0.47. Hence the serum urea levels can be easily converted to BUN
by multiplying it by 0.47.
A rise in blood nitrogen level is known as azotemia.

16.7 CALCIUM
This test measures the amount of Calcium in your blood, not the calcium in your
bones. The body needs it to build and fix bones and teeth, help nerves work,
make muscles contraction, help blood clot, and help the heart to work. The
Calcium test screens for problems with the parathyroid glands or kidneys, certain
types of cancers and bone problems, inflammation of the pancreas (pancreatitis),
and kidney stones.
Normal Results: 8.5 to 10.2 mg/dl

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Biochemistry
16.8 PHOSPHORUS
Phosphorus is a mineral that makes up 1% of a person’s total body weight. The
body needs phosphorus to build and repair bones and teeth, help nerves function,
and make muscles contract. The Kidneys help control the amount of phosphate
in the blood. Extra phosphate is filtered by the kidneys and passes out of the body
in the urine. It plays an important role in the body’s utilization of carbohydrates
and fats and in the synthesis of protein for the growth, maintenance, and repair Notes
of cells and tissues.
High levels of phosphorus in blood only occur in people with severe kidney
disease or severe dysfunction of their calcium regulation. Excessively high
levels of phosphorus in the blood, although rare, can combine with calcium to
form deposits in soft tissues such as muscle.
Normal Results: Standard range not available

16.9 PROTEIN
Protein in urine is noticeably increased in renal disease of any etiology, except
obstruction, and is therefore a very sensitive, general screening test for renal
disease, though not specific. The extent of proteinuria also provides useful
information. The greatest degree of proteinuria is found in the nephrotic
syndrome (> 3 - 4 g/day). In renal disease with the nephritic syndrome, the
urinary protein excretion rate is usually about 1 - 2 g/day. In tubulo-interstitial
disease, urine protein is generally less than 1 g/day. Only in the nephrotic
syndrome is the urine protein loss sufficiently great to result in hypoproteinemia.
Protein in serum can generally be maintained at concentrations above the lower
limit of normal by increased hepatic protein synthesis so long as protein loss is
less than about 3 g/day

16.10 SERUM CREATININE LEVEL

Creatine is a small tripeptide found in the muscles. It stays in its phosphorylated
form and releases energy for any burst of muscular activity. It is released from
the muscles during regular wear and tear and is converted to creatinine (its
internal anhydride). It is to be remembered that unlike urea, creatinine is not a
toxic waste. It is simply used as a marker of renal function.
Creatinine is freely filtered at the glomerulus and is also to a very small extent
secreted into the tubules. So any problem with gromerular filtrations has a
significant effect on the excretion of creatinine resulting in a much substantial
rise in serum creatinine level.

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Biochemistry Normal serum creatinine level is 0.6 to 1.5 mg/dl. Serum creatinine is a better
indicator of renal function and more specifically glomerular function than urea.
For a particular individual the creatinine level is dependent on the muscle mass
and muscle wear and tear. There may be significant difference in creatinine level
of individuals with vastly differing muscle mass. For example a body builder
or athlete will have higher creatinine levels than a sedentary desk worker.
Similarly creatinine level will also increase in case of any muscle trauma or
Notes excessive wear and tear as seems in athletes and people involved in hard physical
labor.
Creatinine is most commonly measured in laboratories calorimetrically by
Jaffe’s method.

16.11 UREA CLEARANCE

Urea clearance is the hypothetical amount of blood from which kidney clears
urea in one minute. This is measured by measuring the concentration of urea in
blood, concentration of urea in urine and amount of urine excreted over a one
hour interval.
Urea clearance is less than its glomerular filtration as some of the urea that is
filtered at the glomerulus is reabsorbed at the tubules.
To measure urea clearance first the patient is made to void urine and then the
made to drink two glasses of water. Then the urine is collected after an hour and
a blood specimen is also collected at the same time. Then the patients urine
sample is collected after another hour. The urea level in the two urine samples
and the blood sample is measured. The urine volume is calculated as urine output
per minute.
If the urine output is more than 2 ml/minute then urea clearance (in ml/ minute)
is measured as:

(Urine urea conc. × Urine volume per minute)

Urea conc. in serum
If urine output is less than 2 ml/minute then urea clearance (in ml/min) is
measured as:

(Urine urea conc. × urine volume ml/min)

Urea conc. in serum
Maximum urea clearance of an average individual or body surface area of 1.73
sq m is 75 ml/ min and a standard urea clearance is 54 ml/min. A urea clearance
below 60% of standard is considered impaired.

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16.12 CREATININE CLEARANCE RATE
Creatinine is filtered at the glomerulus and its reabsorption at the tubular level
is insignificant. Because of this creatinine clearance can be used to measure
Glomerular Filtration Rate (GFR).
It is measured over a period of 24 hrs. For this urine is collected over a 24 hour
period and blood sample is also collected. The concentration of creatinine is
measured both in the urine as well as the serum sample. Notes

Creatinine clearance is measured by the following method:

(Conc. of creatinine in urine × volume of urine)

× 1440
Conc. of creatinine in serum
The normal range of creatinine clearance is:
Males : 100 – 120 ml/ min
Females : 95 – 105 ml/min
This is very close to the glomerular filtration rate.

16.13 INULIN CLEARANCE

Inulin is a small polysaccharide of low molecular weight made up of fructose.
To measure glomerular filtrate the substance used should have the following
qualities:
(a) It should be non toxic.
(b) Should not be metabolized in the body.
(c) Should be completely filtered at the glomerulus.
(d) Should neither be secreted or reabsorbed at the tubules.
Inulin meets all these criteria and hence makes for a suitable candidate to
measure GFR. Inulin clearance hence equals to GFR. GFR is the amount of
blood that passes though and is filtered through the glomerulus in a minute.
To measure Inulin clearance first Inulin is introduced in the blood by means of
a slow continuous infusion to maintain a steady conc. of Inulin in the blood. This
is done by first infusing 30 ml of 10% inulin in 250 ml of normal saline infused
at a rate of 20 ml/ min to achieve desired concentration.
Then 70 ml of 10% inulin in 500 ml saline in infused at a rate of 4 ml/ min to
maintain the desired concentration.

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Biochemistry The patient is asked to micturate 20 minutes after the second infusion and the
urine in discarded and the time noted. After exactly 60 minutes, take another
sample of urine and blood is collected. Measure the volume of urine and the
conc. of inulin in both the serum and urine.
Thereafter the inulin clearance is measured by the formulae:
(Conc. of Inulin in urine × volume of Inulin)
Notes Conc. of Inulin in serum
Normal inulin clearance is 120 to 130 ml/minute for an average person with a
body surface area of 1.73 sq m. This is a close approximation of the GFR.
A below normal inulin clearance shows an impaired glomerular function.

16.14 CONCENTRATION TEST

In case of water shortage in the body the kidney is able to concentrate urine and
conserve water. This is done by increasing the reabsoption of water from the
glomerular filtrate at the tubular level. So in effect the measure of the ability of
the kidney to conserve water and concentrate urine is a measure of tubular
function.
For this test the patient is not allowed to take any food or water after the evening
meal. The first three urine samples passed in the morning are collected and their
specific gravity measured. In a normal person the specific gravity of atleast one
of the samples should be above 1.025 or above. If the specific gravity remains
below 1.025 then it is a sign of tubular dysfunction.

16.15 DILUTION TEST

Like the concentration test the dilution test is also a measure of functioning of
the tubules. In cases of fluid overload of our body the tubules reabsorb lesser
amounts of water resulting in excretion of diluted urine.
For this test the subject is put on overnight fast and then in the morning the
subject is made to drink 1200 ml of water over a time period of 30 minutes. Then
the urine samples are collected every hour for 4 hours. The specific gravity of
the samples is measured and atleast one of the samples should have a specific
gravity of 1.003 or less. If none of the samples have the specific gravity of 1.003
or less this is a sign of tubular dysfunction.

16.16 ELECTROLYTES
The purpose of the kidney is not just water balance and excretion but also to
maintain the electrolyte balance of our body. Kidneys actively reabsorb or

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excrete electrolytes to maintain the electrolyte balance of the body. Owing to Biochemistry
their small size almost all electrolytes are filtered at the glomerulus. After
filtration most of the electrolytes are absorbed back at the tubular level but any
problem at the tubular level will result in non absorption and excessive loss of
electrolytes in urine.
Serum electrolytes that are measured for this purpose are:
Serum Sodium levels (Na+) : 135 to 145 mmols/liter Notes

Serum Pottasium level (K+) : 3.5 to 5 mmols/liter

Serum Chloride level (Cl- ) : 95 to 105 mmols/liter

INTEXT QUESTIONS 16.1

1. The functional unit of kidney is ..................
2. Kidney functional test measure .................. & .................. function
3. Urea level rises when the flomerular function is reduced below ..................
4. Normal serum urea level is ..................
5. A rise in blood nitrogen level is known as ..................
6. Normal serum creatinine level is ..................
7. Creatinine is commonly measured in laboratories calorimetrically by ..................
method
8. .................. rate can be used to measure Glomerular Filteration rate

WHAT HAVE YOU LEARNT

z The main function of the kidney is excretion of water soluble waste products
from our body. The kidney has various filtration, excretion and secretary
functions.
z Derangement of any of these function would result in either decreased
excretion of waste products and hence their accumulation in the body or
loss of some vital nutrient from the body.
z The functional unit of the kidney is nephron. It consists of two main parts,
the glomerulus and the tubular system.
z Components of Kidney function test are tests that measure the Glomerular
function and tubular function.

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Biochemistry The tests that are part of the Kidney Function test panel are:
(a) Urine examination
(b) Serum Urea
(c) Serum creatinine
(d) Blood urea nitrogen (BUN)
(e) Urea clearance
Notes
(f) Creatinine clearance
(g) Inulin clearance
(h) Dilution and concentration test
(i) Serum electrolyte levels
z Urine examination consists of a physical examination where the colour,
odour, quantity, specifc gravity etc of the urine is noted.
z Microscopic examination of urine is done to rule out any pus cells, Rbcs,
casts, Crystals.
z Urea is the end product of protein catabolism and Urea undergoes filtrations
at the glomerulus as well as secretion and re absorption at the tubular level.
z The normal serum urea level is between 20-45 mg/dl. But the level may
also be affected by diet as well as certain non kidney related disorders.
z Blood Urea Nitrogen can be easily calculated from the serum urea level and
the serum urea levels can be easily converted to BUN by multiplying it by
0.47.
z Creatine is a small tripeptide found in the muscles and is simply used as
a marker of renal function.
z Normal serum creatinine level is 0.6 to 1.5 mg/dl. Serum creatinine is a
better indicator of renal function and more specifically glomerular function
than urea.
z Creatinine is filtered at the glomerulus and its reabsorption at the tubular
level is insignificant. Because of this creatinine clearance can be used to
measure Glomerular Filtration Rate (GFR).
z The purpose of the kidney is not just water balance and excretion but also
to maintain the electrolyte balance of our body.

TERMINAL QUESTIONS
1. What is the functional unit of the kidney? Draw a diagram showing its
various components.

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2. What is the purpose of a Kidney Function Test? Biochemistry

3. What is urea? How is it excreted by the kidney?

4. What is urea clearance? How is it measured?
5. What is Creatinine? How is its clearance measured?
6. On what conditions other than renal function does the level of creatinine
in blood depend upon?
Notes
7. What is GFR (Glomerular filtration rate)? How is it measured?
8. What are the ideal characteristics of a substance used to measure the GFR?

ANSWERS TO INTEXT QUESTIONS

16.1
1. Kidney
2. Glomerular & Tubular
3. 50%
4. 20-45 mg/dl
5. Azotemia
6. 0.6 - 1.5 mg/dl
7. Jaffe’s
8. Creatinine Clearance

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Biochemistry

17
Notes
LIVER FUNCTION TESTS

17.1 INTRODUCTION
Liver function tests are a group of tests done to assess the functional capacity
of the liver as well as any cellular damage to the liver cells. To assess all
functional capabilities of the liver such as:
(a) Its Synthetic ability: By measuring the various plasma proteins such as
albumin and prothrombin that are synthesized by the liver. Also lipids
which are also synthesized in the liver.
(b) Its secretory/excretory abilities: By measuring the serum billirubin level

OBJECTIVES
After reading this lesson, you will be able to:
z enumerate the various tests undertaken to measure liver function and
damage.
z describe the various tests are read to reach a conclusion as to the type of
damage inflicted on the liver.

17.2 THE COMMON TESTS THAT FORM PART OF THE

LIVER FUNCTION TEST PROFILE
(a) Serum Bilirubin: both conjugated and unconjugated.
(b) Total serum proteins and albumin globulin ratio.
(c) Liver enzymes : Transaminases :AST (SGOT), ALT (SGPT). Others: ALP,
GGT, LDH.
(d) Prothrombin time

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Biochemistry
17.3 SERUM BILIRUBIN
Bilirubin is one of the end products of haem metabolism and is derived from
the haem part of the hemoglobin molecule. It is a yellow coloured pigment.
Liver plays an important role in the metabolism of bilirubin. After the breakdown
of haem portion of the hemoglobin molecule ‘unconjugated bilirubin’ is
insoluble in water. It is transferred from the site of RBC and haem breakdown
such as the spleen to the liver for ‘conjugation’ bound to albumin. At the liver Notes
it is conjugated with glucoronic acid with the help of enzyme glucuronyl
transferase. This conjugation makes bilirubin water soluble and this conjugated
bilirubin is excreted into the bile.
While measuring bilirubin we measure total and conjugated bilirubin (Direct
bilirubin) and calculate the indirect bilirubin by substracting the direct from the
total.
The normal range of bilirubin is:
Total Bilirubin: 0.2 to 1 mg/dl
Unconjugated Bilirubin: 0.1 to 0.6 mg/dl
Conjugated bilirubin: 0.1 to 0.4 mg/dl
A rise of bilirubin level to that of 2 mg/dl results in the symptoms of jaundice
which is marked by deposition of bilirubin in the various mucous membranes.
Jaundice is divided into three types depending on its etiology:
(a) Pre hepatic jaundice: In this case the cause of jaundice is at the level of
bilirubin processing before it reaches the liver. Most common cause is over
production of bilirubin due to hemolytic disorders. In this case the rise in
the level of unconjugated bilirubin is more than conjugated bilirubin hence
there is a rise in total and indirect bilirubin.
(b) Hepatic jaundice: This is caused by cellular dysfunction of the liver hence
is also called hepatocellular jaundice. It is caused by the inability of the
liver cells to process and excrete the bilirubin in the system. It is seen in
hepatitis, cirrhosis of liver etc. In this jaundice there is rise in total, direct
as well as indirect bilirubin levels.
(c) Post hepatic jaundice: This is also known as obstructive jaundice as it is
caused by obstruction to the outflow of bile resulting is reabsoption of
conjugated bilirubin and it making an appearance in the serum. It cause
by carcinoma of the mouth of gall bladder, stone in the bile duct etc. In
this type of jaundice we see a rise in total as well as direct (conjugated)
bilirubin.

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Biochemistry The common method of measuring serum bilirubin level is the Diazo method
using Diazotized sufanilic acid to convert bilirubin into a azobilirubin the color
intensity of which is measured colorimetrically at a wavelength between 555
nm (550 to 580 nm).

The conjugated bilirubin reacts directly with the Diazo sulfanilic acid in an
aqueous medium and hence also called direct bilirubin. For unconjugated or free
Notes bilirubin which is not water soluble we need to dissolve it into DMSO for the
reaction to occur. This method is the ‘indirect method’ for measuring bilirubin
levels and it measures both conjugated and unconjugated bilirubin i.e. total
serum bilirubin.

INTEXT QUESTIONS 17.1

1. Liver function test assesses ..................... of liver
2. The synthetic ability of liver is assessed by measuring .....................
3. The secretory ability is assessed by measuring .....................
4. Indirect bilirubin is calculated by substracting ..................... from .....................
5. Serum bilirubin is commonly measured by ..................... method

17.4 SERUM ALBUMIN AND ALBUMIN GLOBULIN RATIO

Serum albumin is an important serum protein vital for maintaining the plasma
oncotic pressure as well as acts as a carrier for various biological substances and
drugs. Serum albumin is exclusively synthesized by liver and hence the level
of serum albumin gives us a stock of the synthetic ability of the liver.

Another cause of fall of serum albumin maybe protein malnutrition but in that
case the fall of all serum proteins including globulins will be seen.

The normal total protein level is 5 to 8.5 gm/dl. The total serum albumin level
is 3.5 to 5 gm/dl. The total plasma globulin level is calculated by subtracting
the plasma albumin from the total protein level and is normally in the range of
2 to 2.5 gm/dl.

The normal range for albumin : globulin ratio is 1.2 to 1.5. But with hepatic
dysfunction this ratio recedes towards 1 as the synthetic function of liver is
compromised. The reversal of the ratio i.e if the value recedes below 1, it is an
ominous sign and may mark an infective/inflammatory pathology marked by rise
in serum globulin level and fall in serum albumin levels.

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To measure serum albumin the Bromocresol green method is used. Albumin in Biochemistry
the presence of bromocresol green at a slightly acidic pH gives a yellow green
to blue geen colour. The intensity of this colour is dependent on the concentration
of albumin in the sample. This intensity is read at a wavelength of 630 nm.

To measure the total protein content of the sample the biuret method it used. In
this method the cupric ions of copper (II) sulphate, present in the biuret reagent,
form a violet coloured complex with the proteins in a slightly alkaline medium. Notes
The intensity of the colour formed is measured at a wavelength of 540 nm (530
to 550 nm).

INTEXT QUESTIONS 17.2

Match the following
1. Total Bilirubin (a) 5 - 8.5 mg.dl
2. Conjugated Bilirubin (b) Serum albumin
3. Total Protein (c) 0.2 - 1 mg/dl
4. Total serum albumin (d) Total protein
5. Bromocresol green method (e) 0.1 - 0.4 mg/dl
6. Biuret method (f) 3.5 - 5 gm/dl

17.5 PROTHROMBIN TIME

Prothrombin is a clotting factor (clotting factor II) and it forms an important
part of both the intrinsic and extrinsic pathway. Its active form is Thrombin (also
clotting factor IIa). It is a serine peptidase which converts fibrinogen to fibrin.
Prothrombin is synthesized in the liver. And hence prothrombin activity in
plasma is used to measure the synthetic function of liver.
Prothrombin time is measured by taking human plasma from blood that has been
collected in tube containing citrate as an anticoagulant. The plasma in put in
an automated machine which adds an excess of calcium to reverse the
anticoagulant effects of citrates and measures the time taken for fibrinogen to
be converted to fibrin hence measures the activity of thrombin in the plasma.
The prothrombin time differs in accordance to the analytical method used. Hence
to compensate for this International normalized ratio (INR) is used. In this the
manufacturers of the kit assign an International sensitivity index (ISI) value. This
shows the amount of tissue factor present in the kit as against an internationally
accepted standard. The ISI value is generally 1 to 2.

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Biochemistry The following is the method to calculate INR:

⎛ PT test sam ple ⎞

IN R = ⎜ ⎟ ISI
⎝ PT control ⎠
A ratio of 0.8 to 1.2 is considered normal for patients not on warfarin. For
individuals on warfarin for any disorder an INR of 2.0 to 3.0 is the target.
Notes
17.6 LIVER ENZYMES
Liver enzymes along with bilirubin are the most commonly measured parameter
measured in the liver function test. These enzymes are hepatic in origin and they
are leaked into the serum with the destruction of hepatic cells. Liver enzymes
are measured to get an idea of the cellular insult on the liver and are increased
in a wide variety of conditions such as viral hepatitis, toxic hepatitis, cirrhosis
of liver etc.

The commonly measured enzymes are:

(a) Transaminases: AST (SGOT), ALT (SGPT)

(b) Transpeptidases: GGT
(c) Phosphatase: ALP.

(a) Transaminases: They are a group of enzymes that transfer the amino
group from an amino acid to α keto acid converting the α keto acid into
an amino acid while converting the amino acid into a keto acid.
The transaminases that are measured in the liver function test are ALT and
AST.

Alanine transaminase (ALT) catalyses the following reaction:

ALT
Alanine + α keto Glutarate ⎯⎯⎯ → Pyruvate + Glutamate

Aspartate transaminase (AST) catalyses the following reaction:

AST
Aspartate + α keto Glutarate ⎯⎯⎯ → Oxaloacetate + Glutamate

z The normal level of ALT in serum is 7 to 40 IU/L.

z The normal level of AST in serum is 8 to 40 IU/L.
An increase in AST or ALT levels hints at an insult to the liver parenchyma
tissue. ALT is a more specific marker of hepatic injury than AST as AST
elevation is also seen in cardiac tissue injury, haemolysis and muscle tissue.

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To measure the level of transaminases the reaction catalysed by them is Biochemistry
coupled to a reaction in which NADH is used up resulting in change in the
photometric intensity when read in the UV range at 340 nm. It is a UV
kinetic method.
For ALT (SGPT):

ALT
Alanine + α Keto glutarate ⎯⎯⎯ → Pyruvate + Glutamate Notes
LDH
Pyruvate + NADH + H+ ⎯⎯⎯⎯⎯⎯⎯⎯→
(Lactate dehydrogenase)
Lactate + NAD+

For AST (SGOT):

Aspartate + α Keto glutarate ⎯⎯⎯

AST
→ Oxaloacetate + Glutamate

MDH
Oxaloacetate + NADH + H+ ⎯⎯⎯⎯⎯⎯⎯⎯→
(Malate dehydrogenase) Malate + NAD+

(b) Alkaline Phosphatase: It is a hydrolase that removes phosphates from all

kinds of molecules such as proteins, nucleotides etc.
It is found in cells lining the billiary system hence a rise in it level is
indicative of damage to the billiary tree due to cholestasis. It maybe due to
stone blocking the large ducts or intrahepatic obstruction, inflammation of
the biliary channels.
Alkailine phosphatase is also found in placenta and bones. Hence the level
is also increased in growing children in whom bones undergo remodeling
and in Paget’s disease in adults.
Normal level of alkaline phosphatase is between 45 to 115 IU/L.
The method for measuring the level of alkaline phosphatase is a kinetic
method using p-nitrophenylphosphate as substrate for the enzyme and
measuring rate of formation of the colored substrate (p- nitrophenol) formed
from the reaction. This measurement of the color intensity is done
colorimetrically at a wavelength of 405 nm.

p-Nitrophenylphosphate + H2O ⎯⎯⎯ ALP

→ p- Nitrophenol + Phosphate
(c) Gamma glutamyl transpeptidase: It is another enzyme specific to the
biliary tree and a more specific indicator of cholestasis and damage to the
biliary tree. It is also a highly specific marker and is raised in even minute
and subclinical damage to the biliary tree.
Its normal range is in between 0 to 42 IU/l.

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INTEXT QUESTIONS 17.3

Match the following
1. AST (a) 45 - 115 IU/L
2. Alkaline Phosphatase (b) 0 - 42 IU/L
Notes
3. ALT (c) 8 - 40 IU/L
4. Gamma glutamyl transpeptidase (d) 7 - 40 IU/L

WHAT HAVE YOU LEARNT

z Liver function tests are a group of tests done to assess the functional
capacity of the liver
z Synthetic ability of the liver is assessed by measuring the various plasma
proteins
z Secretory/excretory ability is assessed by measuring the serum billirubin
level
z The common tests that form part of the liver function test profile are Serum
Bilirubin both conjugated and unconjugated, Total serum proteins and
albumin globulin ratio, Liver enzymes and Prothrombin time
z While measuring bilirubin we measure total and conjugated bilirubin
(Direct bilirubin) and calculate the indirect bilirubin by substraction the
direct from the total.
z The common method of measuring serum bilirubin level is the Diazo
method
z The normal total protein level is 5 to 8.5 gm/dl. The total serum albumin
level is 3.5 to 5 gm/dl and the normal range for albumin : globulin ratio
is 1.2 to 1.5
z Serum albumin is measured by Bromocresol green method and total protein
is measured by biuret method
z Prothrombin is a clotting factor (clotting factor II) and it forms an important
part of both the intrinsic and extrinsic pathway
z International normalized ratio (INR) is used for measuring the Prothrombin
time
z The commonly measured enzymes are Transaminases: AST (SGOT), ALT
(SGPT), Transpeptidases: GGT, Phosphatase: ALP.

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TERMINAL QUESTIONS
1. What are functional aspects of liver?
2. Write a short note on conjugated and unconjugated bilirubin.
3. Write short note on the laboratory method to measure serum bilirubin level
both direct and indirect. Notes
4. What is the method used to measure albumin in serum?
5. What is the method used to measure total protein in serum?
6. What is the importance of INR and how is it calculated?

ANSWERS TO INTEXT QUESTIONS

17.1
1. Functional capacity
2. Plasma protein
3. Serum bilirubin
4. Direct, total bilirubin
5. Diazo

17.2
1. (c) 2. (e) 3. (a) 4. (f) 5. (b) 6. (d)

17.3
1. (c) 2. (a) 3. (d) 4. (b)

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Biochemistry

18
Notes
SPECTROPHOTOMETRY,
LIGHT EMISSION AND
SCATTERING ANALYTICAL
TECHNIQUE

18.1 INTRODUCTION
Measuring light emission, transmittance and scattering are few of the most
important techniques used in modern biochemistry laboratory. They are used in
spectrophotometer, colorimetry, chemiluminescence, ELISA etc. The ease and
accuracy of measurement of light emission or transmittance makes it a method
of choice in a large number of analytical techniques.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the functional aspects of a colorimeter.

z describe Spectrophotometry.

z explain the functional aspects of chemiluminescence.

z explain the concepts of incident, transmitted, absorbed and scattered light.

z describe the Beer Lambert’s law.

18.2 EXPLAINING THE TERMS USED

As we study further we will be encountering some terms such as incident light,
absorbed light or absorbance, transmitter light or transmittance, scattered light
etc. Here we try to explain the terms:

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(a) Incident light: Incident light refers to the beam of light that is directed at
the cuvette (or reaction vessel) from a light source.
(b) Transmitted light: Transmitted light is the light that passes through the
cuvette to emerge on the other side.
(c) Absorbed light: Absorbance is the term used to describe the light that is
absorbed by the reaction mixture and it depends on the intensity of the color
of a reaction product in the reaction vessel. Notes
Absorbance (A) = Incident light (I) – Transmitted light (T)
In conditions where scatterence is zero.
(d) Scattered light: It is the light that is not absorbed but is scattered or reflected
back by opaque substances in the reaction vessel. This is used to measure
turbidity in reaction such as immuno turbidity reactions.

18.3 COLORIMETRY
Colorimetry is a method employing the measurement of absorbance of a reaction
mixture. It is the simplest method used and is employed by a large number of
instruments such as a colorimeter, semiauto analyzer, auto analyzer. The most
important thing for this method is that the metabolite or chemical we intend to
measure should undergo a reaction in the cuvette to produce a colored product
or there should be certain change in absorbance before and after the reaction as
in cases of UV kinetic methods. The intensity of the color developed or change
in absorbance will depend on the concentration of the metabolite in the sample.
The method is based on passing a beam of light of a certain wavelength through
the reaction vessel or cuvette containing the colored product of the reaction. The
incident light as well as the transmitted light of the particular wavelength is
measured and the absorbance calculated. The absorbance is plotted on a graph
which has been drawn by plotting a curve of absorbance of the reaction mixture
as against a serial dilution of the metabolite or chemical the reaction mixture
contains.
Absorbance numerically is the log of the ratio of incident light upon transmitted
light.
A = log10( I/ T )
This same principle is used in all colorimeters as well as autoanalyzers, semi
auto analyzers. The difference being that in auto analyzers the machine itself
transfers a pre determined amount of the sample and reagents into a reaction
vessel and reads the absorbance and then plots the absorbance on a graph pre
plotted during calibration while in a semi auto analyzer the reagent and sample

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Biochemistry are mixed in a test tube and fed into the semi auto analyzer to be read and the
value is plotted on a pre stored curve plot during calibration. In colorimeter the
reading is taken manually and then the concentration is calculated by manually
plotting the absorbance on a calibration curve.
In all these instruments the basic architecture is the same. A source of light is
used from which the light is passed through a filter which blocks all wavelengths
Notes except the wavelength at which we need the sample to be read. This light passes
through a cuvette which holds the sample post the color producing reaction.
After passing through the sample the light beam falls onto a photo voltaic cell.
This photo voltaic cell produced a current the strength of which is directly
proportional to the amount of light falling on it which will be inversely
proportional to the absorbance. In reality the photo voltaic cell measures incident
light and based on this calculated the absorbed light or absorbance. The
absorbance will be directly proportional to the amount of the colored particles
in the reaction mixture and hence directly proportional to the metabolite or
chemical we are trying to evaluate in the sample.

Fig. 18.1: A Simplified diagram of a colorimeter

18.4 BEER LAMBERT’S LAW

The Beer Lamberts law describes the co relation between the intensity of
incident light, the path the light has to travel and the absorbance of the medium
it passes through.
Absorbance is directly proportional to length of the path travelled by the light
beam and the optical density of the colored solution. In a colorimeter the length
of the path is fixed and this factor is negated when we set zero in the colorimeter

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with only distilled water in the cuvette. Then the only factor affecting the Biochemistry
absorbance is the optical density of the solution in the cuvette.
A=εbc
A = absorbance (-)
ε = molar absorbtivity with units of L /mol/cm
Notes
b = path length of the sample (cuvette)
c = concentration of the compound in solution, expressed in mol / L

18.5 SPECTROPHOTOMETRY
The difference between colorimetry and spectrophotometry is that in spectro-
photometry instead of taking reading at a set wavelength we read the absorbance
over a range of wavelength taking readings at every 5 or 10 nm range. It instead
of giving a specific absorbance reading gives a data stream which contain
absorbance for every wavelength made incident on the sample. Or it can also
give a spectra reading or a graph that plots absorbance against the increasing
wavelength of incident light.

Fig. 18.2: Spectophotometry

This is an example of spectra reading where the absorbance of the sample is

plotted over a wavelength between 400 to 750 nm.

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18.6 LIGHT EMISSION TECHNOLOGIES OR
CHEMILUMINESCENCE
Chemiluminescence is the production of light during a chemical reaction. This
is seen in nature in various natural reactions such as the one happening in fire
flies.
The chemical reaction can be displayed like this:
Notes
[A] + [B] → [] → [Products] + light
One such reaction is the reaction between luminol and hydrogen per oxide:
luminol + H2O2 → 3-APA[] → 3-APA + light
z where 3-APA is 3-aminophthalate
z 3-APA[] is the excited state fluorescing as it decays to a lower energy
level.
Immunochemilumnisence is a method in which a chemiluminescence reaction
is used by conjugating an enzyme, catalyzing the chemiluminescence, reaction
to an antibody specific to another antibody which is bound to the antigen of
interest. This enzyme converts a substrate to a product and during the process
releasing light energy. This photon of light is detected by a sensor and an accurate
measurement of the number of chemiluminescence reactions occurring during
a specific time period can be made. This directly proportional to the number of
enzyme molecules present which is directly proportional to the antigen
molecules present in the sample.

Fig. 18.3: Chemiluminescence

Chemiluminescence is an extremely accurate method with its accuracy reaching

to the gold standard tests such as Radio immune assays (RIA). It is used to
measure hormone levels in serum.

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18.7 FLUORESCENCE
It is the ability of a substance to emit visible light after absorbing light from the
visible or UV range. When these substances absorb light in the UV range of the
spectra and emit light in the visible range, this property is called UV florescence
and is responsible for certain substances like Vitamin B2, quinine, certain
minerals glowing in the dark.
The principle behind fluorescence is that of excitation of an electron. The Notes
incoming light commonly UV light provides the electron in an orbital energy
hence helping it shift to a higher energy orbital. Some of the energy in the
incident light is used up in this movement of electron. After sometime the
electron returns to its original lower energy orbital and the remaining energy is
released in form of light. This light has lesser energy than the incident light and
hence has a larger wavelength. This results in the phenomenon of high energy
UV light being absorbed by a substance and low energy visible light being
emitted in its place.

Fig. 18.4: Fluorescence

Principal of fluorescence
In laboratory fluorescence spectroscopy is used to analyze organic compounds.
Tryptophan is an aromatic amino acid which displays fluorescence. It is present
in a large number of proteins. The fluorescence of the tryptophan residue of any
protein is effected by the environment of the tryptophan residue. Hence in cases
of aberrant proteins or in cases of aberrant folding of the amino acid chain in
any protein the tryptophan residue’s immediate environment is changed and
hence a measureable change in fluorescence is seen.
The fluorospectrometer consists of a light source, a filter, a monochromatizer,
a sensor to measure the fluorescent light placed at 90% to the incident beam of

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Biochemistry light to prevent reflected light from reaching the sensor. A quartz cuvette is used
as a high grade quartz cuvette does not absorb the light in the wavelength range
of fluorescence.

Notes

Fig. 18.5: Simple diagram of the architecture of

a fluorescence spectrophotometer

Apart from this spectro photometer the flame photometer also works on the
principal of excitation of an electron so as to move it out of its orbit and then
read the visual spectra of the light emitted when the electron moves back to its
original orbit. The only difference being that the excitation energy is provided
by the flame into which the metal ions are sprayed into in form of a fine spary
or mist.

INTEXT QUESTIONS 18.1

1. Beam of light directed to the cuvette is ...................
2. Absorbance of a reaction mixture is measured by ...................
3. ................... is used to measure turbidity in reaction
4. Absorbance over a range of wavelength is taken in ...................
5. The production of light during a chemical reaction is ...................

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WHAT HAVE YOU LEARNT

z Measuring light emission, transmittance and scattering are few of the most
important techniques used in modern biochemistry laboratory.
z They are used in spectrophotometer, colorimetry, chemiluminescence,
ELISA etc.
Notes
z The ease and accuracy of measurement of light emission or transmittance
makes it a method of choice in a large number of analytical techniques
z Incident light refers to the beam of light that is directed at the cuvette (or
reaction vessel) from a light source.
z Transmitted light is the light that passes through the cuvette to emerge other
side.
z Absorbance is the term used to describe the light that is absorbed by the
reaction mixture and it depends on the intensity of the color of a reaction
product in the reaction vessel.
z Scattered light is the light that is not absorbed but is scattered or reflected
back by opaque substances in the reaction vessel and is used to measure
turbidity in reaction such as immune turbidity reactions.
z Colorimetry is a method employing the measurement of absorbance of a
reaction mixture. It is the simplest method used and is employed by a large
number of instruments such as a colorimeter, semiauto analyzer, auto
analyzer
z The difference between colorimetry and spectrophotometry is that in
spectrophotometry instead of taking reading at a set wavelength we read
the absorbance over a range of wavelength taking readings
z Chemiluminescence is the production of light during a chemical reaction
z Immunochemilumnisence is a method in which a chemiluminescence
reaction is used by conjugating an enzyme, catalyzing the chemiluminescence,
reaction to an antibody specific to another antibody which is bound to the
antigen of interest
z Fluorescence is the ability of a substance to emit visible light after absorbing
light from the visible or UV range

TERMINAL QUESTIONS
1. Enumerate the various diagnostic methods that use emitted, transmitted or
generated light for measurement.

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Biochemistry 2. What is the beer lambert law? What are the correlations derived from it.
3. What is the principle of colorimetry? Draw a simple labelled diagram of
a colorimeter.
4. Write a short note on spectrophotometry.
5. What is chemiluminescence? What are its uses in diagnostic?
6. What is fluorescence? How is it used in diagnostics?
Notes
7. What is the principal of a flame photometer?

ANSWERS TO INTEXT QUESTIONS

18.1
1. Incidence light
2. Colorimetry
3. Scattered light
4. Spectrophotometry
5. Chemiluminescence

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Basic Principles of Radioactive Measurements MODULE
Biochemistry

19
Notes
BASIC PRINCIPLES OF
RADIOACTIVE MEASUREMENTS

19.1 INTRODUCTION
Radioactivity is defined as the process in which unstable atomic nuclei loses
energy by emitting radiation in the form particles or electromagnetic waves.
These radiations are able to ionize the atoms and molecules along their track.
These radiations are able to cause cancer and death. Therefore these radiations
are of health and safety concern.

To understand radioactivity we should be able to understand certain basic

concepts like atom and atomic stability.

OBJECTIVES
After reading this lesson, you will be able to:

z describe Atom
z explain radioactive delay
z describe units of radioactivity
z explain the characteristics of radioactive emissions

19.2 ATOM
An atom is defined as the smallest component of an element having the chemical
properties of the element. It consists of positively charged nucleus surrounded
by negatively charged electrons. The nucleus is made up of positively charged
protons and uncharged neutrons.

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Notes

Fig. 19.1: Structure of an atom

In an atom the number of protons (P) and number of electrons (E) are equal. This
number is termed as atomic number (Z).
Atomic number (Z) = (P) = (E)
The atomic number is independent of neutrons. Therefore it does not affect the
chemical properties of an atom. The mass of the proton and neutron is equal and
is1850 times more than that of electron. Hence the mass of an atom is
concentrated in its nucleus. Mass number of an atom (A) is given by the sum
of number of protons and number of neutrons in a given nucleus.
Mass number (A) = (P) + (N)
The number of neutrons for a given atom of an element may vary which gives
rise to atoms with different mass number. Isotopes are defined as atoms with
same atomic number but different mass number.
Eg: There are 3 isotopes for hydrogen- 1H, 2H, 3H

19.2.1 Atomic stability and radiation

The stability of an atom depends upon the neutron to proton ratio (N: P)(also
known as N:Z) in its nuclei. For elements with lower atomic number, N: P is
1. For elements with higher atomic number, N: P is greater than1. If this ratio
is altered then the atom becomes unstable. By emitting particles or electromagnetic
radiation these atoms tend to become stable. This process is known as
Radioactivity or radioactive decay.

19.3 RADIOACTIVE DECAY

There are several types of radioactive decay. The most relevant to biochemistry
are:

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1. Decay by negatron emission Biochemistry

2. Decay by positron emission

3. Decay by alpha particle emission
4. Decay by emission of gamma rays
5. Decay by electron capture.
Notes
1. Decay by negatron emission
In this type of decay a neutron is converted into a proton by ejection of a
negatively charged beta (β) particle called a negatron (β –ve)
Neutron ⎯⎯→ Proton + Negatron (β –ve)
Negatron is nothing but an electron of nuclear origin. As a result of this emission
the nucleus gains a proton but loses a neutron. Here N: P ratio decreases, Z
increases by 1 and A remains constant. An isotope frequently used in biological
work that decays by negatron emission is 14C.
14 C
6 ⎯⎯→ 14 N
7 + (β –ve)
Negatron emission is important because most of the radioactive substances used
in biochemistry decay by this mechanism.
3H &14C are used label any organic compound.35S for methionine & 32 P for
nucleic acid.

2. Decay by positron emission

In this type of decay a proton is converted into a neutron by ejection of a
positively charged beta (β) particle called as positron (β +ve)
Proton ⎯⎯→ Neutron + Positron (β+ve)
Positron is extremely unstable with transient existence. After losing their energy
they interact with electrons and get destroyed. The mass and energy of these two
particles gets converted into two gamma rays (γ) emitted at 180° to each other
this is known as back to back emission.
As a result of this emission the nucleus gains a neutron but loses a proton. Here
N: P ratio increases, Z decreases by 1and A remains constant. An isotope that
decays by positron emission is 22 Na.
22 Na
11 ⎯⎯→ 14 N
7 + (β –ve)
Positron emission tomography (PET),a brain scanning technique is used in
finding out the active and inactive areas of the brain.

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Biochemistry 3. Decay by alpha particle emission

Isotopes of elements with high atomic number usually decay by alpha (α)
particle emission. As a result of this emission the nucleus loses 2 protons and
2 neutrons, which is similar to helium nucleus (42He)
Here N: P ratio remains constant, Z decreases by 2 and A decreases by 4. Alpha
emitters are rarely used in biological work they are highly toxic due to its large
Notes mass and ionizing power.

4. Decay by emission of gamma rays

This is an electromagnetic radiation as a consequence of electron excitation that
accompanies alpha and beta particle emission. It does not lead to change in
atomic number or mass. Gamma radiation has low ionizing but high penetrating
power.
131
63 I ⎯⎯→ 131I
64 + (β –ve) + γ

5. Decay by electron capture

In this type of decay a proton captures an electron orbiting in the innermost k
shell. The proton becomes a neutron and an electromagnetic radiation (X-ray)
is given out.
Neutron + Electron ⎯⎯→ Proton +X-rays
. 125 I 125 Te
53 52

19.3.1 Radioactive decay energy

The radioactive decay energy is expressed as electron volt. One electron volt is
the energy acquired by one electron accelerating through a potential difference
of 1volt and is equivalent to 1.6 × 10-9 J. For isotopes million or mega electron
volts is applicable. Alpha particles are more energetic falling in the range 4-8
Mev. Beta and gamma emitters have decay energies less than 3 Mev.

19.3.2 Rate of radioactive decay

Radioactive decay is a spontaneous process, which takes place in an exponential
way. Different isotopes decay at different rate. The number of atoms disintegrating
in a given time depends upon number of isotopes present (N) at that time (t) and
λ is decay constant. This λ is specific for a given isotope and is defined as
number of atoms disintegrating in unit time (t – 1).
dN/dt = - λN
This equation can be converted into logarithmic form
Ln Nt/N0 = - λt

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Nt is the number of radioactive atoms present at time t,and N0 is the number Biochemistry
of radioactive atoms present originally. In practice decay constant is expressed
in terms of half life t1/2.
The half-life of an radioactive material is defined as the time taken to become
half of its original value
So Nt/No becomes ½ at t becomes t1/2. Now the above said equation becomes
Notes
Ln1/2 = - λt1/2

1
0.9
Amount of Radioactive Nuclel

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
First t½ Second t½ Third t½ Four t½
Time

Fig. 19.2: Radioactive decay

Conversion of ln to log it becomes

2.303 log10(1/2) = - λt1/2
Finally, t1/2 = 0.693/ λ
The values of t1/2 varies from 10 19 years for lead (204 pb) and 3 × 10-7 s for
polonium (212Po).
Half life of some isotopes used in biological studies

Isotopes Half-life
3H 12.26 years
14C 5760 years
32P 14.20 days
35S 87.20 days
125I 60 days

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19.4 UNITS OF RADIOACTIVITY
The radioactivity of a substance can be measured using different units. They are
z Becquerel is the SI unit for measurement of radioactivity. It is defined as
the number of disintegration per second (d.p.s.)
1 Becquerel (Bq) = 1 dps
Notes Terra Bq = 1012 Bq
Giga Bq = 109 Bq
Mega Bq = 106 Bq
z Curie is the commonly used unit. It is defined as the quantity of radioactive
material having nuclear disintegration similar to that of 1g of radium i.e.
3.7 × 1010 dps (or 37 Giga Bq)
1 Curie (Ci) = 3.7 × 1010 dps
Milli Ci = Ci × 10-3
Micro Ci = Ci × 10-6
z Specific activity of a substance is defined as activity per unit weight or
volume (e.g., Bq/gm or B/l).
z Counts per minute (c.p.m) is disintegration detected by a radiation counter.

19.5 CHARACTERSTICS OF RADIOACTIVE EMISSIONS

19.5.1 Alpha Particles
z Alpha particles have helium nucleus with double positive charge
z These are high energy particles (3-8 Mev) with less speed
z They interact with matter in 2 ways
1. Excitation: In this electrons of nearby atoms are shifted to higher orbitals
2. Ionisation: It removes the orbital electron completely from nearby atoms
z Their penetrating power is less

19.5.2 Negatrons
z Negatrons are very small and rapidly moving particles
z They also cause excitation and ionization but lesser than alpha particle
z They have more penetrating power
z These are low energy particles (0.018-4.81 Mev)

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19.5.3 Gamma Rays Biochemistry

z These are electromagnetic rays with no mass and charge.

z They have very high penetrating power
z They lead to production of secondary electrons which in turn cause
excitation and ionization.

Notes

INTEXT QUESTIONS 19.1

1. Match the following:
1. Alpha particles (a) Electromagnetic radiation
2. Negatron (b) Electron capture
3. Positron (c) High energy radiation
4. Gamma rays (d) Low energy radiation
5. X-rays (e) Extremely unstable radiation
2. True or false:
1. Curie is SI unit of radioactivity.
2. One Becquerel is number of disintegrations equal to 1 gram of
Radium.
3. Alpha particles have Helium nucleus.
4. Negatrons have high penetrating power than gamma rays.
5. Positron emission tomography is used for studying active regions of
the brain.

WHAT YOU HAVE LEARNT

z Radioactivity is defined as the process in which unstable atomic nuclei loses
energy by emitting radiation in the form particles or electromagnetic waves
z The half-life of an radioactive material is defined as the time taken to
become half of its original value
z Different types of radioactive decay are:
1. Decay by negatron emission
2. Decay by positron emission
3. Decay by alpha particle emission
4. Decay by emission of gamma rays

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Biochemistry

TERMINAL QUESTIONS
1. What is Radioactivity?
2. What are the different types of radioactive decay?
3. What is half-life?
Notes 4. What are the characteristics of various radioactive emissions?
5. Write a note on units of radioactivity.

ANSWERS TO INTEXT QUESTIONS

18.1
1. 1. (a) 2. (b) 3. (c) 4. (d) 5. (e)
2. 1. F 2. F 3. F 4. F 5. F

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Biochemistry

20
Notes
ELECTROCHEMISTRY

20.1 INTRODUCTION
Electrochemistry is the study of interchange between chemical energy and
electrical energy. Many significant chemical reactions are electrochemical in
nature. Electrochemical reactions are chemical reactions in which electrons are
transferred. To understand electrochemical reactions, it is necessary to understand
the terms and concepts of electricity and extend these to apply to electrochemical
relationships.

OBJECTIVES
After reading this lesson, you will be able to:
z describe the basic concepts of Oxidation and Reduction
z explain Electrochemical cells
z describe Electrochemical potentials
z describe the application of Electrochemistry

20.2 BASIC CONCEPTS OF OXIDATION AND

REDUCTION
Electrons are always transferred from one atom or molecule to another in an
electrochemical reaction. There are three different types of electrochemical
reactions based on the changes in oxidation state that occur in them. They are
1. Oxidation reactions: atoms of the element(s) involved in the reaction lose
electrons. The charge on these atoms must then become more positive.
Eg: Fe2+ (aq) ⎯→ Fe3+(aq) + e-

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Biochemistry 2. Reduction reactions: In reduction reactions, atoms of the elements

involved gain electrons.
Eg: Zn2+(aq) + 2e- ⎯→ Zn(s)
3. Redox reactions : A redox reaction is an electrochemical reaction in which
both reduction and oxidation take place together. The electrons lost in an
oxidation component are gained in a reduction component.
Notes Eg: Fe3+ + Cu+ ⎯→ Fe2+ + Cu2+
These redox reactions are of two types they are:
(a) Direct redox reaction: In a direct redox reaction, both oxidation and
reduction reactions take place in the same vessel. Chemical energy is
converted to heat energy in a direct redox reaction.
(b) Indirect redox reaction: In indirect redox reactions, oxidation and reduction
take place in different vessels. In an indirect redox reaction, chemical energy
is converted into electrical energy.

20.3 ELECTROLYTE AND ELECTRODES

Compounds, molecules, and atoms that do not carry any charge are referred to
as neutral species. Eg KCl, O2.
z Ion is an atom or molecule that has an electrical charge.
z Cation: An ion that carries a positive charge is called a cation.
z Anion: An ion that carries a negative charge is called an anion.
z Electrolyte: A solution that contains ions is called an electrolyte solution,
or an electrolyte. They conduct electricity as charged ions can move through
them.
z Electrodes: A solid electric conductor through which an electric current
enters or leaves an electrolytic cell or other medium. In electrochemical
reactions oxidation or reduction reaction takes place at the electrodes. These
are called electrode reactions, or half-reactions.
z A half-reaction can be either a reaction in which electrons appear as
products (oxidation) or a reaction in which electrons appear as reactants
(reduction). A combination of two half reaction forms the complete reaction.
z Cathode: An electrode in which reduction takes place.
z Anode: An electrode where oxidation takes place.

20.4 ELECTROCHEMICAL CELLS

Electrochemical cell is a device which converts chemical energy into electrical
energy in an indirect redox reaction.

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An electrochemical cell can either drive an external electrical device (load) or Biochemistry
be driven by it (power supply), depending upon the relative electromotive forces
applied by the cell and the device. It is of three types galvanic, reversible, or
electrolytic:
z A galvanic cell is a cell in which current flows, power is produced, and the
cell reaction is proceeding spontaneously.
z An electrolytic cell is a cell in which current flows, power is consumed,and Notes
the cell reaction being driven is the reverse of the spontaneous cell reaction.
z A reversible cell is a cell in which no current flows (and therefore no power
is involved, because P = EI. The cell reaction in a reversible cell is neither
spontaneous nor nonspontaneous; it is called reversible because an
infinitesimal change in the cell potential can cause it to proceed in either
direction.
In an electrochemical cell, the half cell where oxidation takes place is known
as oxidation half cell and the half cell where reduction takes place is known as
reduction half cell. Oxidation takes place at anode which is negatively charged
and reduction takes place at cathode which is positively charged. During this
process transfer of electrons takes place from anode to cathode while electric
current flows in the opposite direction. An electrode is made by dipping the metal
plate into the electrolytic solution of its soluble salt. A salt bridge is a U shaped
tube containing an inert electrolyte in agar-agar and gelatine.
A salt bridge maintains electrical neutrality and allows the flow of electric
current by completing the electrical circuit.

20.4.1 Representation of an electrochemical cell

While representing an electrochemical cell Anode is written on the left while
the cathode is written on the right.
Anode represents the oxidation half cell and is written as:
Metal/Metal ion (Concentration) Cathode represents the reduction half cell and
is written as: Metal ion (Concentration)/Metal. Salt bridge is indicated by
placing double vertical lines between the anode and the cathode. The physical
state of all components like solid, liquid, gas, aqueous (s, l, g, aq, etc.) should
be mentioned.
Example: An aqueous cell that operates spontaneously using the following
reaction:
2Cu(s) + Zn2+ ⎯→ 2Cu+ + Zn(s)

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Biochemistry would be written in cell notation as

Cu(s)/Cu+//Zn2+/Zn(s)
The Zn2+ is being reduced at the cathode, so the Zinc electrode couple is written
on the right.

Notes

Fig. 20.1

20.5 ELECTROCHEMICAL POTENTIALS

It is the potential difference measured between two electrodes. Voltmeters can
be used to measure the potential differences across electrochemical cells but
cannot measure directly the actual potential of any single electrode.

20.5.1 Electrode potential

Electrode potential is the potential difference that develops between the
electrode and its electrolyte. The separation of charges at the equilibrium state
results in the potential difference between the metal and the solution of its ions.
It is the measure of tendency of an electrode in the half cell to lose or gain
electrons.

There are 2 types of electrode potentials: Oxidation potential and reduction

potential. Oxidation potential is the tendency of an electrode to lose electrons
or get oxidized. Reduction potential is the tendency of an electrode to gain
electrons or get reduced. Oxidation potential is the reverse of reduction potential.
The electrode having a higher reduction potential has a higher tendency to gain
electrons. So, it acts as a cathode. The electrode having a lower reduction
potential acts as an anode.

20.5.2 Standard electrode potentials

When the concentration of all the species involved in a half cell is unity, then
the electrode potential is known as standard electrode potential. It is denoted as

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EÈ. For solid or liquid compounds or elements, standard conditions are the pure Biochemistry
compound or element; for gases they are 100 kiloPascal (kPa) pressure, and for
solutes they are the ideal 1 molar (mol/liter) concentration.
The standard electrode potential of an electrode cannot be measured in isolation.
Tables of standard electrode potentials can be obtained for all elements if any
one electrode, operated under standard conditions, is designated as the standard
electrode or standard reference electrode with which all other electrodes will be Notes
compared. According to convention, the Standard Hydrogen Electrode,
abbreviated SHE is taken as a reference electrode and it is assigned a zero
potential at all temperatures.
The potential difference across a reversible cell made up of any electrode and
a SHE is called the reversible potential of that electrode, E. If this other electrode
is also being operated under standard conditions of pressure and concentration,
the reversible potential difference across the cell is the standard electrode
potential EΘ of that electrode.
In many practical potential measurements, the standard hydrogen electrode
cannot be used because hydrogen reacts with other substances in the cell or
because other substances in the cell react with the catalytic platinum electrode
surface upon which the H+/H2 potential is established. It is often much more
convenient to use alternative electrodes whose potentials are precisely known
with respect to the SHE.
Two of the electrodes most commonly used as reference electrodes are the silver/
silver chloride electrode with EΘ = +0.2224 V, and the saturated calomel
electrode (SCE) at 0.241 V. The effect of changing the reference electrode is to
change the zero of a potential scale while leaving the relative positions of all
of the potentials unchanged.

20.5.3 Standard hydrogen electrode

Standard hydrogen electrode consists of a platinum wire sealed in a glass tube
and carrying a platinum foil at one end. The electrode is placed in a beaker
containing an aqueous solution of an acid having 1 Molar concentration of
hydrogen ions. Hydrogen gas at 1 bar pressure is continuously bubbled through
the solution at 298 K. The oxidation or reduction takes place at the Platinum
foil. The standard hydrogen electrode can act as both anode and cathode.
If the standard hydrogen electrode acts as an anode:
H2 (g) ⎯→ 2H+ (aq) + 2e-
If the standard hydrogen electrode acts as a cathode:
2H+ (aq) + 2e- ⎯→ H2 (g)

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Notes

Fig. 20.2
In the electrochemical series, various elements are arranged as per their standard
reduction potential values. A substance with higher reduction potential value
means that it has a higher tendency to get reduced. So, it acts as a good oxidising
agent. A substance with lower reduction potential value means that it has a higher
tendency to get oxidised. So, it acts as a good reducing agent. The electrode with
higher reduction potential acts as a cathode while the electrode with a lower
reduction potential acts as an anode. The potential difference between the 2
electrodes of a galvanic cell is called cell potential and is measured in Volts.
The cell potential is the difference between the reduction potential of cathode
and anode.
E cell =E cathode –E anode

Cell potential is called the electromotive force of the cell (EMF) when no current
is drawn through the cell.

20.5.4 Electrode potential at Non-Standard conditions

Nernst studied the variation of electrode potential of an electrode with
temperature and concentration of electrolyte. Nernst formulated a relationship
between standard electrode potential EΘ and electrode potential E.
When cell is not at standard conditions, use Nernst Equation:
RT
E = E° – lnQ
nF
R = Gas constant 8.315 J/K•mol
F = Faraday constant

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Q = reaction quotient [products] coefficient/[reactants] coefficient Biochemistry

E = Energy produced by reaction

T = Temperature in Kelvins
n = number of electrons exchanged in BALANCED redox equation
Electrode potential increases with increase in the concentration of the electrolyte
Notes
and decrease with decrease in temperature.
Nernst equation when applied to a cell:

2.303 RT log [Anode ion]

Ecell = EΘcell –
nF [Cathode ion]

This helps in calculating the cell potential. At equilibrium, cell potential Ecell
becomes zero

20.6 APPLICATION OF ELECTROCHEMISTRY IN

BIOCHEMISTRY
Based on the electrochemical principle several analytes of biochemical importance
can be detected. Some of the devices are explained below

20.6.1 Ion selective electrodes: (I.S.E.)

Principle
An ideal I.S.E. consists of a thin membrane across which only the ion to be
measured can be transported. The transport of ions from a high conc. to a low
one through a selective binding with some sites within the membrane creates
a potential difference. This forms the basis of ion selective electrode.

Types of I.S.E.
A wide variety of analytes can be detected using ISE by varying the membrane
as given below:
1. Glass membrane (i.e. H+ electrode)
2. Solid-state electrode (e.g. F- electrode uses a Eu2 +-doped LaF3 crystal)
3. Liquid-based electrode (e.g. Ca2+ electrode uses a liquid chelator)
4. Compound electrode (e.g. CO2 gas sensing electrode)

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Biochemistry Advantages and limitations of I.S.E.

Advantages
1. Linear response: It shows linear response for wide concentration range of
the analyte.
2. Non-destructive: The analyte is not consumed at the end of the reaction.

Notes 3. Non-contaminating.: The sample remains unchanged at the end of the

reaction and can be used for other analysis
4. Short response time: The analyte can be determined within seconds or minutes
5. Unaffected by color or turbidity: Presence of color and turbidity does not
affect the analysis as in spectrophotometry.

Limitations
1. Precision is rarely better than 1%.
2. Electrodes can be fouled by proteins or other organic solutes.
3. Interference by other ions.
4. Electrodes are fragile and have limited shelf life.
5. Electrodes respond to the activity of uncomplexed ion. So ligands must be
absent or masked.

20.6.2 pH Electrodes
Principle: The H+ ions either diffuse out or into the gel layer of the glass
membrane depending on the pH value of the measured solution. Since the glass
electrode has an internal buffer with a constant pH value, the potential at the
inner surface of the membrane is constant during the measurement. The total
membrane potential is a result of the difference between the inner and outer
charge. From the potential the H+ ions concentration can be determined using
Nernst equation.

Instrumentation
The whole pH measuring circuit consists of a measuring electrode (glass
electrode) and a reference electrode, which are both immersed in the same
solution. In order to obtain a definite pH value the reference electrode must have
a defined stable potential which is independent of the measured solution. Every
reference electrode consists of a reference element which is immersed in a
defined electrolyte.
This electrolyte must be in contact with the measured solution. This contact most
commonly occurs through a porous ceramic junction. Of the many reference

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systems, only the mercury/calomel and the silver/silver chloride systems, along Biochemistry
with certain modifications of them, have attained practical importance.
Due to environmental considerations, however, the mercury electrode is rarely
used today. The potential of the reference electrode system is defined by the
reference electrolyte and the reference element (e.g. silver/silver chloride). Here
it is important that the reference electrolyte has a high ion concentration which
results in a low electrical resistance. Ideally no reaction between the reference
Notes
electrolyte and the measuring solution should occur over a wide temperature
range.

Combination electrodes
Since the combination electrode is much easier to handle than the separate
electrodes, the former is used almost exclusively today. In the combination
electrode the glass electrode is concentrically surrounded by the reference
electrolyte.
Only when the different parts of the electrode are expected to have very different
life expectancies is the use of separate electrodes recommended instead of a
single combination electrode.
Three-in-one electrodes A recent innovation is the addition of a temperature
sensor to the pH combination electrode. By housing the temperature sensor in
the same body as the pH and reference elements, temperature compensated
readings can easily be made with a single probe.

Application
Most of the biochemical reactions are pH dependent.
Eg. Enzyme assays

Fig. 20.3

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Biochemistry 20.6.3 pO2 (Clark) Electrode

Principle
Binding of oxygen to the polypropylene membrane creates a potential which is
directly propotinal to pO2

Instrumentation
Notes This electrode is known as “Clark Type” after their inventor, Dr. Leland Clark.
The Clark electrode consists of an anode and cathode in contact with an
electrolyte solution. It is covered at the tip by a semi-permeable membrane
usually polypropylene membrane, which is permeable to gases but not
contaminants and reducible ions of the sample. The cathode is in a glass
envelope in the body of the electrode. The anode has a larger surface that
provides stability and guards against drift due to concentration of the pO2
electrolyte (usually potassium chloride, 0.1 M). This silver/ silver chloride (Ag/
AgCl) anode provides electrons for the cathode reaction. The Clark (pO2)
electrode measures oxygen tension amperometrically. That is the pO2 electrode
produces a current, at a constant polarizing voltage (usually -0.6 V vs. Ag/AgCl)
which is directly proportional to the partial pressure of oxygen (pO2) diffusing
to the reactive surface of the electrode. Silver at the anode becomes oxidized.
Ag anode : 4Ag + 4 Cl– 4AgCl + 4e–
Pt cathode : O2 + 4H+ + 4e– 2H2O
Reduction of oxygen occurs at the surface cathode which is exposed at the tip
of the electrode. Oxygen molecules diffuse through the semi-permeable
membrane and combine with the KCl electrolyte solution. The current produced
is a result of the following reduction of oxygen at the cathode.

Fig. 20.4

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Production of four electrons accompanies each molecule reduced. The pO2 Biochemistry
channel measures this flow of electrons and the resulting microvoltage is
displayed as pO2. Therefore, pO2 is measured amperometrically; the pO2
electrode produces a current at a constant polarizing voltage (0.6 V) which is
directly proportional to the partial pressure of oxygen diffusing to the reactive
surface of the electrode.

Notes
20.6.4 Biosensors

Principle
Biosensors are analytical instruments that convert biochemical signal into
quantifiable electrical signal.

Instrumentation
It consists of a biocatalyst which can be an enzyme, cell or tissue. This is
immobilized on a membrane or gel. It is held in close contact with a transducer
which converts biochemical signal into quantifiable electrical signal.

Advantages
1. Highly specific & sensitive
2. Detect wide range of molecules

Fig. 20.4

Uses
1. It has wide range of uses in clinical, environmental and Industrial fields
Eg. Glucose sensors used in the detection of blood glucose for diabetes
management

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Biochemistry Types
Based on the intimacy between the biocatalyst and the transducer. They are
classified into
1. First generation
2. Second generaion
3. Third generation sensors
Notes
In first generation, the biocatalyst and transducer can be separated easily. Both
can function separately without the other.
In second generation, the biocatalyst and transducer cannot function separately.
In third generation, biochip is used.
Analyte Biocatalyst Transducer
Alcohol Alcohol oxidase O2
Glucose Glucose oxidase O2
Urea Urease NH4+
Glutamate Glutamate decarboxylase CO2

20.6.4.1 Micro-potentiometric sensors

z These sensors are very small and are used to measure analytes in micro litre
volumes of sample.
z These are initially developed for studying pH in living cells.
z These units are also used for monitoring organ perfusion.
z It can measure ions like H+, Na+, K+, Ca2+ in 10 µL volumes as in 96-well
microtiter plates
z It can be used in flow-through systems, such as HPLC detector with 50 µL
volumes.

20.6.5 Electrochemical Detectors

Introduction
Electrochemical detection (ECD) technique used for High Performance Liquid
chromatography (HPLC) is an extremely selective and sensitive detection
technique.

Detection principle
In amperometric electrochemical detection the electrical current is measured
resulting from oxidation or reduction reactions. A sample is introduced in HPLC

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and separated on the chromatographic column. The column is connected to an Biochemistry
ECD cell, an electrochemical sensor where a reaction takes place at an electrode.
Electrochemically active substances that elute from the column undergo an
electrochemical reaction, electrons are transferred resulting in an electrical
current. The electrodes are connected to an electronic circuitry with a powerful
-low noise- amplifier that converts a pico- or nanoampere current in a signal in
the range of ± 1 Volt which is commonly used in data acquisition.
Notes
Uses
z It is useful in analyses of several compounds such as aromatic substances,
drugs, catechol amines, neurotransmitters etc.
z Concentrations as low as 50 pmole/L and as high as 100 µmole/L or more
can be detected using this technique.

INTEXT QUESTIONS 20.1

1. Fill in the blanks
1. Oxidation is ................ of electrons
2. Reduction is ................ of electrons
3. Both reduction and oxidation takes place in ................ reactions.
4. The potential of Standard Hydrogen electrode is ................
5. In cathode ................ reaction takes place.
2. State True or false:
1. In pH electrode polypropylene membrane is used.
2. Silver/ Silver chloride electrode can be used as standard electrode.
3. Micropotentiometric sensors use larger volume for detection of ions.
4. Glutamate detection biosensor uses CO2 as transducer
5. First generation biosensors, the biocatalyst and the transducer cannot
be separated.

WHAT YOU HAVE LEARNT

z Oxidation is defined as a loss of electrons while reduction is defined as a
gain of electrons.

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Biochemistry z In a redox reaction, both oxidation and reduction reaction takes place
simultaneously.
z In an electrochemical cell: Oxidation takes place at anode which is
negatively charged and reduction takes place at cathode which is positively
charged. Transfer of electrons takes place from anode to cathode while
electric current flows in the opposite direction. An electrode is made by
dipping the metal plate into the electrolytic solution of its soluble salt. A
Notes
salt bridge is a U shaped tube containing an inert electrolyte in agar-agar
and gelatine.
z Electrode potential is the potential difference that develops between the
electrode and its electrolyte. The separation of charges at the equilibrium
state results in the potential difference between the metal and the solution
of its ions. It is the measure of tendency of an electrode in the half cell to
lose or gain electrons.
z According to convention, the Standard Hydrogen Electrode is taken as a
reference electrode and it is assigned a zero potential at all temperatures.
Standard calomel electrode can also be used as a reference electrode.
z The cell potential is the difference between the reduction potential of
cathode and anode. Ecell = Ecathode – Eanode
z Ion selective electrodes are useful for detecting wide range of bioanalytes.

TERMINAL QUESTIONS
1. What is Redox reaction?
2. Write short notes on redox potential.
3. Write about electrode potential and Nernst equation
4. Write about biosensors, its types and applications
5. What is an electrochemical cell and how to represent it?
6. Write a note on Standard hydrogen electrode
7. Write about different types of ion sensitive electrodes and its uses
8. Write a note on Clarks oxygen electrode
9. Write a note on pH electrode
10. What are electrochemical detectors and its applications?

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Biochemistry

21
Notes
ELECTROPHORESIS

21.1 INTRODUCTION
The movement of particles under spatially uniform electric field in a fluid is
called electrophoresis. In 1807, Ferdinand Frederic Reuss observed clay
particles dispersed in water to migrate on applying constant electric field for the
first time. It is caused by a charged interface present between the particle surface
and the surrounding fluid. The rate of migration of particle depends on the
strength of the field, on the net charge size and shape of the molecules and also
on the ionic strength, viscosity and temperature of medium in which the
molecules are moving. As an analytical tool, electrophoresis is simple, rapid and
highly sensitive. It is used analytically to study the properties of a single charged
species and as a separation technique. It provides the basis for a number of
analytical techniques used for separating molecules by size, charge, or binding
affinity, example- for the separation of deoxyribonucleic acid (DNA), ribonucleic
acid (RNA), or protein molecules using an electric field applied to a gel matrix.
Gel matrix used mainly is polyacrylamide and agarose. DNA Gel electrophoresis
is usually performed for analytical purposes, often after amplification of DNA
via PCR, but may be used as a preparative technique prior to use of other
methods such as mass spectrometry, RFLP, PCR, cloning, DNA sequencing, or
Southern blotting for further characterization.

OBJECTIVES
After reading this lesson, you will be able to:
z define the electrophoresis
z describe the principle and important types of electrophoretic methods
z explain the principle and components of a electrophoresis
z explain various uses of electrophoresis

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21.2 PRINCIPLE
The surface adsorbed sample strongly affects suspended particles by applying
electric surface charge, on which an external electric field exerts an electrostatic
coulomb force. According to the double layer theory, all surface charges in fluids
are screened by a diffuse layer of ions, which has the same absolute charge but
opposite sign with respect to that of the surface charge. The electric field also
exerts a force on the ions in the diffuse layer which has direction opposite to that
Notes
acting on the surface charge This force is not actually applied to the particle, but
to the ions in the diffuse layer located at some distance from the particle surface,
and part of it is transferred all the way to the particle surface through viscous
stress. This part of the force is also called electrophoretic retardation force. When
the electric field is applied and the charged particle to be analyzed is at steady
movement through the diffuse layer, the total resulting force is zero:
Ftoto = 0 = Fel + Ff + Fret
Considering the drag force on the moving particles due to the viscosity of the
dispersant, in the case of low turbulence and moderate electric charge strength E,
the velocity of a dispersed particle ν is simply proportional to the applied field,
which leaves the electrophoretic mobility μe defined as:
ν
μe =
E
The most known and widely used theory of electrophoresis was developed in
1903 by Smoluchowsky
εr ε0 ζ
μe = ,
η
where εr is the dielectric constant of the dispersion, ε0 is the permittivity of free
space (C² N–11 m–2), η is dynamic viscosity of the dispersion medium (Pa s),
and ζ is zeta potential (i.e., the electrokinetic potential of the slipping plane in
the double layer).
The Smoluchowski theory is very powerful because it works for dispersed
particles of any shape at any concentration. Unfortunately, it has limitations on
its validity. It follows, for instance, from the fact that it does not include Debye
length κ–1. However, Debye length must be important for electrophoresis, as
follows immediately from the Figure on the right. Increasing thickness of the
double layer (DL) leads to removing point of retardation force further from the
particle surface. The thicker DL, the smaller retardation force must be.
Detailed theoretical analysis proved that the Smoluchowski theory is valid only
for sufficiently thin DL, when particle radius a is much greater than the Debye
length :

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aκ >> 1 Biochemistry

This model of “thin Double Layer” offers tremendous simplifications not only
for electrophoresis theory but for many other electrokinetic theories. This model
is valid for most aqueous systems because the Debye length is only a
few nanometers there. It breaks only for nano-colloids in solution with ionic
strength close to water.
The Smoluchowski theory also neglects contribution of surface conductivity. Notes
This is expressed in modern theory as condition of small Dukhin number.
Du << 1
In the effort of expanding the range of validity of electrophoretic theories, the
opposite asymptotic case was considered, when Debye length is larger than
particle radius:
aκ < 1
Under this condition of a “thick Double Layer”, Huckel predicted the following
relation for electrophoretic mobility:

2εr ε0 ζ
μe =
3η

This model can be useful for some nanoparticles and non-polar fluids, where
Debye length is much larger than in the usual cases. There are several analytical
theories that incorporate surface conductivity and eliminate the restriction of a
small Dukhin number pioneered by Overbeek and Booth. Modern, rigorous
theories valid for any zeta potential and often any aκ stem mostly from Dukhin-
Semenikhin theory. In the thin Double Layer limit, these theories confirm the
numerical solution to the problem provided by O’Brien and White.

INTEXT QUESTIONS 21.1

1. Define electrophoresis.
2. The gel matrix widely used in electrophoresis is mainly ................ and
................
3. The widely used theory of electrophoresis was developed in ................ by
................
4. The part of the force that is transferred to the particle surface through
viscous stress is called ................

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21.3 PREPARING AND RUNNING STANDARD
AGAROSE GEL
The equipment and supplies necessary for conducting agarose gel electrophoresis
are relatively simple (Fig 21.1) and include:
1. An electrophoresis chamber and power supply
Notes 2. Gel casting trays, which are available in a variety of sizes and composed
of UV-transparent plastic. The open ends of the trays are closed with tape
while the Gel is being cast, then removed prior to electrophoresis.
3. Sample combs, around which molten agarose is poured to form sample
wells in the gel.
4. Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-
EDTA (TBE).
5. Loading buffer, which contains something dense (e.g. glycerol) to allow the
sample to “fall” into the sample wells, and one or two tracking dyes, which
migrate in the gel and allow visual monitoring or how far the electrophoresis
has proceeded.
6. Ethidium bromide, a fluorescent dye used for staining nucleic acids. NOTE:
Ethidium bromide is a known mutagen and should be handled as a
hazardous chemical - wear gloves while handling.
7. Transilluminator (an ultraviolet light box), which is used to visualize
Ethidium bromide-stained DNA in gels. NOTE: always wear protective
eyewear when observing DNA on a transilluminator to prevent damage to
the eyes from UV light.

Fig. 21: Gel Electrophoresis unit

To pour a gel, agarose powder is mixed with electrophoresis buffer to the desired
concentration, and then heated in a microwave oven until completely melted.
Most commonly, Ethidium bromide is added to the gel (final concentration 0.5
µg/ml) at this point to facilitate visualization of DNA after electrophoresis. After

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cooling the solution to about 6°C, it is poured into a casting tray containing a Biochemistry
sample comb and allowed to solidify at room temperature.
After the gel has solidified, the comb is removed, using care not to rip the bottom
of the wells. The gel, still in its plastic tray, is inserted horizontally into the
electrophoresis chamber and just covered with buffer. Samples containing DNA
mixed with loading buffer are then pipeted into the sample wells, the lid and
power leads are placed on the apparatus, and a current is applied. You can Notes
confirm that current is flowing by observing bubbles coming off the electrodes.
DNA will migrate towards the positive electrode, which is usually colored red.
The distance DNA has migrated in the gel can be judged by visually monitoring
migration of the tracking dyes. Bromophenol blue and xylene cyanol dyes
migrate through agarose gel at roughly the same rate as double-stranded DNA
fragments of 300 and 4000 bp, respectively.
When adequate migration has occurred, DNA fragments are visualized by
staining with Ethidium bromide. This fluorescent dye intercalates between bases
of DNA and RNA. It is often incorporated into the gel so that staining occurs
during electrophoresis, but the gel can also be stained after electrophoresis by
soaking in a dilute solution of Ethidium bromide. To visualize DNA or RNA,
the gel is placed on a ultraviolet transilluminator.
Fragments of linear DNA migrate through agarose gel with a mobility that is
inversely proportional to the log10 of their molecular weight. In other words, if
you plot the distance from the well that DNA fragments have migrated against
the log10 of either their molecular weights or number of base pairs, a roughly
straight line will appear.

21.3.1 Requirements
Electrophoretic unit, conical flask, measuring cylinder, power pack, micropipette,
micro tips (1X) TAE buffer, Gel loading dye, EtBr, Agarose.

21.3.2 Steps
1. For preparing 0.8% agarose gel, 0.14g of Agarose was dissolved in 20ml
of TAE (1X).
2. Mixture was boiled till a clear solution was obtained.
3. Left at room temperature till suspension reaches 40-45°C.
4. Then 2µl of 1% EtBr was added.
5. Seal the casting tray properly and placed the combs at appropriate place.

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Biochemistry 6. Pour the gel and leave at room temperature for 45-50 minutes to solidify
the gel.
7. Fill the buffer tank with TAE (1X) so that the gel was dipped.
8. 5 µl of the sample was loaded into the well by mixing with 1µl of 6X
loading dye containing Bromophenol blue.
9. Switch on the power supply at the rate of 5V/cm (Fig. 21.2)
Notes
10. When the electrophoretic front reaches bottom of the gel power supply was
switched off. The gel was placed over transilluminator and observed under
UV light.

Fig. 21.2: Gel Electrophoresis tank with power unit

INTEXT QUESTIONS 21.2

1. The fluorescent dye used in the gel electrophoresis is ..................
2. Give the function of a transilluminator.
3. Which two dyes migrate through the gel at same rate to help in visualizing
the sample that is loaded in the gel?
.................. and ..................
4. The electrophoresis buffer used is mostly ..................or ..................

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Biochemistry
21.4 TYPES OF ELECTROPHORESIS

21.4.1 Affinity electrophoresis

The methods include the mobility shift electrophoresis, charge shift electrophoresis
and affinity capillary electrophoresis. The methods are based on changes in the
electrophoretic pattern of molecules (mainly macromolecules) through biospecific
interaction or complex formation. The interaction or binding of a molecule, Notes
charged or uncharged, will normally change the electrophoretic properties of a
molecule. Membrane proteins may be identified by a shift in mobility induced
by a charged detergent. Nucleic acids or nucleic acid fragments may be
characterized by their affinity to other molecules. The methods has been used
for estimation of binding constants, as for instance in lectin affinity electrophoresis
or characterization of molecules with specific features like glycan content or
ligand binding. For enzymes and other ligand-binding proteins, one dimensional
electrophoresis similar to counter electrophoresis or to “rocket
immunoelectrophoresis”, affinity electrophoresis may be used as an alternative
quantification of the protein. Some of the methods are similar to affinity
chromatography by use of immobilized ligands.

21.4.2 Capillary electrophoresis

Capillary electrophoresis (CE), can be used to separate ionic species by their
charge and frictional forces and hydrodynamic radius. In traditional
electrophoresis, electrically charged analytes move in a conductive liquid
medium under the influence of an electric field (Fig. 21.3). Introduced in the
1960s, the technique of capillary electrophoresis (CE) was designed to
separate species based on their size to charge ratio in the interior of a small
capillary filled with an electrolyte.

Fig. 21.3: Capillary Electrophoresis

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21.4.3 Immunoelectrophoresis
Immunoelectrophoresis is a general name for a number of biochemical methods
for separation and characterization of proteins based on electrophoresis and
reaction with antibodies. All variants of immunoelectrophoresis require
immunoglobulins, also known as antibodies reacting with the proteins to be
separated or characterized.
Notes 1. Rocket immunoelectrophoresis is one-dimensional quantitative
immunoelectrophoresis
2. Fused rocket immunoelectrophoresis is a modification of one-dimensional
quantitative immunoelectrophorsis used for detailed measurement of proteins
in fractions from protein separation experiments.
3. Affinity immunoelectrophoresis is based on changes in the electrophoretic
pattern of proteins through specific interaction or complex formation with
other macromolecules or ligands.

21.4.4 Pulsed field gel electrophoresis

Pulsed field gel electrophoresis is a technique used for the separation of large
deoxyribonucleic acid (DNA) molecules by applying to a gel matrix an electric
field that periodically changes direction.

While in general small fragments can find their way through the gel matrix more
easily than large DNA fragments, a threshold length exists above 30–50 kb
where all large fragments will run at the same rate, and appear in a gel as a single
large diffuse band. However, with periodic changing of field direction, the
various lengths of DNA react to the change at differing rates. That is, larger
pieces of DNA will be slower to realign their charge when field direction is
changed, while smaller pieces will be quicker. Over the course of time with the
consistent changing of directions, each band will begin to separate more and
more even at very large lengths. Thus separation of very large DNA pieces using
PFGE is made possible.

21.4.5 SDS-PAGE
One of the most common means of analyzing proteins by electrophoresis is by
using Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis. SDS
is a detergent which denatures proteins by binding to the hydrophobic regions
and essentially coating the linear protein sequence with a set of SDS molecules.
The SDS is negatively charged and thus becomes the dominant charge of the

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complex. The number of SDS molecules that bind is simply proportional to the Biochemistry

size of the protein. Therefore the charge to mass ratio should not change with
size. In solution (water), in principle all different sized proteins covered with
SDS would run at about the same mobility. However, the proteins are not run
through water. Instead they are run through an inert polymer, polyacrylamide.
The density and pore size of this polymer can be varied by just how you make
it (concentration of monomer and of cross-linking agent). Thus, the size of
Notes
molecules that can pass through the matrix can be varied. This determines in
what molecular weight range the gel will have the highest resolving power.

21.4.6 Native Gels

It is also possible to run protein gels without the SDS. These are called native
gels in that one does not purposely denature the protein. Here, the native charge
on the protein (divided by its mass) determines how fast the protein will travel
and in what direction.

21.4.7 Electrofocusing Gels

Another variation of gel electrophoresis is to pour a gel that purposely has a pH
gradient from one end to the other. As the protein travels through this pH
gradient, its various ionizable groups with either pick up or lose protons.
Eventually, it will find a pH where its charge is zero and it will get stuck
(focused) at that point.

21.4.8 DNA Agarose Gels

A simple way of separating fairly large fragments of DNA from one another by
size is to use an agarose gel. Agarose is another type of matrix used for many
purposes (such as the support for the growth of bacteria on plates). DNA does
not need a detergent, since it already has a large under of negative phosphate
groups evenly spaced. Thus, as with SDS-PAGE, the charge to mass ratio is
constant. Also like SDS-PAGE, the separation results from the matrix itself. The
range of size sensitivity can be varied by changing the density of the agarose.

DNA denaturing polyacrylamide gels (often called sequencing gels). To look at

smaller DNA molecules with much higher resolution, people generally denature
the DNA via heat and run it through a thin polyacrylamide gel that is also kept
near the denaturing temperature. These gels usually contain additional denaturing
compounds such as Urea. Two pieces of DNA that differ in size by 1 base can
be distinguished from each other this way.

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INTEXT QUESTIONS 21.3

1. Expand the following abbreviations:
z SDS
z PAGE
Notes
z CE
2. DNA denaturing gels are also called as ....................
3. Mention the correct type of electrophoresis for the following:
z Gel has a pH gradient
z Separate large DNA fragments
z Separate protein without SDS
z Separate proteins based on electrophoresis and reaction with antibodies
z separate ionic species by charge, friction force and hydrodynamic
radius

21.5 APPLICATIONS
Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology
and biochemistry. The results can be analyzed quantitatively by visualizing the
gel with UV light and a gel imaging device. The image is recorded with a
computer operated camera, and the intensity of the band or spot of interest is
measured and compared against standard or markers loaded on the same gel.
Depending on the type of analysis being performed, other techniques are often
implemented in conjunction with the results of gel electrophoresis, providing a
wide range of field-specific applications.

WHAT HAVE YOU LEARNT

z Electrophoresis plays a vital role in the separation of nucleic acids and
proteins in the field of genomics and proteomics
z The techniques are very simple but has its role in advanced studies
z The electrophoretic devices are economical and can be explored to the core
to analyze the complexity of biomolecules

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ANSWERS TO INTEXT QUESTIONS

21.1
1. The movement of particles under spatially uniform electric field in a fluid
is called electrophoresis.
Notes
2. Polyacrylamide and agarose
3. 1903, Smoluchowsky
4. electrophoretic retardation force

21.2
1. ethidium bromide (Et Br)
2. It is used to visualize the Et Br stained DNA or RNA in gel through
ultraviolet light of specific wavelength.
3. Bromophenol blue and xylene cyanol
4. TAE (tris Acetate EDTA) or TBE (Tris Borate EDTA)

21.3
1. SDS – Sodium dodecyl sulphate
PAGE – polyacrylamide gel electrophoresis
CE – capillary electrophoresis
2. Sequencing gels
3. z Gel has a pH gradient electrofocusing gels
z Separate large DNA fragments Agarose gels
z Separate protein without SDS Native gels
z Separate proteins based on Immunoelectrophoresis
electrophoresis and reaction
with antibodies
z separate ionic species by charge, capillary electrophoresis
friction force and hydrodynamic
radius

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22
Notes
CHROMATOGRAPHY AND
MASS SPECTROMETER

22.1 INTRODUCTION
We know that the biochemistry or biological chemistry deals with the study of
molecules present in organisms. These molecules are called as biomolecules and
they form the basic unit of every cell. These include carbohydrates, proteins,
lipids and nucleic acids. To study the biomolecules and to know their function,
they have to be obtained in purified form. Purification of the biomolecules
includes many physical and chemical methods. This topic gives about two of
the commonly used methods namely, chromatography and mass spectrometry.
These methods deals with purification and separation of biomolecules namely,
protein and nucleic acids.

OBJECTIVES
After reading this lesson, you will be able to:
z define the chromatography and mass spectrometry
z describe the principle and important types of chromatographic methods
z describe the principle and components of a mass spectrometer
z enlist types of mass spectrometer
z describe various uses of mass spectrometry

22.2 CHROMATOGRAPHY
When we have a mixture of colored small beads, it is easily separated by visual
examination. The same holds true for many chemical molecules. In 1903,

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Mikhail, a botanist (person studies plants) described the separation of leaf Biochemistry
pigments (different colors) in solution by using solid adsorbents. He named this
method of separation called chromatography. It comes from two Greek words:
chroma – colour
graphein – to write/detect
Modern separation methods are based on different types of chromatographic
methods. The basic principle of any chromatography is due to presence of two Notes
phases:
z Mobile phase – substances to be separated are mixed with this fluid; it may
be gas or liquid; it continues moves through the chromatographic instrument
z Stationary phase – it does not move; it is packed inside a column; it is a
porous matrix that helps in separation of substances present in sample. It
works on simple physical process called adsorption.

22.2.1 Components of Chromatography

The equipment used for separation of mixture in a sample is called a
chromatograph. The major components of a chromatograph:

Fig. 22.1

z Mobile phase source – separate chambers or containers having either gas

or liquid
z Analyte – sample to be separated
z Sample injection – chamber to inject the sample
z Separating column – having the stationary phase

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Biochemistry z Detector – identify the change in separation of a molecule; this calculates

the time between injection and detection of a sample, which is called as
retention time.
z Recorder and analyser – usually a computer attached to the chromatograph.
This records the response of detector as a graph called chromatogram

22.2.2 Types
Notes
There are many types of chromatographic methods present today. Some of the
important methods are described as follows:
z Gas chromatography
z Liquid chromatography
z Gel filtration chromatography
z Ion exchange chromatography
z Affinity chromatography
z Others- hydrophobic interaction chromatography (HIC), Isochromatic
focusing (ICF), etc.

22.2.2.1 Gas Chromatography

The gas chromatography principle involves separation of the components of the
sample due to separation in between the gaseous mobile phase and stationary
phase (usually silica). The components separated into gas comes out first while
other comes later. It detects compounds like fatty acids, essential organic
solvents, flavored oils, etc.

Soap-bubble meter
Reccorder

Syfinge
Detector Electrometer
or
Two-stage bridge
pressure Flow Injector
regulator Rotometer splitter
+
+
–
–

– ADC
Flow
controller
Carrier Data
gas Column system
supply

Column oven

Fig. 22.2: Gas chromatography

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In GC, a liquid sample is injected into the column. The GC column is usually Biochemistry
coated with stationary phase and placed inside an oven chamber. The sample
is vaporized as it passes the column which is more than its boiling point. The
sample compounds are carried to column by gas (usually helium or nitrogen)
and then to a detector. The detector signals a chart recorder for chromatogram
(Fig.22.2)

Notes
22.2.2.2 Liquid Chromatography
It is separation technique where the mobile phase is a liquid. It can be performed
in a column or a plane surface. This has been advanced to many types :
z HPLC – high performance liquid chromatography (Fig.22.3)
z FPLC – fast protein liquid chromatography

Data System

Solvent

Columa Waste

Solvent System Injector

Delivery Sample Detector (s)

Fig. 22.3: HPLC

22.2.2.3 Gel Filtration Chromatography

This chromatographic technique is also known as size exclusion chromatography
or molecular sieve chromatography. It involves the separation of molecules
based on their molecular weight and size. (Fig.22.4) It involves:
z Mobile phase – liquid mixed with sample mixture
z Stationary phase – gel matrix containing a particular pore size to allow
smaller molecules to easily pass through or vice versa.
It well suitable for separation of biomolecules that are sensitive to environmental
conditions like temperature, pH, etc.

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Notes

Fig. 22.4: Gel Filtration Chromatography

22.2.2.4 Ion Exchange Chromatography

Every chemical molecule that is capable of forming into ions (different charges
– positive, cations and negative, anions) is separated by using this chromatography
(Fig. 22.5). In this:
z Mobile phase – liquid
z Stationary phase – matrix of beads with either positive charge (cationic) to
separate anionic sample or negative charge (anionic) to separate cationic
sample.

Fig. 22.5: Ion Exchange Chromatography

It is used to separate charged proteins and peptides in a given sample.

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22.2.2.5 Affinity Chromatography Biochemistry

Affinity chromatography separates proteins on the basis of a specific interaction

between the molecules in the sample with a compound called ligand in the
column (Fig.22.6). Some of the ligands used in matrix are:
z enzymes
z antibody
Notes
z metal ions like Nickel,etc.

Fig. 22.6: Affinity Chromatography

This technique is highly specific and is used mostly for purification of proteins
and peptides.

22.3 MASS SPECTROMETRY

The mass spectrometer is an instrument that measures the masses and relative
concentrations of atoms and molecules. It makes use of the basic magnetic force
or velocity on a moving charged particle.

22.3.1 Basic Principle

Suppose you had a cannonball travelling past you and you wanted to deflect
(change of direction) it. All you’ve got is a jet of water from a hose-pipe. But,
it’s not going to make a lot of difference! Because the cannonball is so heavy,
it will hardly be deflected at all from its original course.

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Biochemistry If suppose instead of water, you try to deflect the table tennis ball travelling at
same speed using jet of water. Because the ball is so light, it will be deflected
easily.
Thus from the above examples, we can understand that amount of deflection
depends on the weight (mass) of the object. The same principle holds true for
atom-sized molecules.
Notes
22.3.1.1 Working Principle
“The technique for studying the masses of atoms or molecules or fragments of
molecules separated by their mass-to-charge ratio (m/z)”.

22.3.2 Components
There are many different kinds of mass spectrometers, but all use magnetic and/
or electric fields to exert forces on the charged particles produced from the
chemicals to be analyzed (Fig. 22.7). A basic mass spectrometer consists of three
parts:

Fig. 22.7: Mass Spectrometry - components

1. Source - in which ions are produced from the chemical substances to be
analyzed.
2. Analyzer - in which ions are separated according to mass.
3. Detector - which produces a signal from the separated ions.
A magnetic field or electric field separates ions according to their momentum
(mass x velocity)

22.3.3 Types of Mass Spectrometry

There are many different kinds of mass spectrometers based on the type of ion
source, mass analyzer and detector used. Let us see the various types of ion
source and mass analyzers:
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22.3.3.1 ESI – TQ Mass Spectrometer Biochemistry

The basic workflow of ESI tandem MS is as follows:

z the proteins pass through the source become ions;
z then the ions pass through the first analyzer and some specific ions are
selected;
z then these selected ions are broken up by a procedure called collision- Notes
induced dissociation (CID);
z finally the second analyzer is used to catch the ions produced after CID.
ESI – Electro Spray Ionization – helps in ionizing all molecules of a given
sample (Fig. 22. 8)

Sample Small Droplets dry Mass

⎯⎯⎯⎯→ ⎯⎯→ ions ⎯⎯
→
flow Capillary with ions analyzer

Fig. 22.8: Work flow of ESI ion source

Mass Analyzer – it may be of:

z Triple Quadrapole – consists of three single quadrupole mass analyzers Q1,
Q2 and Q3.
z Ion Trap – traps specific molecules/ions with a particular m/z ratio and
leaves all other to pass through; voltage is then suddenly increased leading
to colliding of molecules/ions and then detection (Fig. 22.9)

Fig. 22.9: ESI Tandem Mass Spectrometer

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Biochemistry 22.3.3.2 MALDI TOF Mass Spectrometer

MALDI – Matrix Assisted Laser Desorption and Ionisation, is a soft ionization
technique used for analysis of biomolecules (Fig.22.10)

Notes

Fig. 22.10: Ion source of MALDI

TOF – Time Off Flight analyzer - An ion of known electrical charge and
unknown mass enters a mass spectrometer and is accelerated by an electrical
field of known strength. This acceleration results in any given ion having the
same kinetic energy as any other ion given that they all have the same charge.
The velocity of the ion will depend however on the m/z ratio (Fig.22.11).

Fig. 22.11: TOF Analyzer and Detector

The result given by the detector is in the form of a graph called as a mass
spectrum based on the different molecules present in the sample (Fig. 22.12).

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15000 1340,71
Linear TOF-MS
10000 Resolution · 000 (FWHM)

1000

0
1335 1340 1345 1350 1355 1360
MASS (m/z)
Notes
Fig. 22.12: Mass Spectrum

22.3.4 Uses
z Helps in identification of similar fragments of a molecule
z It can easily combine different ion source and mass analyzers.
z Has high resolution, friendly and robust; highly sensitive
z It is used for all kinds of chemical analyses, ranging from environmental
analysis of petroleum products, trace metals and biological materials.

INTEXT QUESTIONS 22.1

1. Phases of chromatography are ................... & ...................
2. Types of liquid chromatography are ................... & ...................
3. Gel filtration chromatography separates molecules based on ................... &
...................
4. The instrument that measures the masses and relative concentration of atom
and molecules is ...................

WHAT HAVE YOU LEARNT

z Among the different methods to separate large and complex biomolecules,
chromatography and mass spectrometry are important today.
z Each method can be used either individually or together to find out the
composition of a large molecule.
z Based on the different properties and characters of molecules, differences
in chromatography and mass spectrometry are discussed in the text.
z Finally, the need to study and importance of each of the methods are
analyzed.

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TERMINAL QUESTIONS
1. Why is it necessary to separate and study molecules?
2. Define chromatography and the principle on which it works.
3. Discuss the major components of a chromatographic system.
Notes 4. What are the different chromatographic techniques?
5. Explain the basic principle of mass spectrometry.
6. Discuss the difference between TQ and TOF mass analyzers.

ANSWERS TO INTEXT QUESTIONS

22.1
1. Mobile and Stationary
2. High performance & Fast Protein
3. Molecular weight and Size
4. Mass Spectrometry

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Clinical Enzymology MODULE
Biochemistry

23
Notes
CLINICAL ENZYMOLOGY

23.1 INTRODUCTION
Enzymes are catalysts that increase the rate or velocity of physiologic reactions.
Each and every reaction in our body takes place with the help of an enzyme. In
general, most enzymes are present in cells at much higher concentrations than
in plasma. Measurement of their levels in plasma indicates whether their tissue
of origin is damaged leading to the release of intracellular components into the
blood. This forms the basis of clinical enzymology. Thus clinical enzymology
refers to measurement of enzyme activity for the diagnosis and treatment of
diseases.

OBJECTIVES
After reading this lesson, you will be able to:
z describe plasma enzymes
z explain about the assessment of cell damage and proliferation
z describe the role of enzymes in health and diseases

23.2 PLASMA ENZYMES

Enzymes present in plasma can be classified into 2 types, they are
z Functional Plasma enzymes and
z Non-functional plasma enzymes
Functional plasma enzymes:
z Present in plasma at higher concentration than tissues

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Biochemistry z They function in plasma.

z Mostly synthesized by the liver
z Usually decreased in disease conditions
z Eg. Clotting enzymes, lipoprotein lipase
Non-functional plasma enzymes:
Notes z Present in plasma at lower concentration than tissues
z Do not have any function in plasma
z Mostly synthesized by liver, skeletal muscle, heart, brain etc
z Usually increased in disease conditions
z Eg. Creatine kinase, Alanine transaminase etc
z Measurement of these enzymes in plasma can be used to assess cell damage
and proliferation i.e. diagnosis of disease.

23.2.1 Assessment of Cell Damage and Proliferation

Plasma enzyme activities can be used in the diagnosis of disease and prognosis
of treatment.
Plasma enzyme levels depend on balance between the rate of influx of active
enzyme into the circulation and its eventual clearance from the blood. The rate
of influx is determined by the rate of release from damaged cells and altered rate
of enzyme synthesis.

23.2.2 Localization of Damage

Enzymes used to measure tissue damage are present in nearly all cells with
varying concentration. So the measurement may indicate an abnormality, but the
specific diagnosis cannot be made. For example if there is circulatory failure
after a cardiac arrest very high plasma levels of enzymes originating from many
tissues may occur because of hypoxic damage to cells and reduced rates of
clearance: the raised plasma levels of ‘cardiac’ enzymes do not necessarily mean
that a myocardial infarct caused the arrest.
The diagnostic precision of plasma enzyme analysis may be improved by
1. Estimation of more than one enzyme. Many enzymes are widely distributed,
but their relative concentrations may vary in different tissues. For eg.
Alanine and aspartate transaminases are abundant in the liver, the
concentration of aspartate transaminase is much greater than that of alanine
transaminase in heart muscle

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2. Isoenzyme determination. Some enzymes exist in more than one form: these Biochemistry
isoenzymes may be separated by their different physical or chemical
properties. If they originate in different tissues such identification will give
more information than the measurement of plasma total enzyme activity:
for example, creatine kinase may be derived from skeletal or cardiac muscle,
but one of its isoenzymes is found predominantly in the myocardium
3. Serial enzyme estimations. The rate of change of plasma enzyme activity
Notes
is related to a balance between the rate of entry and the rate of removal
from the circulation. A persistently raised plasma enzyme activity is
suggestive of a chronic disorder or occasionally of impaired clearance. The
distribution of enzymes within cells may differ. Alanine transaminase and
lactate dehydrogenase are predominantly located in cytoplasm and glutamate
dehydrogenase in mitochondria, whereas aspartate transaminase occurs in
both these cellular compartments. Different disease processes in the same
tissue may affect the cell in different ways, causing alteration in the relative
plasma enzyme activities

23.2.3 Isoenzymes
z Isoenzymes (also known as isozymes) are enzymes that differ in amino acid
sequence but catalyze the same chemical reaction
z Believed to be originating from closely linked genes or from multiple gene
loci
z Evolution from a single form possibly due to long-term mutations
z They vary with respect to their kinetic parameters, electrophoretic mobility,
and localization
z They all have independent action
z Eg.Lactate dehydrogenase have 5 isoenzymes (LDH1, LDH2, LDH3,
LDH4 & LDH5)
z They can be used to identify the specific affected tissues
z They can be differentiated from each other and can be clinically quantified
in the lab

23.3 ENZYMES IN HEALTH AND DISEASES

Estimation of enzymes activities in the serum has many applications in the
diagnosis, differential diagnosis (e.g. in myocardial infarction both AST and
LDH are increased in the serum but in case of pulmonary embolism AST is
normal but LDH is increased), assessing prognosis of diseases, and early
detection of disease (e.g. increase level of ALT in serum in viral hepatitis before

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Biochemistry the occurrence of jaundice). Some important enzymes of clinical significances

are discussed below:
Distribution and application of clinically important enzymes
Enzymes Tissues Clinical applications

Alanineamino Liver Hepato parenchymal diseases

transferase
Notes
Alkaline phosphatase Liver, bone, intestinal Liver and bone diseases
mucosa, Placenta

Amylase Salivary glands, Pancreatic diseases

Pancreas

Aspartate amino Liver, Skeletal muscle, Hepatic parenchymal disease,

transferase Heart, Erythrocytes Muscle disease

Cholinesterase Liver Organophosphorus insecticide

poisoning, Hepatic parenchymal
diseases

Creatine kinase Skeletal muscle,Heart Muscle diseases

Gamma glutamyl Liver Hepatobiliary diseases, Marker

transferase of alcohol abuse

Lipase Pancreas Pancreatic diseases

Lactate Heart, liver, skeletal Hepatic parenchymal diseases,

dehydrogenase muscle erythrocytes, muscle diseases Hemolysis,
lymph nodes, Platelets tumor marker

5’nucleotidase Liver Hepatobiliary diseases

Trypsin Pancreas Pancreatic diseases

23.3.1 Pancreatic enzymes

23.3.1.1. a-Amylase: (EC3.2.1.1; 1,4- a-D-glucan glucanohydrolase; AML)
belongs to hydrolyase class that catalyzes the hydrolysis of 1,4- a-glycosidic
linkages in polysaccharides. They are low molecular weight proteins (54 to 62
kDa) that can pass the glomeruli of the kidneys. It is the only plasma enzyme
physiologically found in urine. The AMY activity present in normal serum and
urine is of pancreatic (P-AMY) and salivary gland (S-AMY)origin.

Clinical Significance
Normal values of amylase: 28-100 U/L = 0.48-1.7 ì kat/L

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Causes of Raised Plasma Amylase Activity Biochemistry

1. Marked increase (five to 10 times the upper reference limit):

z Acute pancreatitis
z Severe glomerular impairment
2. Moderate increase (up to five times the upper reference limit):
z Perforated peptic ulcer Notes
z Acute cholecystitis
z Intestinal obstruction
z Salivary gland disorders like mumps, salivary calculi

23.3.1.2 Lipase: (EC 3.1.1.3; triacylglycerol acylhydrolase; LPS) is a single

–chain glycoprotein with molecular weight of 48 kDa.

Clinical Significance
Normal values: 40-200 U/L
z Plasma lipase levels are elevated in acute pancreatitis and carcinoma of the
pancreas.
z serum amylase is increased in mumps, pancreatic disease or due to some
other cause, whereas lipase is increased only in pancreatitis. Therefore, the
determination of both amylase and lipase together helps in the diagnosis
of acute pancreatitis.

23.3.1.3 Trypsin: (EC 3.4.21.4; no systemic name; TRY) is a serine

proteinase that hydrolyze the peptide bonds formed by the carboxyl groups of
lysine arginine with other amino acids.

Clinical Significance
Normal values of trypsin: 25 ± 5.3 μ g/L
Increased in pancreatic disease. But as there is no distinct role of trypsin
estimation in the routine management of patients with acute pancreatitis, this test
is therefore considered of limited clinical value.

23.3.2 Liver enzymes

The assay of serum enzymes is very useful for the differential diagnosis and
monitoring of various heptobiliy disorders.

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Biochemistry There are three types of enzymes:

1. Enzymes which are normally present inside the hepatocytes released into
the blood when there is a hepatocellular damage= markers of hepatocellular
damage.
2. Enzymes which are primary membrane bound (plasma membrane or side
of hepatocytes) = markers of cholestasis
Notes 3. Enzymes which are synthesized in the hepatocyte = indicates disturbances
in the hepatocellular synthesis.

23.3.2.1 Markers of hepatocellular damage

1. Aminotransferases/Transaminases
The transaminases are enzymes involved in the transfer of an amino group from
a 2-amino- to a 2-oxoacid: they need the cofactor, pyridoxal phosphate for
optimal activity. They are widely distributed in the body.
The 2-oxoglutarate/L-glutamate couple serves as one amino group acceptor and
donor pair in all amino-transfer reactions; the specificity of the individual
enzymes derives from the particular amino acid that serves as the other donor
of an amino group. Thus AST catalyzes the reaction:

COOƟ COOƟ COOƟ COOƟ

AST, P-S-P
H C NH2 + C O C O + H C NH2
CH2 CH2 CH2 CH2
COOƟ CH2 COOƟ CH2
COOƟ COOƟ
L-Aspartate 2-Oxoglutarate Oxaloacetate L-Glutamafe

ALT catalyzes the analogous reaction:

COOƟ COOƟ COOƟ COOƟ

ALT, P-S-P
H C NH2 + C O C O + H C NH2
CH3 CH2 CH3 CH2
CH2 CH2
COOƟ COOƟ
L-Alanine 2-Oxoglutarate Pyruvate L-Glutamafe

The reactions are reversible, but the equilibrium of AST and ALT reactions favor
formation of aspartate and alanine respectively.

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z In the liver, the concentration of ALT per unit weight of the tissue is more Biochemistry
than AST.
z AST and ALT enzymes are more important in assessing and monitoring the
degree of liver cell inflammation and necrosis.
z Elevated plasma ALT are considered to be relatively specific for liver
disease.
z AST may be elevated in other forms of tissue damage, such as myocardial Notes
infarction, muscle necrosis and renal disorders.
z In liver disease, the ALT level is increased markedly compared to AST.
In acute viral hepatitis there is a 100-1000 times increase in both ALT and
AST but ALT level is increased more than that of AST
(a) Aspartate Transaminase (EC 2.6.1.1; L-aspartate:2-oxoglutarate
aminotransferase; AST)
Clinical Significance
Normal values of AST: Male: <35 U/L = <0.60 mkat/L
Female: <31 U/L = <0.53 mkat/L
(b) Alanine Transaminase (EC 2.6.1.2; L-alanine:2-oxoglutarate
aminotransferase; ALT)
Clinical Significance
Normal values of ALT: Male: <45 U/L = <0.77 mkat/L
Female: <34 U/L = <0.58 mkat/L

23.3.2.2 Markers of cholestasis

I. Alkaline phosphatase (EC 3.1.3.1; orthophosphoric-monoester
phosphohydrolase
[alkaline optimum]; ALP). Half-life= 10 days

Clinical Significance
The alkaline phosphatases are a group of enzymes that hydrolyse organic
phosphates at high pH. They are present in most tissues but are in particularly
high concentration in the osteoblasts of bone and the cells of the hepatobiliary
tract, intestinal wall, renal tubules and placenta. The exact metabolic function
of ALP is unknown but it is probably important for calcification of bone In adults
plasma ALP is derived mainly from bone and liver in approximately equal
proportions: the proportion due to the bone fraction is increased when there is
increased osteoblastic activity that may be physiological

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Biochemistry Causes of raised Plasma ALP activity

1. Physiological: There is a gradual increase in the proportion of liver ALP
with age: in the elderly the plasma bone isoenzyme activity may increase
slightly.
2. Bone ailment: rickets and osteomalacia
3. Liver disease:
Notes
4. Malignancy bone or liver involvement or direct tumor production.

Possible Causes of Low Plasma ALP Activity

z Arrested bone growth
z Hypophosphatasia: an autosomal recessive disorder, associated with rickets
or osteomalacia.

Isoenzymes of Alkaline Phosphatase

Bone disease with increased osteoblastic activity, or liver disease with involvement
of the biliary tracts, are the commonest causes of an increased total alkaline
phosphatase activity. The isoenzymes originating from cells of bone, liver,
intestine and placenta may be separated by electrophoresis, but interpretation
may be difficult if the total activity is only marginally raised.
Assays for ALP isoenzymes are needed when:
I. The source of an elevated ALP in serum is not obvious and should be
clarified.
II. The main clinical question is concerned with detecting the presence of liver
or bone involvement
III. In the case of metabolic bone disorders, to ascertain any modifications in
the activity of osteblastes to monitor the disease activity and the effect of
appropriate therapies.
2. Gamma-glutamyl-transferase (EC 2.3.2.21; γ-glutamyl-peptide: amino acid
γ-glutamyletransferase; GGT): catalyzes the transfere of the γ–glutamyl
group from peptides and compounds that contain it to an acceptor Gamma-
glutamyl transferase occurs mainly in the cells of liver, kidneys, pancreas
and prostate. Plasma GGT activity is higher in men than in women.

Clinical Significance
Normal values for GGT Male: <55 U/L = <0.94 μkat/L
Female: <38 U/L = <0.65 μkat/L

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Causes of raised plasma GGT activity Biochemistry

z Induction of enzyme synthesis, without cell damage, by drugs or alcohol.

z Hepatocellular damage, such as that due to infectious hepatitis:

23.3.2.3 Other enzymes

1. Cholinesterase (EC 3.1.1.7, acetylecholine acetylhydrolase), which is
called true cholinesterase or choline esterase I. found in: Notes

(a) erythrocytes
(b) lung and spleen
(c) nerve endings
(d) the gray matter of the brain.
Normal values for CHE: 4.9-11.9 U/mL
Measurements of CHE activity in serum are used:
1. as a test of liver function
2. as an indicator of possible insecticide poisoning
Causes of decreased plasma cholinesterase activity
1. Hepatic parenchymal disease (reduced synthesis)
2. Ingestion or absorption through the skin, of such anticholinesterases as
organophosphates.
Causes of increased plasma cholinesterase activity
1. Recovery from liver damage (actively growing hepatocytes)
2. Nephrotic syndrome

2. Glutamate dehydrogenase (EC 1.4.1.3; L-glutamate: NAD(P)+

oxidoreductase, deaminating; GLD) is a mitochondrial enzyme found
mainly in the:
(a) liver
(b) heart muscle
(c) kidneys but small amounts occur in other tissue, including
(d) brain
(e) skeletal muscle tissue
(f) leukocytes

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Biochemistry Clinical significance

GLD is increased in serum of patients with hepatocellular damage offering
differential diagnostic potential in the investigation of liver disease, particularly
when interpreted in conjunction with other enzyme test results. The key to this
differential diagnostic potential is to be found in the intraorgan and intracellular
distribution of the enzyme. As an exclusively mitochondrial enzyme, GLD is
released from necrotic cells and is of value in estimation of the severity of liver
Notes
cell damage. GLD activity in serum is stable at 4ºC for 48 hours and at -20ºC
for several weeks. The GLD upper reference limits are 6U/L (women) and 8U/
L (men), when a method optimized at 37ºC is used.

23.3.3 Muscle enzymes

23.3.3.1 Creatine Kinase (EC 2.7.3.2; adenosine triphosphate: creatine
Nphosphotransferase CK) CK is most abundant in cells of cardiac and skeletal
muscle and in brain, but also occurs in other tissues such as smooth muscle.The
concentration gradients between some human tissues and serum for creatine
kinase. The concentration gradient is logarithmic

Clinical significance
Normal range for total CK: Male : 46-171 U/L= 0.78-2.90 μkat/L
Female: 34-145 U/L= 0.58-2.47 μkat/L
Serum CK activity is greatly elevated in all types of muscular dystrophy. In
progressive muscular dystrophy (particularly Duchenne sex-linked muscular
dystrophy), enzyme activity in serum is highest in infancy and childhood (7-10
years of age) and may increase long before the disease is clinically apparent.
Serum CK activity characteristically falls as patients get older and as the mass
functioning muscle diminishes with the progression of the disease. About 50%-
80% of the asymptomatic female carriers of Duchenne dystrophy show threefold
to six-fold increase of CK activity. Quite high values of Ck are noted in viral
myositis, polymyositis and similar muscle disease. However in neurogenic
muscle disease, such as:
(a) Myasthenia gravis
(b) Multiple sclerosis
(c) Polimyeltis
(d) Parkinsonism

Serum enzyme activity is normal

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Isoenzymes of CK Biochemistry

CK consists of two protein subunits, M (for muscle) and B (for brain), which
combine to form three isoenzymes. BB (CK-1), MB (CK-2) and MM (CK-3).
CK-MM is the predominant isoenzyme in skeletal and cardiac muscle and is
detectable in the plasma of normal subjects.

CK-MB accounts for about 35 per cent of the total CK activity in cardiac muscle
and less than five per cent in skeletal muscle: its plasma activity is always high Notes
after myocardial infarction. It may be detectable in the plasma of patients with
a variety of other disorders in whom the total CK activity is raised, but this
accounts for less than six per cent of the total. CK-BB is present in high
concentrations in the brain and in the smooth muscle of the gastrointestinal and
genital tracts. Although they have also been reported after brain damage and in
association with malignant tumours of the bronchus, prostate and breast,
measurement is not of proven value for diagnosing these conditions. In
malignant disease plasma total CK activity is usually normal. Approximate
concentrations of tissue CK activity (expressed as multiple activity concentrations
in serum and cytoplasmic isoenzyme composition

23.3.3.2 Lactate Dehydrogenase (EC 1.1.1.27; L-lactate: NAD+

oxidoreductase; LD) catalyses the reversible interconversion of lactate and
pyruvate. The enzyme is widely distributed in the body, with high concentrations
in cells of cardiac and skeletal muscle, liver, kidney, brain and erythrocytes:
measurement of plasma total LD activity is therefore a non-specific marker of
cell damage.

LD has a molecular weight of 134 kDa and is composed of four peptide chains
of two types:

M (or A)
H (or B)
Each under separate genetic control

The subunit compositions of the five isoenzymes are listed below in order of
their decreasing anodal mobility in an alkaline medium.

LD-1 (HHHH; H4) = migrates fastest towards the anode

LD-2 (HHHM; H3M)
LD-3 (HHMM; H2M2)
LD-4 (HMMM; HM3)
LD-5 (MMMM; M4)

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Biochemistry Clinical significance

Normal range of total LDH: 180-360 U/L= 3.1-6.1 μkat/L
It is increased in plasma in Myocardial injury, acute leukemias, generalized
carcinomatosis and in acute hepatitis. Estimation of its isoenzymes in more
useful in clinching diagnosis between hepatic disease and Myocardial.Injury.

Notes Causes of Raised Plasma Total LD Activity

z Artefactual:
Due to in vitro haemolysis or delayed separation of plasma from whole
blood.
z Marked increase (more than 5 times the upper reference limit in adults):
z Circulatory failure with ‘shock’ and hypoxia:
z Myocardial infarction
z Some haematological disorders. In blood diseases such as megaloblastic
anaemia, acute leukaemias and lymphomas. very high levels (up to 20 times
the upper reference limit in adults) may be found.
z Moderate increase. viral hepatitis: malignancy of any tissue: skeletal muscle
disease: pulmonary embolism: infectious mononucleosis.

Isoenzymes of LD
LD1 fraction predominates in cells of cardiac muscle, erythrocytes and kidneys.
LD5 is the most abundant form in the liver and in skeletal muscle. \Whereas in
many conditions there is an increase in all fractions, the finding of certain
patterns is of diagnostic value.
z Predominant elevation of LD1 and LD5. (LD1 greater than LD5 occurs after
myocardial infarction, in megaloblastic anaemia and after renal infarction.
z Predominant elevation of LD2 and LD3 occurs in acute leukaemia: LD3
is the main isoenzyme elevated due to malignancy of many tissues.
z Elevation of LD5 occurs after damage to the liver or skeletal muscle.
Other clinically important enzymes
23.3.3.3 Acid Phosphatase (EC 3.1.3.2; orthophosphoric acid-monoester
phosphohydrolase [acid optimum]; ACP)
Acid phosphatase is present in lysosomes, which are organelles present in all
cells with the possible exception of erythrocytes. Extra lysosomal ACPs are also
present in many cells:

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(a) prostate, Biochemistry

(b) bone (osteoclasts),

(c) spleen
(d) platelets
(e) erythrocytes.
The lysosomal and prostatic enzymes are strongly inhibited by d-tartrate ions Notes
(tartrate-labile ACP), whereas the erythrocyte and bone isoenzymes are not (TR-
ACP)
Normal range of TR-ACP: 1.5-4.5 U/L= 0.03-0.08 ì kat/L
Elevated TR-ACP
(a) Paget disease
(b) Hyperparathyroidism with skeletal involvement
(c) Presence of malignant invasion of bones by cancers
The only nonbone condition in which elevated activities of TR-ACP are found
in serum is Gaucher disease of the spleen, a lysosome storage disease.
The main indications for estimation are to help diagnose prostatic carcinoma and
to monitor its treatment. The estimation is gradually being replaced by the
measurement of plasma prostate specific antigen (PSA) a protein derived from
the prostate. This test is more specific and sensitive for diagnosis and monitoring
treatment. However, it may be raised in similar circumstances to those affecting
prostatic ACP and is more expensive to estimate. ACP is more useful for
monitoring the treatment of a known case of disseminated prostatic carcinoma
than for making the diagnosis.
3.3.4 Glucose -6-phosphate Dehydrogenase (EC 1.1.1.49); D-Glucose -6-
phosphate:
NADP+ oxidoreductase; G6PD) is expressed in all cells and catalyzes the first
step in the hexose monophosphate pathway, the conversion of glucose-6-
phosphate to 6-phosphogluconate, generating NADPH. G6PD deficiency is the
most common ezymeopaty , affecting 400 million people worldwide. More than
400 different types of G6PD variants have been described, leading to different
enzyme activities associated with a wide range of biochemical and clinical
phenotypes.
The majority of G6PD – deficient individuals develop hemolysis only when
oxidative stress occurs, as with infections and after ingestion of certain drugs
or fava beans. Outside these periods, they are usually asymptomatic; however,

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Biochemistry G6PD deficiency also leads to mild to sever chronic hemolysis, exacerbated by
oxidative stress.
The reference interval for G6PD on erythrocytes is 8-14U/g Hb. Values >18 U/
g Hb are often encountered in any condition associated with younger than normal
RBCs but are of no clinical significance

Notes
INTEXT QUESTIONS 23.1
1. Fill in the blanks
1. Clotting enzymes are plasma ................... enzymes.
2. Lactate dehydrogenase has ................... isoenzymes.
3. ................... isoenzyme is increased in myocardial infarction.
4. Specific marker for liver damage is ...................
5. Acid phosphatase is a marker for ...................
2. Match the Following:
1. Lactate dehydrogenase (a) Muscle disease
2. Creatine kinase (b) Cholestasis
3. Alanine transaminase (c) Hemolysis
4. Lipase (d) Hepatitis
5. Alkaline phosphatase (e) pancreatitis

WHAT HAVE YOU LEARNT

z Enzyme concentrations are high in cells. Enzymes are released into the
plasma due to cellular damage.
z Assays of selected enzymes may help to identify the damaged tissues and
isoenzyme studies may increase the specificity. In general, knowledge of
the patterns of enzyme changes, together with the clinical and other
findings, are needed if a useful interpretation is to be made.
z Non-specific causes of raised enzyme activities include peripheral circulatory
insufficiency, trauma, malignancy and surgery.
z Artefactual increases may occur in haemolysed samples.

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z Enzyme estimations may be of value in the diagnosis and monitoring of: Biochemistry

(a) Myocardial infarction (CK. LD and its isoenzymes and sometimes

AST);
(b) Liver disease (transaminases. ALP and sometimes GGT):
(c) Bone disease (ALP):
(d) Prosratic carcinoma (tartrate-labile ACP);
Notes
(e) Acute pancreatitis (á-amylase):
(f) Muscle disorders (CK)
z Plasma enzyme patterns in diseases
(a) Muscle Disease
In the muscular dystrophies -Plasma levels of the muscle enzymes,
CK and the transaminases are increased, probably because of leakage
from the diseased cells.
(b) Hematological Disorders
Very high activities of LD (HBD) may he found in megaloblastic
anaemias and leukemias and in other conditions in which bone
marrow activity is abnormal. Typically there is much less change in
the plasma AST than in the LD (HBD) activities.
(c) Myocardial Infarction
All plasma enzyme activities (including that of CK-MB) may be
normal until at least four hours after the onset of chest pain due to
a myocardial infarction; blood should not be taken for enzyme assay
until this time has elapsed. The simultaneous measurement of plasma
CK-MB activity, which is shown to exceed six per cent of the total
CK activity, may occasionally help in the early diagnosis: a raised
plasma CK-MB activity or concentration alone is not diagnostic of
an infarction.Most of the CK released after a myocardial infarction
is the MM isoenzyme, which is found in both skeletal and myocardial
muscle and has a longer half-life than the MB fraction. After about
24 hours the finding of a high MM and undetectable MB does not
exclude myocardial damage as a cause of high total CK activities:by
this time the plasma HBD activity is usually raised. In most cases of
suspected myocardial infarction measurement of plasma total CK and
LD1 (HBD) activities, together with the clinical and ECG findings,
are adequate to make a diagnosis. Plasma total CK activity alone can
be very misleading.

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 MODULE Clinical Enzymology

Biochemistry A raised plasma total CK activity, due entirely to the MM isoenzvme,

may follow recent intramuscular injection, exercise or surgery, this
is more likely if associated with normal plasma LDl (HBD) or AST
activity.
Newer markers for myocardial infarctions: troponin T and troponin
I are regulatory proteins involved in myocardial contractility. both
being evaluated as an early and specific marker of acute myocardial
Notes
infarction. Elevated serum troponins are more predictive of adverse
outcomes in unstable angina or myocardial infarction than the
conventional assay of CK2.
(d) Enzymes in Malignancy
Plasma total enzyme activities may be raised or an abnormal isoenzyme
detected, in several neoplastic disorders.
z Serum prostatic (tartrate-labile) acid phosphatase activity rises in some
cases of malignancy of the prostate gland.
z Any malignancy may be associated with a non-specific increase in plasma
LD1 (HBD) and occasionally, transaminase activity.
z Plasma transaminase and alkaline phosphatase estimations may be of value
to monitor treatment of malignant disease. Raised levels may indicate
secondary deposits in liver or of alkaline phosphatase, in bone. Liver
deposits may also cause an increase in plasma LD or GGT.
z Tumors occasionally produce a number of enzymes, such as the ‘Regan’
ALP isoenzyme.’LD (HBD) or CK-BB. assays of which may be used as
an aid to diagnosis or for monitoring treatment.

TERMINAL QUESTIONS
1. Write a note on plasma enzyme types
2. What are isoenzymes. Add a note on its clinical significance
3. Write short notes on the following enzymes:
(a) Lactate dehydrogenase
(b) Creatine kinase
(c) Alanine transaminase
(d) Alkaline phosphatase
4. Explain in detail about the enzyme changes in liver diseases
5. Explain in detail about the enzyme changes in myocardial infarction

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Biochemistry

ANSWERS TO INTEXT QUESTIONS

23.1
1. 1. Functional
2. Five
Notes
3. CK-MB
4. ALT
5. Prostatic carcinoma
2. 1. (c)
2. (a)
3. (b)
4. (e)
5. (d)

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 MODULE Immunochemical Techniques

Biochemistry

24
Notes
IMMUNOCHEMICAL
TECHNIQUES

24.1 INTRODUCTION
All vertebrates have advanced immune system. The more complex the organism
the more advanced the immune system. The immune system of mammals has
evolved over a million years; it provides an incredible protection system capable
of responding to infective challenges that arise in the body. Immunity in our body
is monitored or controlled by specific cells from stem cells in bone marrow. The
most important cell types are B and T lymphocytes, which have the ability to
act against bacterial and other viruses. B-cells releases antibody against
stimulation of a foreign substance into the body. An antigen is a foreign
substance capable of an immune response leading to the production of
antibodies. They are the targets to which antibodies bind. So antibodies are
antigen specific (bind to the antigen that initiated its production). This unit
focuses on the identification and diagnostic chemical techniques on the response
of an antibody to a specific antigen. Immunochemical methods are based on
the selective, reversible and non-covalent binding of antigens by antibodies.
These methods are employed to detect or quantify either antigens or antibodies.

OBJECTIVES
After reading this lesson, you will be able to:
z define immunochemical techniques
z explain the characteristic features and roles of antigen – antibody reactions
z describe and distinguish the types of immunochemical techniques

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Immunochemical Techniques MODULE
Biochemistry
24.2 FACTORS CONTROLLING THE IMMUNOCHEMICAL
TECHNIQUES
All the techniques cannot be used for the identification of a specific antigen or
antibody. In order to perform the immunochemical techniques, several controlling
criteria are appropriate:
z Experimental conditions – nature and the place of work, type of sample
collected. Notes
z Nature of Reagents – the quality is studied, standardized and analyzed
z Sensitivity and selectivity of technique to the particular sample

24.3 IMMUNOCHEMICAL TECHNIQUES

Immunochemistry is an advanced area of immunology. It deals with the chemical
components and chemistry (chemical reactions) of immunological phenomena,
that is of antibody and antigen. Immunochemical methods are processes utilizing
the highly specific affinity of an antibody for its antigen. It detects the
distribution of a given protein or antigen in tissues or cells. The methods used
for the immunochemical analysis are called Immunochemical techniques; they
are highly important in diagnostic and clinical context, as now even normal cell
with many proteins are altered in diseased state (in cancer).

24.3.1 Characteristics and Role

24.3.1.1 Characteristics
z Simple, rapid and robust
z Highly sensitive
z Easily automated – applicable to regular clinical diagnostic laboratories
z Does not require extensive and easily destructible sample preparation
z Do not require expensive instrumentation
z Mostly based on simple photo-, fluoro- and luminometric detection
z Measurements may be either qualitative or quantitative.

24.3.1.2 Roles
The characteristic features of these techniques are helpful for the following:
z Function of newly identified or novel proteins can be identified
z Importance of uncharacterized protein in their natural environment can be
analyzed

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 MODULE Immunochemical Techniques

Biochemistry z Determination of species or tissues in which the protein or residues is

expressed
z The cell type or sub-cellular compartment in which the protein can be found
z Detect whether there is any variation in expression of protein during
development of the organism
z Some alteration in the normal expression pattern of a particular protein may
Notes indicate a particular disease state; Gives information that may be useful for
diagnosis and treatment.

24.4 METHODS OF ANALYSIS

All immunochemical methods are based on a highly specific and sensitive
reaction between an antigen and an antibody. Antibodies are immunoglobins,
belonging to a family of glycoproteins – IgG, IgA, IgD, IgM and IgE. Structurally,
antibodies are often visualized as Y-shaped molecules, each containing 4
polypeptides – 2 identical polypeptide units called heavy chains and another
2 called light chains. It has a domain called Fab, the site where it binds to an
antigen. The region of an antigen binding to an antibody is called an epitope
(Fig. 24.1).

Fig. 24.1: Antigen-Antibody reaction

The measure of the strength of the binding is called affinity, and it is usually
expressed in terms of the concentration of an antibody-antigen complex
measured at equilibrium. It is measured by quantitative precipitin curve (basis
for many immunochemical techniques) proposed by Heidelberger and Kendall
in 1935.

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Quantitative precipitin curve: it describes the relationship between the antigen Biochemistry
concentration and the amount of precipitate for a constant quantity of an
antibody. Three zones can be distinguished from the precipitin curve: (Fig. 24.2)

Notes

Fig. 24.2: Precipitin curve

(i) antibody excess zone – first phase where less antigen is present in sample
(ii) equivalence zone – both antigen and antibody are cross-linked forming
precipitate; no free antigen or antigen is present
(iii) antigen excess zone – amount of precipitate reduces due to high antigen
concentration
The precipitin curve forms the basis of most of the immunochemical techniques
that can be performed in clinical laboratories.

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Biochemistry

INTEXT QUESTIONS 24.1

1. .................... cells releases antibody against stimulation of a foreign substance
into the body
2. .................... is a foreign substance capable of inducing the production of
Notes antibodies
3. .................... methods detect or quantify either antigen or antibodies
4. The measure of the strength of the binding is called ....................

24.4.1 Types of Antibody Used

The antibodies used for the methods are produced by various ways:
1. Monoclonal antibody – products of single clone of plasma cells by B-
lymphocytes; mostly prepared in laboratory. They are directed against single
epitope – identical copies with same structure and antigen specificity. They
have excellent specificity but poor ability to precipitate antigen.
2. Polyclonal antibody – they are conventional, i.e., produced by immunization
of animals with antigen. Thus antibody consists of mixture of monoclonal
antibodies having specificity for complex antigens.
Sometimes monoclonal is called as “monovalent” and polyclonal as “polyvalent”
which indicates the antigen specificity.

24.4.2 Types
Based on the type of reaction performed, reagents and samples used, the
techniques are categorized as follows:
z Particle methods – where the antigen-antibody interaction is observed. It
includes:
z Agglutination
z Immunoprecipitation
z Immunoelectrophoresis
z Immunofixation
z Immunoturbidimetry
z Immunonephlometry
z Label methods – either antigen or antibody is labeled and through the label
concentration, the antigen-antibody reaction is observed. This includes:

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z Immunoassay Biochemistry

z Competitive binding
z Other methods – includes immunofluoroscence, immunoelectron microscopy,
etc.

24.4.2.1 Agglutination
Agglutination (from Latin, agglutino – to glue/ attach) is a process of formation Notes
of clumping of cells; it occurs due to reaction of antibody on a particulate antigen
(Fig. 24.3). Among all other antibodies, IgM is a good agglutinin, since it has
high affinity to different antigens.

Fig. 24.3: Agglutination reaction

They may be either:

z Qualitative test – to identify the presence of an antigen or an antibody.

z Quantitative test – to measure the level of antibodies to particulate antigens;
serial dilutions of sample is used.
There are many variations in agglutination method:

z Direct – antigen present on cell surface is directly agglutinated by specific

antibodies.
Eg: in hematology – determination of blood group (ABO typing) diagnosis
of Salmonella typhi – Widal Test
z Indirect (reverse agglutination) – uses particles already coated with antigens
(antibodies) to determine the antibody (antigen) in given sample. Eg:
Pregnancy test – using hormone secretion (human chorionic gonadotropin),
diagnosis of rheumatoid arthritis
z Agglutination inhibition – where competitive binding of antigen occurs;
determine the concentration of soluble antigen in sample.
z Haemagglutination – involve reactions using red blood cells; detection of
diseases and other viruses, blood typing, etc.

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Biochemistry

INTEXT QUESTIONS 24.2

1. Types of antibody used are .................... & ....................
2. .................... is a process of formation of clumping cells
3. Example of Direct agglutination is .................... typing
Notes
4. Example of Indirect agglutination is .................... test

24.4.2.2 Immunoprecipitation
Immunoprecipitation methods include flocculation and precipitation reactions.
When a solution of an antigen is mixed with its corresponding antibody under
suitable conditions, the reactants form flocculating or precipitating aggregates.
They can be assessed visually by the formation of precipitin line at the region
of equivalence – where equivalent amount of antigen and antibody are present.
It may be either:
z Simple – reaction of one antigen and an Precipitin line antibody
z Complex – when many unrelated reactants are used

Aga Aga

Precipitin line

Aba

Fig. 24.4: Precipitation reaction

In simple methods, a concentration gradient is established between the antigen

and antibody; it includes:
z Single radial immune diffusion (SRID) – developed by Mancini; simple
technique where the circular precipitin area originating from antigen well
in gel is equal to amount of antigen concentration.
z Double immune diffusion – developed by Orjan Ouchterlony; both antigen
and antibody diffuse from separate wells in a gel to form precipitin line.
In complex methods, different antigens are compared with an antibody and vice
versa. The result is based on the presence or absence of precipitin line.

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24.4.2.3 Immunoelectrophoresis Biochemistry

Immunoelectrophoresis is a qualitative technique that combines of two methods

one after another:

z Gel electrophoresis – separation of components by charge (Fig. 24.5a)

z Immunodiffusion – diffusion of both antibody and antigen toward each
other produces precipitin line (Fig. 24.5b)
Notes

+ –

direction of electrophoretic separation

migration migration
of antibodies of antigens

Fig. 24.5: (a) electrophoresis, (b) immunodiffusion

It is strictly qualitatively technique to detect antibody and antigen in sample. But

also a quantitative technique exist called rocket immunoelectrophoresis to
measure the antigen level in a sample.

Another variation called as counter-current immunoelectrophoresis where it is

similar to double immunodiffusion.

24.4.2.4 Immunofixation
Immunofixation is used for detection and identifying antibody in serum, urine
and other body fluids (Fig. 24.6). The principle is similar to
immunoelectrophoresis, involves two stages:

z Electrophoresis separation of antibody proteins

z Immunoprecipitation separation with specific antibodies.
It is easy to perform, more sensitive and easily evaluated. In clinical laboratories,
is used for detection for cancerous myeloma through specific antibody.

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Notes

Fig. 24.6: Immunofixation – serum and urine samples

24.4.2.5 Immunoturbidimetry
The precipitin line or curve that forms in a gel, can also be formed in a solution.
When an antigen solution is combined with a specific antibody solution, the
clumping formed results in a cloudy appearance. This is called turbidity; it forms
the principle of immunoturbidimetry. It is measured based on the intensity/
amount of light that pass through the sample. It is performed by an instrument
called spectrophotometer (Fig. 24.7-A).

cuvette with tested sample cuvette with tested sample

incoming light detector incoming light

45°
detector 90°

Fig. 24.7: A – turbidimetry, B – nephelometry

24.4.2.5 Immunonephelometry
Immunonephelometry works on the principle similar to turbidimetry, but this
method measures the intensity of scattered light from sample directly. It uses
laser light and is measured by using a nephelometer (Fig. 24.7-B).

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Both the methods are faster but are more expensive. Increased sensitivity of Biochemistry
antigen and antibody can be got by automation of these techniques.

24.4.2.7 Immunoassay
Immunoassay technique works on the principle of labeling either the antigen or
antibody before the reaction. This increases the sensitivity of detection in the
experiment than the formation of immunoprecipitate. Notes
The label can be of:
z Radio isotope – called as Radio Immunoassay (RIA)
z Enzyme – reaction known as Enzyme Immunoassay (EIA); it also includes
ELISA (Enzyme Linked Immuno Sorbent Assay)
z Fluorescent substance
z Chemilumniscent substance
RIA – first immunoassay technique; based on the measurement of radioactivity
associated with antigen-antibody complexes.
ELISA – safer and efficient method; based on the measurement of an enzyme
action associated with antigen-antibody complexes (Fig. 24.8) Most commonly
used enzymes – biotin, alkaline phosphatase, etc.

Fig. 24.8: ELISA – sandwich type

24.4.2.8 Competitive Binding

Another method to measure the amounts of antigen is competitive binding; in
this technique, it works on the principle of competitive binding:
z Antibody is first incubated in solution having antigen
z This mixture is added to antigen coated reaction well
The more the antigen present in sample, the less the antibody will bind to the
coated antigen. To this, when labeled antibody is added, we can measure the
amount of antigen present in sample.

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Biochemistry 24.4.2.9 Other Methods

z Immunofluorescence – an antibody labeled with a fluorescent molecule
(fluorescein or rhodamine or one of many other fluorescent dyes) is used
to detect the presence of an antigen in or on a cell or tissue by the
fluorescence emitted by the bound antibody.
z Immunoelectron microscopy – uses the specificity of antibody to view
Notes tisssues and its components through a microscope.
z Immunostaining – use of antibody labeled with enzyme or dye to detect
specific protein in a sample. Most important staining method is Western
Blot method that includes three processes:
z Electrophoretic separation of proteins in sample
z Immunoblotting – transfer of proteins from gel to a membrane
z Immunodetection – identification using labeled antibody

24.5 DETECTION
In order to detect and diagnose diseases and also certain body conditions
properly, there are many fast immunochemical techniques. Some examples are:

z Detection of Salmonella typhi (causing typhoid) in blood

z Detection of narcotics in urine and saliva
z Detection of protein hormones like insulin, thyroxine, etc.
z Pregnancy test for level of human choriogonadotropic hormone (hCGH)

WHAT HAVE YOU LEARNT

z Immunity in our body is monitored or controlled by specific cells from stem
cells in bone marrow.
z The most important cell types are B and T lymphocytes, which have the
ability to act against bacterial and other viruses.
z B-cells releases antibody against stimulation of a foreign substance into the
body.
z An antigen is a foreign substance capable of an immune response leading
to the production of antibodies
z Immunochemical methods are based on the selective, reversible and non-
covalent binding of antigens by antibodies

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z The methods used for the immunochemical analysis are called Biochemistry
Immunochemical techniques
z All immunochemical methods are based on a highly specific and sensitive
reaction between an antigen and an antibody
z Antibodies are immunoglobins, belonging to a family of glycoproteins –
IgG, IgA, IgD, IgM and IgE
z The measure of the strength of the binding is called affinity Notes

z Quantitative precipitin curve describes the relationship between the antigen

concentration and the amount of precipitate for a constant quantity of an
antibody
z Types of antibodies used are Monoclonal antibody and Polyclonal
antibody

z Based on the type of reaction performed, reagents and samples used, the
techniques are categorized as follows:
z Particle methods – where the antigen-antibody interaction is observed. It
includes:
„ Agglutination
„ Immunoprecipitation
„ Immunoelectrophoresis
„ Immunofixation
„ Immunoturbidimetry
„ Immunonephlometry
z Label methods – either antigen or antibody is labeled and through the label
concentration, the antigen-antibody reaction is observed. This includes:
„ Immunoassay
„ Competitive binding
z Other methods – includes immunofluoroscence, immunoelectron microscopy,
etc.

TERMINAL QUESTIONS 24.1

1. Give an account of characteristics of immunochemical techniques.
2. What are the different methods of analysis?

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Biochemistry 3. Discuss the various methods of agglutination.

4. Enumerate the differences between immunoturbidimetry and
immunonephlometry.
5. Compare immunoprecipitation and immunoelectrophoresis.
6. Give the importance of immunoassay. Mention its types.

Notes

ANSWERS TO INTEXT QUESTIONS

24.1
1. B
2. Antigen
3. Immunochemical
4. Affinity

24.2
1. Monoclonal & Polyclona
2.Agglutination
3. ABO
4. Pregnancy

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Automation in Clinical Laboratory MODULE
Biochemistry

25
Notes
AUTOMATION IN CLINICAL
LABORATORY

25.1 INTRODUCTION
The word automation is inspired by word automatic. Automatic means
exercising control without interference. So automation means getting work done
by machines which can run on their own without our continuous monitoring.
Automation refers to machines with intelligence and adaptability which reduces
our workload and need for nonstop supervision.

OBJECTIVES
After reading this lesson, you will be able to:
z define automation

z list the uses of automation in clinical lab

z discuss automation at each step of analysis

z describe different types of auto analyzers

z enlist the advantages and disadvantages of automation

25.2 AUTOMATION IN CLINICLA LABORATORY

There are several individual steps in the analysis process as a whole in a
laboratory such as:
1. Identifying the patient
2. Getting the correct sample
3. Identifying and proper labeling of the sample
4. Delivery of sample in proper storage condition and within time

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Biochemistry 5. Preparation of sample for test

6. Sample loading/aspirating
7. Analysis
8. Reporting
9. Entering in register

Notes Imagine if you are asked to add from no 1 to 100 and write the result throughout
the day. How would you feel? Can you assure that after 2 hours of repeating the
same task you will not be bored and make mistakes? But an automated machine
will never feel tired nor make mistake as often as you will.
Automation has a lot of benefits for the laboratory personnel.
1. Reduces the workload
2. Increases turnaround time (Saves time used per analysis)
3. Increases total number of tests done in less time
4. Eliminates repetition and monotony from human life so decreases human
error, improves accuracy
5. Improves reproducibility (repeatability)
6. Uses minimum amount of sample and reagent
In a clinical laboratory set up automation is useful in routine chemistry,
hematology, immunological assay, and daily processing of large number of
samples etc. Usefulness of automation in advanced and well equipped clinical
laboratory can be also extended to
1. Transport of specimen
2. Processing of specimen
3. Loading of specimen into auto analyzer
4. Assessment of results of performed tasks
However, if above steps are not automated due to lack of finance and
infrastructure, still any ordinary laboratory at least go for automation in its
analysis step. The routine techniques and procedures that are done manually by
technicians are replaced by automated analyzers called AUTOANALYZER.

INTEXT QUESTIONS 25.1

State true or false
1. One of the benefits of automation is that it saves reagent
2. One of the benefits of automation is that it reduces turnaround time

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3. The total number of tests is decreased by automation Biochemistry

4. Automation improves reproducibility

5. Automation is has no use in hematology
6. Transport of sample is not part of automation of laboratory

In the beginning of this lesson we enlisted 9 number of steps involved in entire

analytical process for a laboratory. Let us see the possibility of use of automation Notes
in some of these steps.

25.2.1 Sample collection

The use of glucometer is one such example where just by pressing a button on
finger tip, the finger is pricked with least pain and analysis is also done then and
there.
However, not all tests can be done in glucometer. So, automation in sample
collection mainly refers to improved, faster and least discomfort causing
techniques of collection. Such as: robotic system, vacutainers etc.

Fig. 25.1: Blood collection with vacutainer

Blood collected using a vacutainer. Here the phlebotomist need not pull the
syringe, blood gets sucked in due to negative pressure filling the vaccum

Fig. 25.2: Vacutainer

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Biochemistry Different types of vacutainer for serum collection and for plasma collection
using different types of anticoagulants. The identification is done with the colour
of caps used.

25.2.2 Sample identification by Labeling and bar coding

The laboratory information system comes into take part here. It first generates
a unique identity or hospital number for each new patient. A record is maintained
Notes
thereafter for him/her. All sample collected have to bear the name and details
of the patient along with this unique identity (hospital number). The same is used
while entering the details into auto analyzer software and result is also published
with this number and other details.
Some advanced labs are using computer generated bar coding technology for
labeling samples. It has the advantage that it can be scanned and read by bar
code reader accurately so transcriptional error (mistake in writing manually)
is avoided.

Fig. 25.3: An example of stickers with Fig. 25.4: Different samples with different
bar code bar codes pasted on the tube

Fig. 25.5: Bar coding of patient

Bar coding of the patient,unique sample identity is attached to his/her wrist.

Even when patient is sleeping or unconscious, nurse can read the code for patient
identification.

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Notes

Fig. 25.6: Bar code in Blood collection

Bar coding of blood bag in hematology, stores entire detail of type of blood, its
components, storage condition, time of storage etc just by bar codes

25.2.3 Sample delivery

Most of the laboratories rely on human pick up system or conveyer belt system.
Though cheaper, it may lead to human error, delays etc.
Pneumatic tube systems (use of pressurized gas to move the tubes containing
samples) are used in some laboratories. However, care has to be taken that
acceleration and deceleration should not damage any sample.
In very advanced laboratory mobile robots are used.

Fig. 25.7: A sealed sample container at left Fig.25.8: A container to transport blood
culture

The pneumatic tube systems at right sample which can fit into all pneumatic
tube systems

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Biochemistry 25.2.4 Sample preparation

Most of the labs depend on technicians for sample processing (such as serum
separation) as soon as sample arrives. However introduction of automation can
reduce the workload on technicians and save their time and expertise for analysis
purpose. Therefore, now days many semi automated devices are developed
which can analyze whole blood itself. For example, automated ion selective
electrode, use of dry chemistry etc.
Notes
The automated sample processors come in 2 types:
z Stand alone automated sample processors and
z Independent automated sample processors.
These automated sample processors can do the following tasks: sorting of
samples, removing caps, separating samples, bar coding etc. having an
automated sample processor solves the task of bar coding and sample delivery
via conveyer belt system.

Hematology / Pathology lab

Automated sample processors Biochemistry lab

Microbiology lab

Fig. 25.9

The arrow mark represents pneumatic tube system or Conveyer belt

INTEXT QUESTIONS 25.2

Match the following
1. Sample collection (a) Pneumatic tube systems
2. Sample identification (b) Autoanalyzer
3. Sample delivery (c) Different coloured caps
4. Plasma (d) Bar coding
5. Sample analysis (e) Vacutainer

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25.3 SAMPLE ANALYSIS
Now let us study briefly about each aspect of automation in the analysis process
itself which is the minimum essential aspect of automation for any laboratory.
For automation in analysis process we have invented auto analyzer.

25.4 TYPES OF AUTO ANALYZERS

Notes
Auto analyzers based on above principle can be divided into two types
z Open system
z Closed system

25.4.1 Open system

In this system, the operator has the advantage that he can purchase reagents from
any company so he can save money by buying reagents which are cheaper
thereby reducing the cost per test.
In a modular design: An auto analyzer is designed by assembling pulling together
of all parts. This increases the flexibility of machine according to customer’s
demand.
For example: Ion selective electrode. Due to modular approach, the facility for
analysis of sodium, potassium and chloride can be built into the system or can
be added later on.

25.4.2 Closed system

In this system the operator has to buy chemicals only from a particular company,
because reagents from different companies will simply not be accepted by the
machine, so machine will not run. So operator can not reduce the cost per test.
Next, reagents have to be provided in unique containers or format as prescribed
by its manufacturer. This too adds to cost per test.
However, it allows high degree of automation.
Laboratory can be managed by just one or two well trained technical assistants.
The automated machines are designed to function as either a modular
system or an integrated system.

Modular system in auto analyzer

Compare it with modular kitchen where there are many smaller cabinets fitted
according to its use. So if there is a problem with any part, only that part is

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Biochemistry replaced without disrupting the rest of the kitchen. Similarly, in a lab when
whole automated system is subdivided into multiple useful parts it becomes a
modular designed automation. Each part is created separately in such a way that
they can fit into different systems. It is useful because if one part of machine
is out of order, it can be replaced without affecting the entire machine. One such
example is: Roche Modular P auto analyzer.

Notes

Fig. 25.10: A modular kitchen Fig. 25.11: A modular kitchen see the different
types of cabinets

Integrated system in auto analyzer

In integrated system, the entire equipment is designed in such a way that every
part in an essential part of the machine. Basically it is possible due to the merging
of the functionality of different softwares into one integrated solution.
It also comes with the disadvantage that service and maintainance may be
difficult and expensive too. We have to rely on company engineers every time
even for a minor service.
However, it offers a lot of advantages such as
z Improved communication
z Improved data exchange between different softwares and the
machine
Now let us understand different types of processing by auto analyzer. It is
broadly of 2 types:
(a) Continuous flow processing: Based on this principle a continuous flow
analyzer (CFA) is made of different modules, such as:
Sampler

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Pump Biochemistry

Mixing coil
Heater/incubator
Sample treatment chamber (dialysis, distillation etc)
Signal detector
Read out device (data generator) Notes
This provides one analysis per analyte for one sample at a time. The flowing
carrier solution passes through small tubes continuously. This is the main
principle of Continuous flow processing. Here sample is injected into a
flowing carrier solution. The sample mixes with diluents and reagent and it is
sent through the tubing and mixing coils. The machine prevents carry over
effect between different samples by injecting bubbles of air. The air bubbles
literally create separate space for different reactions to take place inside the
tubing and mixing coils.
The tubing passes the samples from one apparatus to the other. There are
different apparatus for different functions, such as ion exchange, heating,
incubation, and finally recording of the signal.

Fig. 25.12: Note the complex pattern of tubing system in a CFA

The flow conditions are regulated. When reaction is taking place, the optical
density of the colour formed is read and results are obtained. So we do not have
to wait till the reaction ends. It has separate heater for promoting enzymatic
reaction and colour development.
Let us take one example for better understanding. In a nephrotic syndrome
patient you want to analyze total protein, albumin and creatinine. In case of
continuous flow processing analyzer the patient sample will be sucked by the
instrument and injected into the tubing with reagents for protein, and diluents

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Biochemistry (if needed). Next air bubbles will be injected and patient sample will be sucked
again. This time instrument will inject reagent for albumin. Mixing will be done
inside the tubing and mixing coils. Again the process will be repeated for
creatinine estimation. The 3 reactions will occur inside the same long tubing but
they will remain separate due to air bubbles in between.
As the sample and standard are treated in the same manner, mixed in same
Notes condition, travel the same length of tubing, it removes the difference
between the two. So the difference in reading for test-tube from that of standard
gives the answer.
Even though originally, CFA was designed to process only colorimetric
reactions, later on CFA were designed to read reactions based on ion selective
electrode, flame photometry, flurometry etc. depending on the need of the
laboratories.
The major uses is for batch analysis such as liver profile, lipid profile, renal
profile assay etc.
There is certain disadvantage in this system:
z Even when there is no test to be done, reagents are drawn to maintain
the flow. This adds to the cost per test.
z Maintainance of instrument is required more frequently.
z The probe and internal tubings must be free of colgs. When there is no
sample the probe must be dipped in distilled water to avoid blockage
or precipitation.
z The machine itself occupies large space.

Fig. 25.13: Image of an aut analyzer based on continuous flow analysis

(b) Discrete processing: in this type of auto analyzer, each sample is provided
a discrete space. It means each analysis even for same analyte or sample
takes place at different cups. This is the main principle of discrete
processing.

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Notes

Fig. 25.14

Let us take the previous example of the nephrotic syndrome patient again. You
want to analyze the same 3 parameters: total protein, albumin and creatinine.
Now, in case of discrete processing analyzer the same patient sample will be
sucked by the instrument and poured into 3 different cups. Then reagents for
protein, albumin and creatinine and diluents (if needed) will be added. Mixing
will be done. Cups will be read at different times to give results.
Exact amount of sample and reagent is aspirated and mixed. So there is no loss
of excess reagents used for flow as in continuous flow processing. As each
analysis is done in different cups and read in different cuvetes, there is no carry
over effect at all. So literally each analysis is discrete from each other.
z This is more useful
z Saves reagent cost and hence popular than continuous flow analysis.
z No sample carry over effects
Based on this principle the auto analyzers developed into two different varieties
such as centrifugal analyzer and random access analyzer.

25.4.2.1 Centrifugal analyzer

Sample and reagents is pipette into different chambers on a rotor. The centrifugal
force is used for transfer and mixing of sample and reagents. Rotor moves the
final product upto the optical system for final reading. This is time saving for
batch analysis because all cuvets can be read at a time. But its disadvantage is
that only one test can be performed at a time.

25.4.2.2 Random access analyzer

Each sample can be analyzed for multiple tests, and multiple samples for one
test also can be done by giving appropriate commands to computer software. It

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Biochemistry is the most versatile of all type analyzers. Let us say we have 3 different
samples. First one needs renal profile, second one needs only glucose and urea
and third one need triglyceride, albumin and calcium. So the technician has to
simple take 3 different sample cups and loads 3 samples. Then he has to enter
sample number, cup number and the tests required. And when he presses the start
button tests will be done automatically.

Notes

Fig. 25.15

INTEXT QUESTIONS 25.3

Match the following
1. Modular design (a) No carry over effect of samples
2. CFA (b) Air bubbles separate samples
3. Integrated design (c) Most versatile analyzer
4. Discrete analyzer (d) Removal of parts easy
5. Random access analyzer (e) Improved communication

25.5 REPORTING
The entire hospital information system can be linked to automated instruments
and reporting authorities’ desktops. So as soon as a sample report is ready,
information goes to reporting officer’s computer.

After he/she approves the result, the same is displayed in all computers in the
hospital. So the clinician or nurses awaiting the report can check at regular
intervals for the report and after matching the patient’s unique hospital number
they can directly take a print out of the report.

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Notes

Fig. 25.16

This minimizes the time delay in reporting and manual report delivery.
Some problems faced in automation in lab
1. Lack of trained personnel at every aspect of automation
2. Automation may not work in a system where administration wants to reduce
cost at every step
3. Hidden costs: trained personnel, supply and maintainance, system upgrading,
service
Therefore, till now in many labs of our country you will not find entire system
automated. However, most of the labs use automation in the form of automated
analyzers discussed in this chapter for this purpose.

INTEXT QUESTIONS 25.4

1. An open system analyzer saves money by reducing ...................
2. A modular system is flexible because its ................... can be replaced
without affecting entire machine.
3. The main feature of CFA is that, a flow carrier solution runs through it
...................
4. The main feature of discrete analyzer is that each reaction takes place
discretely ...................
5. Which analyzer is associated with minimum carry over effect? Choose
between CFA and discrete analyzer.
6. One disadvantage of automation is ................... cost What have you learnt

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WHAT HAVE YOU LEARNT

z Analysis in lab can be automated right from the step of patient identification
upto report delivery. However, due to the hidden cost and demand of trained
personnels at each stage most of the labs restrict their automation only at
the laboratory analysis level. This is mainly done by auto analyzer. Modern
Notes auto analyzers run mostly on the principle of discrete analysis where each
analysis takes place in different cuvets, avoiding carry over effect. Random
access analyzers are the most versatile type where multiple tests can be run
at any time. Integrated system in auto analyzers improve efficiency but
increases maintenance tasks and cost per test.

TERMINAL QUESTIONS
1. What are the individual steps in the analysis process as a whole in a lab?
2. How can you introduce automation in patient identification?
3. Discuss the role of automation in sample identification and delivery.
4. Discuss the disadvantages of CFA.
5. How is discrete analyzer better than CFA?

ANSWERS TO INTEXT QUESTIONS

25.1
1. True 2. False 3. False 4. True 5. False

25.2
1. (e) 2. (d) 3. (a) 4. (c) 5. (b)

25.3
1. (d) 2. (b) 3. (e) 4. (a) 5. (c)

25.4
1. Cost per test 2. Damaged parts 3. Continously
4. In different cups 5. Discrete analyzer 6. Hidden

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Laboratory Quality Management MODULE
Biochemistry

26
Notes
LABORATORY QUALITY
MANAGEMENT

26.1 INTRODUCTION
Have ever accompanied your family members to nearby market for buying
vegetables? Have you noticed how many customers lift few vegetables and either
press or break it into two? For example, to know whether a lady’s finger is
supposed to be good if it is found to be firm while breaking off its tail. Many
vegetables and fruits such as brinjal, apples etc. are supposed to be good if they
are felt as firm but not hard at our hands. So what exactly is the customer doing
by checking one or two vegetable or fruit? And is it sufficient if they check just
one or two randomly? Why don’t they touch and check each and every piece?
Well, what they are doing is they are doing a quality check. And yes, most often
if a random piece is good then mostly the whole bunch is usually almost good.
Similarly, in a laboratory set up if you want to know whether the tests that are
being done are of good standard or not you have to run a quality check. And this
is slightly different from random checking of vegetables. It is a methodical
process with certain rules and regulations which is known as Quality control.
Besides the process, laboratory quality control has to be managed following
regularity and policies. This is otherwise known as Laboratory quality
management.

LITY
UA
E D
O V
Q

P R
AP
L

O
CO N T R
Fig. 26.1

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Biochemistry

OBJECTIVES
After reading this lesson, you will be able to:
z define quality control, accuracy and precision
z define laboratory quality management
Notes z differentiate between precision and accuracy

26.2 QUALITY MANAGEMENTCONTENT

The main objective laboratory quality management is to
(a) Identify errors,
(b) Decrease errors
(c) Correct the root cause of errors
(d) Increase the efficiency of the lab
by regular checking of the analytical process at each step sticking to quality
control material or calibration material (internal and external), before releasing
the patients’ reports .
So what is basically quality control?
Quality control otherwise known as QC is a measure of precision.
Precision means how often the measurement system produces the same result
again and again over time and under different operating conditions.
As we are discussing precision you must also know what accuracy is. Accuracy
is how often the measurement system produces the correct result over time and
under different operating conditions.
For example according to internal quality control (level 2; secondary standard)
of the laboratory, the value of creatinine is 2 mg/dl in the quality control material.
If your laboratory measurement system measures this for 6 times by instrument
A and 5 times by instrument B.
Instrument A produced results as:
(a) 4.1
(b) 4.2
(c) 4.1
(d) 4
(e) 4.1
(f) 4.1

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And Instrument B produced results as: Biochemistry

(a) 4
(b) 0.9
(c) 2.1
(d) 2
(e) 1.9 Notes
(f) 2
Then your Instrument A is precise but not accurate. Why? This is so because,
it 4 out of 6 times produced result close to 4. So it is always producing values
close to one result. But it was supposed to produce result close to 2! Serum
creatinine normal range is roughly 0.5 to 1.1 mg/dl in females and 0.6 to 1.2 mg/
dl in males. And our level 2; secondary standard which corresponds to higher
level standard, value should be close to 2. So it is not accurate and this type of
inaccurate results will make laboratory reports unreliable.
On the other hand, Instrument B is accurate but not precise. Why? This is so
because, 4 out of 6 times, it produced result close to 2. So it is always producing
values close to standard value. But it also produces result far away from that is
0.9 and 4. So it is good for the laboratory to produce accurate result 3 times out
of 5. But also in 2 cases out of 6 if value is so far from correct value, laboratory
cannot be sure whether it’s the right value or one of its precision errors.
For extra information on standards and internal quality control refer chapter on
Primary and Secondary standards.

INTEXT QUESTIONS 26.1

1. The main objective laboratory quality management is to decrease errors
2. Accuracy means how often the measurement system produces the same
result
3. Precision means how often the measurement system produces the correct
result

26.3 LABORATORY QUALITY MANAGEMENT

The management of quality in a laboratory follows an order which works in a
cycle. The various parts are
(a) The goal and objectives set by the management to maintain quality in
laboratory

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Biochemistry (b) To provide initial requirements such as instruments, calibration materials,

trained manpower, a competent lab manager etc.
(c) To set the quality control processes
(d) To do quality assessment at required interval and identify error
(e) To take steps to eliminate error and also to improve quality

Notes
Quality
improvement

Quality
control
GOAL &
objectives
Quality
Quality control
assessment processes

Fig. 26.2 : The management of quality in a laboratory

INTEXT QUESTIONS 26.2

1. Arrange the followings in order.
(a) Set the quality control processes
(b) Provide initial requirements
(c) Take steps to eliminate error
(d) Perform the quality assessment at required interval
(e) Set your goals and objectives

26.4 SETTING OF GOAL AND OBJECTIVES FOR QC

This depends from laboratory to laboratory. This depends on the vision of
management and facilities provided. The objectives for QC is guided by the
keeping the following points in mind

z To set the analytical error limit

z To set the critical range for analytes to make medical decisions

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So we should understand the types of errors that can occur in a laboratory. Biochemistry
Basically all types of errors associated with analysis and final reporting of a
patient’s sample can be broadly divided into 3 categories.
1. Pre-analytical error
2. Analytical error
3. Post analytical error
Notes
26.4.1 Pre-analytical error
It consists of all errors that occur before the patient’s sample reaches laboratory.
One should be aware that most of the errors (around 75%) come under this group.
Therefore, even though being a laboratory personnel we are responsible for
analytical error, we should not ignore pre-analytical error. Laboratory personnel
must be aware of all types of pre-analytical error and be concerned with it.
Because if we can decrease pre analytical error then the total load of analysis
by us will also decrease. It means automatically the percentage of correct reports
generated by our laboratory also will increase. Apart from that it will save the
time, money and our energy spent on processing of wrong sample. This in turn
will improve patient care service by laboratory. Some of the common preanalytrical
errors are described below.
(a) Order the test:
z Inappropriate test: Thyroid hormones should not be ordered within
2 weeks of starting therapy
z Wrong patient identification: It can happen if clinician does not verify
the patient his/her name and other details
z Handwriting not readable etc: One of the most common error
laboratory personnel have to deal with! For example if FBS (Fasting
blood sugar) is not written properly can look like RBS (Random blood
sugar). So a value of 130 for FBS (should be <126mg/dL) that should
have drawn attention for RBS (should be <140mg/dL) would go
unnoticed.
(b) Sample collection
z Wrong patient identification: It can happen if clinician does not verify
the patient his/her name and other details before collecting sample
z Wrong tube: Blood sample for serum should not be collected in
anticoagulated tubes
z Inappropriate volume collected: First the phlebotomist should calculate
the total amount of sample needed. This is done by adding the amount
of sample needed for each test ordered. In this way sample collected
would neither be wasted nor be less for any test.

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Biochemistry z Inappropriate sample collected: For example if sample is hemolyzed

due to incorrect way of collection, it should not be sent for potassium
estimation.
z Collection at wrong time: Sample for cortisol should always be
collected before 8am.
z Delayed transport: Delay in transport can decrease the value of sugar
in blood sample at 7% rate. This happens because sugar gets utilized
Notes by RBC and WBC.
If the laboratory personnel identify pre-analytical error, sample should be
rejected.

INTEXT QUESTIONS 26.3

(Being an efficient lab personnel, do you agree or not with the following
statements. Answer with yes or no)
1. The clinicians are so busy. They need not write the diagnosis or patients
name on requisition form
2. We should not fuss over simple mistakes like writing FBS which looks like
RBS
3. Clinicians and nurses are directly dealing with patients and not by us who
are sitting in laboratory. They can never make mistake in identifying their
on patient
4. If a patient who has come from far distance, for giving blood for serum
cortisol at 12 pm I should not feel pity. I should tell him to come again next
day
5. I do not have a serum tube but there are enough heparinized tube with me.
It is alright if I collect in serum tube, later on I can transfer into a heparinized
tube

26.4.2 Analytical error

Most common analytical errors are
z Faulty calibration of instrument or errors not corrected in instrument

z Interfering substance in sample

z Sample mix up (very dangerous and can lead to legal problems) etc

The errors that occur while analyzing samples can be decreased by using
standard equipments such as:
z Using standard quality of water

z Calibrating the pipettes, glasswares etc

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z Maintaining the instrument within their proper working condition etc Biochemistry

z Checking the quality of analysis (most important) by comparing the values

with quality control materials (external and internal QC material).
The QC sample has to be stable, should be divided into different vials. So we
can analyzes the same QC sample from time to time. Either it has to be bought
from laboratories supplying secondary standards as lyophilized powder for
internal calibration. It can also be prepared by pooling of sera and storing kept Notes
in different aliquots at -20 degree for repeated use. The most common method
for comparing the laboratory value observed for true value of standard is by
drawing control charts or by statistics.
Laboratory quality control material is run at the beginning of each shift or
preferably in the morning and in the evening, also, after an instrument is
serviced, or when a new reagent box is opened etc and of course whenever
patient results seem inappropriate.
The easiest chart is Levey Jenings chart. This chart is named after its inventors
Sir Levey and Sir Jennings in 1950. later it was improved by Henry and
Segalove. Here, the days are plotted in X-axis and observed values for standard
are plotted on Y-axis. The mean and one standard deviation (1SD), two standard
deviation (2SD), and three standard deviation (3SD) limits are plotted on the Y-
axis. Comparing the pattern of plotted points gives the simple way to detect the
z Increased random error
z Any shifts in the trends of calibration

Fig. 26.3: Levey-Jennings chart/Graph

In order to draw a Levey Jenings chart, the QC material has to be analyzed for
20 consecutive days. Then calculate its mean and standard deviation. Now draw
the points on the y axis on a graph sheet with no of days in X axis. Now join the

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Biochemistry points. Next draw the mean as a line parallel to X axis. Next draw the lines on
both sides of mean for range of ± 1SD then ± 2SD. Likewise, ± 3SD then ±42SD
also can be drawn. However, the control limit is taken as mean ± 2SD range.
If any observed value falls out of this mean ± 2SD range, on any day, all
laboratory procedure has to be stopped, error has to be corrected. Till then
patient report should not be given out.
Notes Shewhart chart on the other hand is a statistical process. Here, the short term
estimate of sigma is used unlike Levey-Jennings chart where long term estimate
of sigma (standard deviation) is used.

There is a Westgard multirule procedure which helps us decide which

observed values for QC material is to be rejected. For example,

z If one control value exceeds ± 1SD then it’s a warning.

z If one control value exceeds ± 3SD; or if one value exceeds + 2 SD and
the other – 2SD then these are random errors, so test should be rejected.
Go for error detection. Withhold patients’ results.
z If 2 consecutive values exceed ± 2SD; or 4 consecutive values exceed ±
1SD then these are systematic errors, so test should be rejected. Go for
error detection. Withhold patients’ results.
And so on…..

Here we should know what a random error is. It is an error that occurred by
chance. For example, sudden loss of power leading to decreased incubation
temperature. So this would affect test run only at that time and not to any other
test. So error happens randomly.

So what is a systematic error? It is an error that is occurring systematically to

all tests. For example: use of a faulty pipette. So, an incorrect volume of sample
or reagent will be equally added to all tests. So error happens systematically.

INTEXT QUESTIONS 26.4

1. The pipetting error is a type of ................... error
2. Sample mix up is an analysis error which can lead to ................... problems
3. Westgard rule is for ................... of a test.
4. Levey Jennings chart is drawn after minimum ................... days.

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26.4.3 Post analytical error Biochemistry

Most common post-analytical errors are

z Wrong patient identification so wrong reporting
z· Transcription error (wrong entry, writing not clear)
z Delayed reporting
z Previous values not available for comparison etc. Notes
You should also know that delayed reporting is a post analytical error. Still the
lab is held responsible for it because laboratory personnel are responsible for its
reporting.
The laboratory is controlled by a laboratory manager who is the link between
lab technicians and the management. The laboratory manager is responsible for
satisfying customers’ quality requirements and also to meet the regulations set
by the management as well.
In any organization, the people working behind the machines are most important
and not the machine itself. Therefore, irrespective of the quality of instruments
and other facilities provided quality in laboratory can be maintained to its best
possible standard by the dedication of laboratory personnel. Therefore, a positive
attitude among all laboratory personnel, respect for each other and a sincere
desire to solve problems will improve the over all quality of the laboratory
service.

WHAT HAVE YOU LEARNT

z As the laboratory deals with patients’ samples based on which medical
decisions aret taken it is important to run a quality check and control of
quality. The laboratory has to manage control of quality by certain rules in
order to decrease pre analytical, analytical and post analytical errors which
can occur at random or systemically. For error detection labs rely on various
statistical methos or graphs such as Levey Jennings chart with Westgard’s
rules etc. after error detection the source of error has to be removed, till
then no patient report should be given out.

TERMINAL QUESTIONS
1. What are the steps for management of laboratory quality?
2. Discuss various types of pre analytical errors

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Biochemistry 3. Describe Levey Jennigs chart and role of Westgard’s rules in it.
4. Describe various types of errors that can be detected by Levey Jennigs chart.

ANSWERS TO INTEXT QUESTIONS

Notes 26.1
1. True
2. False
3. True

26.2
(e) Set your goal and objectives
(b) Provide initial requirements
(a) Set the quality control processes
(d) Perform the quality assessment at required interval
(c) Take steps to eliminate error

26.3
1. No
2. No
3. No
4. Yes
5. No

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Electrolytes and Blood Gases MODULE
Biochemistry

27
Notes
ELECTROLYTES AND
BLOOD GASES

27.1 INTRODUCTION
Electrolytes are charged particles (ions) dissolved in the various fluid compartments
of the body (intravascular, interstitial, and intracellular) and perform a variety of
functions in the human body. The electrolytes of importance at this point in the
course are: (1) Sodium; (2) Potassium; (3) Calcium; (4) Hydrogen; and
(5) Bicarbonate. Since hydrogen and bicarbonate ions are primarily involved in
pH balance, they are discussed separately.

Sodium, potassium and calcium will be considered here. Sodium is primarily an

extracellular ion, that potassium occurs primarily intracellularly, and that calcium
performs a variety of functions.

27.2 SODIUM
Sodium is the major extracellular cation. In general, sodium balance defines water
balance.

z Na+ is the most abundant electrolyte in the body Transmission and

conduction of nerve impulses
z Responsible for osmolality of vascular fluids
z Regulation of body fluid levels
z Sodium shifts into cells and potassium shifts out of the cells (sodium pump)
z Assists with regulation of acid-base balance by combining with Cl - or HCO3-
to regulate the balance

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Biochemistry
Abnormalities
Hyponatremia: Excessive sodium loss or H2O gain

Causes
z Prolonged diuretic therapy
z Insufficient Na intake
Notes
z GI losses – suctioning, laxatives, vomiting
z Administration of hypotonic fluids
z Alcoholism
Hypernatremia: occurs with excess loss of H2O or excessive retention of Na.
Can lead to death if not treated

Causes
z Vomiting/diarrhea
z Inadequate AntiDiuretic Hormone
z Major burns

27.3 POTASSIUM
Potassium is the most abundant cation in the body cells. About 97% is found in
the intracellular fluid. Normal extracellular K+ is 3.5-5.3mEq/L. A serum K+
level below 2.5 or above 7.0 can cause cardiac arrest

Functions
z Essential for normal membrane excitability for nerve impulse
z Promotes conduction and transmission of nerve impulses
z Contraction of muscle
z Promotes enzyme action
z Assist in the maintenance of acid-base

Abnormalities
Hypokalemia

Causes
z Prolonged diuretic therapy

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z Inadequate intake Biochemistry

z Gastric suctioning, laxative use, vomiting

z Excess insulin
z Hepatic disease
z Lower Potassium concentration dramatically affects excitable membrane
function of muscle and nerve. Levels < 3 produce marked neuromuscular
Notes
symptoms

Hyperkalemia

Causes
z Shock, severe hemolysis, tumor lysis, burns (cellular release)
z Renal failure (decreased excretion)
z Endocrine diseases with mineralocorticoid deficiency
z “Potassium sparing” diuretics
z Acidosis (K exits cells in exchange for H)
z Artifactual hemolysis of blood specimens; K leak in chilled or unprocessed
specimens

Symptoms
z Greater then 5.0, EKG changes, decreased pH
z Acts as myocardial depressant; decreased heart rate, cardiac output
z Symptoms: muscle weakness, nausea, lethargy, arrhythmias, characteristic
EKG changes, cardiac arrest, GI hyperactivity
z levels > 7.5 produce symptoms and levels > 10 are lethal

27.4 CHLORIDE
The major extracellular anion (expected, 118 - 132 meq/l in serum)

Function
z Important in water distribution, osmotic pressure, and anion-cation balance
z Cl-: regulates osmotic pressure and assists in regulating acid-base balance.
Maintains serum osmolality along with Na+
z Helps to maintain acid/base balance

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Biochemistry z Combines with other ions for homeostasis; sodium, hydrochloric acid,
potassium, calcium
z Closely tied to Na+
z Decreased level is most commonly due to GI losses
z Found in ECF
z Changes the serum osmolality
Notes
z Along with Na+ causes retention of water
z Assists with regulation of acid-base balance
z Cl- combines with hydrogen ion to form hydrochloric acid in the stomach

Abnormalities
Hypochloremia

Causes
z Fluid volume expansion (dilution)
z Renal diseases (reabsorption failure)
z Metabolic acidosis with increased “unmeasured anions” (e.g. DKA)
z SIADH
z GI loss (protracted vomiting)

Hyperchloremia

Causes
z Dehydration
z Renal tubular acidosis, acute renal failure
z Bicarbonate loss (diarrhea)
z Mineralocorticoid excess (Cushing’s syndrome and hyperaldosteronism)
z Diabetes insipidus

27.5 CALCIUM
Ca2+: usually combined with phosphorus to form the mineral salts of bones and
teeth, promotes nerve impulse and muscle contraction/relaxation.

Abnormalities
Hypocalcemia:

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Causes Biochemistry

z Abnormalities of the parathyroid gland

z Inadequate intake
z Excessive losses.

Symptoms
Notes
z Can cause skeletal and neuromuscular abnormalities
z Impairs clotting mechanisms
z Affects membrane permeability
z Diagnostic findings:
z EKG changes
z Serum Ca++levels < 8.5 mg/dL
z Prolonged PT and PTT

Hypercalcemia
z Increased serum levels of Ca++
z Symptoms are directly related to degree of elevation
z Clients with metastatic cancer are especially at risk

Causes
z Excessive intake
z Excessive use of antacids with phosphate-binding
z Prolonged immobility
z Excessive vitamin D intake
z Thiazide diuretics
z Cancer
z Thyrotoxicosis

27.6 MAGNESIUM
z Mg2+ plays role in carbohydrate and protein metabolism, storage and use
of intracellular energy and neural transmission.
z Important in the functioning of the heart, nerves, and muscles

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Biochemistry At a glance
z Hyponatremia (sodium deficit < 130mEq/L)
z Hypernatremia (sodium excess >145mEq/L)
z Hypokalemia (potassium deficit <3.5mEq/L)
z Hyperkalemia (potassium excess >5.1mEq/L)
z Chloride imbalance (<98mEq/L or >107mEq/L)
Notes
z Magnesium imbalance (<1.5mEq/L or >2.5mEq/L)

27.7 BLOOD GAS TESTING

z Blood gas analysis may be carried out in a main laboratory, in an ER or
OR lab or using POC devices.
z Provides accurate measurement of PO2, PCO2, pH, hemoglobin saturation,
bicarbonate and hematocrit (some analyzers also offer electrolytes, lactate,
glucose, Hgb, CO-Hgb, met- and sulfHgb).
z Arterial blood, drawn in appropriate equipment and transported quickly (10
- 15 min) on ice to the lab.
z Check ulnar artery patency before withdrawing blood from radial artery .
z “Arterialized capillary blood” (warmed heel or earlobe) may be acceptable
in some Cases.
z Specimen is accompanied by history with FIO2 and clinical status of patient,
and patient temperature (all are important for interpretation and are included
in the returned report).
z Erroneous results from room temp specimens and specimens with air bubbles
or improper capping.

TERMINAL QUESTIONS
1. Enumerate in detail the functions of the electrolytes Sodium and Potassium
and also describe the related abnormalities.
2. What is hypochloremia? Write down the causes.
3. What is hypokalemia? List out the causes and symptoms.
4. What is hypocalcemia? Write briefly on causes and symptoms of hypocalcmia.
5. List out the uses of blood gas analysis.
6. Normal levels of serum Calcium is………………….
7. Hyponatremia is defined as when levels of sodium falls below…………….
8. …………….. artery blood sample is used for blood gas analysis.

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Centrifugation MODULE
Biochemistry

28
Notes
CENTRIFUGATION

28.1 INTRODUCTION
A centrifuge is the equipment generally driven by an electric motor that puts
an object to rotate around fixed axis, and a perpendicular force is applied to axis.
The particles get separated according to their size, shape, density, viscosity of
the medium and rotor speed.

OBJECIVES
After reading this lesson, you will be able to
z describe centrifugation and its principle
z describe centrifugal force
z practice safety measures in the laboratory

28.2 PRINCIPLE
The centrifuge involves principle of sedimentation, where the acceleration at
centripetal force causes denser substances to separate out along the radial
direction at the bottom of the tube. By the same concept lighter objects will tend
to move to the top of the tube; in the rotating picture, move to the center. In a
solution, particles whose density is higher than that of the solvent sink
(sediment), and particles that are lighter than it float to the top. The greater the
difference in density, the faster they move. If there is no difference in density
(isopycnic conditions), the particles stay steady. To take advantage of even tiny
differences in density to separate various particles in a solution, gravity can be
replaced with the much more powerful “centrifugal force” provided by a
centrifuge.

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Biochemistry What happens to a particle (macromolecule) in a centrifugal field?

Consider a particle m in a centrifuge tube filled with a liquid.
The particle (m) is acted on by three forces:
FC: the centrifugal force
FB: the buoyant force
Ff: the frictional force between the particle and the liquid
Notes

r
w
m

Ff FB
FC

Fig. 28.1: Centrifugal field

28.3 SEDIMENTATION PRINCIPLE

Sedimentation is the tendency for particles in suspension to settle out of the
fluid in which they are entrained, and come to rest against a barrier. This is due
to their motion through the fluid in response to the forces acting on them: these
forces can be due to gravity, centrifugal acceleration or electromagnetism.
Settling is the falling of suspended particles through the liquid, whereas
sedimentation is the termination of the settling process.

28.3.1 Svedberg Equation

The single most important advance in the use of centrifugal force to separate
biologically important substances was the coupling of mechanics, optics and
mathematics by T. Svedberg and J.W. Williams in the 1920’s. They initiated the
mathematics and advanced the instrumentation to a point where it was possible
to prove that proteins were large molecules that could be weighed in a centrifuge.
In honor of that work, the value for a molecule’s (or organelle’s) sedimentation

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Centrifugation MODULE
velocity in a centrifugal field is known as its Svedberg constant or S value for Biochemistry
short.
The instrumentation has progressed quite far since the early work of Svedberg
and Williams. Today, any technique employing the quantitative application of
centrifugal force is known as ultracentrifugation. The design of the basic
instruments, the rotors and the optical systems for measurement are too
extensive to cover in this volume. For our purposes, we will concentrate on two Notes
types of rotor, and a few selected parameters to be measured.
Calculation of S:

v M(1 − vρsol )
S= 2
=
ω r N AV f

M = molecular weight (m × NAV)

s = svedberg coefficient
vρ = partial specific volume of the molecule
N = Avogadro’s number
f = frictional coefficient
s = sedimentation coefficient (units: 1 Svedberg = 10-13 sec)
The above equation depends on the size of the molecule (M), however the shape
of the molecule plays an important role in its behavior under centrifugal force
so it is appropriate to take this (f) into account.
This is the Svedberg equation and is used to describe the motion of the particle
in terms of molecular weight (a size term) and frictional coefficient (a shape
term). The equation also relates the motion to the solvent density.
The Svedberg coefficients are not additive. That is, 40S plus 60S does not equal
100S. This is the case for the ribosomal subunits, where the combination of a
40S small subunit and a 60S large subunit produces an 80S complete ribosome.

28.4 CENTRIFUGAL FORCE

Centrifugal force, word from Latin centrum, meaning “center”, and fugere,
means “to flee”, is the apparent force that draws a rotating body away from the
center of rotation. It is caused by the inertia of the body as the body’s path is
continually redirected. In Newtonian mechanics, the term centrifugal force is
used to refer to one of two distinct concepts: an inertial force (also called
a ”fictitious” force) observed in a non-inertial reference frame, and a reaction force
corresponding to a centripetal force.

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Biochemistry The term is also sometimes used in Lagrangian mechanics to describe certain
terms in the generalized force that depend on the choice of generalized coordinates.

The concept of centrifugal force is applied in rotating devices such

as centrifuges, centrifugal pumps. The two different forces are equal in magnitude,
but centrifugal forces is opposite in direction to the centripetal force.

A centrifuge is used to separate particles or macromolecules:

Notes
1. Cells- biological components in tissues and cells are separated by
centrifugation and this principle is widely used in biological laboratory, in
fact it is one of the most essential instrumentation in design of a laboratory.
2. Sub-cellular components- substances like cytoplasmic fluid, nucleus,
mitochondria, golgi bodies are separated by this principle.
(a) Proteins- based on density protein in cells and tissues is separated
using high speed centrifugation.
(b) Nucleic acids- DNA, RNA, snRNA, etc., are separated by this
method.

28.4.1 Basis of separation

z Size: size of the particle matters a lot while application of this principle.
It has the basis that as much lesser the size will be, more the particle will
be towards the base.
z Shape: the shape of particle ex- circular particles will settle down easily
as compared to polygonal shape particles.
z Density: this component is main play of centrifugation principle, denser the
object, lower the settling.

INTEXT QUESTIONS 28.1

1. The centrifuge works on the simple working principle of .................
2. What are the two distinct forces that come under the “centrifugal force”?
3. The ................., ................. is used to describe the motion of a particle on
the basis of molecular weight and frictional coefficient.
4. 1 Svedberg = ................. unit .................
5. What is the difference between settling and sedimentation?

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28.4.2 Methodology Biochemistry

z Utilizes density difference between the particles/macromolecules and the

medium in which these are dispersed
z Dispersed systems are subjected to artificially induced gravitational fields.

28.4.2.1 Principle

Equipment Notes

The acceleration achieved by centrifugation is expressed as a multiple of the

earth’s gravitational force (g = 9.81 m s–2). Bench-top centrifuges can reach
acceleration values of up to 15000 g, while high speed refrigerated centrifuges
can reach 50000 g and ultra-centrifuges, which operate with refrigeration and
in a vacuum, can reach 500000 g. Two types of rotor are available in high-
powered centrifuges: fixed angle rotors and swing-out rotors that have movable
bucket containers. The tubes or buckets used for centrifugation are made of
plastic and have to be very precisely adjusted to avoid any imbalances that could
lead to accidents.

Relative centrifugal force F = M r ω2

M: mass of particle
r: radius of rotation (cm) (ie.distance of particle from axis of rotation)
ω: Average angular velocity (radians/sec), ω = 2π revolutions/60 minutes

Theory
The velocity (v) of particle sedimentation during centrifugation depends on the
angular velocity ω of the rotor, its effective radius (ref, the distance from the axis
of rotation), and the particle’s sedimentation properties. These properties are
expressed as the Sedimentation

Coefficient S (1 Svedberg, = 10–13 s). The sedimentation coefficient depends

on the mass M of the particle, its shape (expressed as the coefficient of friction,
f), and its density (expressed as the reciprocal density v, “partial specific
volume”). At the top right, the diagram shows the densities and sedimentation
coefficients for biomolecules, cell organelles, and viruses. Proteins and protein-
rich structures have densities of around 1.3 g cm–3, while nucleic acids show
densities of up to 2 g cm–3. Equilibrium sedimentation of nucleic acids therefore
requires high-density media – e.g., concentrated solutions of cesium chloride
(CsCl). To allow comparison of S values measured in different media, they are
usually corrected to values for water at 20°C (“S20W’’).

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Biochemistry
28.5 DENSITY GRADIENT CENTRIFUGATION
Density gradient centrifugation is used to separate macromolecules that differ
only slightly in size or density. Two techniques are commonly used. In zonal
centrifugation, the sample being separated (e. g., a cell extract or cells) is placed
on top of the centrifugation solution as a thin layer. During centrifugation, the
particles move through the solution due to their greater density.
Notes The rate of movement basically depends on their molecular mass. Centrifugation
stops before the particles reach the bottom of the tube. Drilling a hole into the
centrifugation tube and allowing the contents to drip out makes it possible to
collect the different particles in separate fractions.
During centrifugation, the solution tube is stabilized in the tube by a density
gradient. This consists of solutions of carbohydrates or colloidal silica gel, the
concentration of which increases from the surface of the tube to the bottom.
Density gradients prevent the formation of convection currents, which would
impair the separation of the particles. Isopycnic centrifugation, which takes
much longer, starts with a CsCl solution in which the sample material (e. g.,
DNA, RNA, or viruses) is homogeneously distributed. A density gradient only
forms during centrifugation, as a result of sedimentation and diffusion processes.
Each particle moves to the region corresponding to its own buoyant density.
Centrifugation stops once equilibrium has been reached. The samples are
obtained by fractionation, and their concentration is measured using the
appropriate methods.

28.6 MOVING BOUNDARY/ZONE CENTRIFUGATION

In moving boundary (or differential centrifugation), the entire tube is filled with
sample and centrifuged. Through centrifugation, one obtains a separation of two
particles but any particle in the mixture may end up in the supernatant or in the
pellet or it may be distributed in both fractions, depending upon it size, shape,

Low Faster Higher Highest

speed speed speed speed

Fig. 28.2: Differential Centrifugation

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density, and conditions of centrifugation. The pellet is a mixture of all of the Biochemistry
sedimented components, and it is contaminated with whatever unsedimented
particles were in the bottom of the tube initially. The only component which is
purified is the slowest sedimenting one, but its yield is often very low. The two
fractions are recovered by decanting the supernatant solution from the pellet. The
supernatant can be recentrifuged at higher speed to obtain further purification,
with the formation of a new pellet and supernatant.
Notes
28.7 RATE ZONAL CENTRIFUGATION
Particles of the same size (M) but different shapes (e.g., linear versus globular)
will separate - the particle with the greater frictional coefficient (f) will move
slower (rod shaped moves slower than globular). This technique is called velocity
gradient centrifugation (a gradient of sucrose is used to linearize the motion of
the particles).

Centrifugal force

Gradient of sucrose
Initial layer concentration, Separated bands
of mixture hence density

Fig. 28.3: Velocity Gradient Centrifugation

In rate zonal centrifugation, the sample is applied in a thin zone at the top of
the centrifuge tube on a density gradient. Under centrifugal force, the particles
will begin sedimenting through the gradient in separate zones according to their
size shape and density. The run must be terminated before any of the separated
particles reach the bottom of the tube.

Sample
Three proteins
separate according
to Si and shape

Fig. 28.4: Rate Zonal Centrifugation

Particles can be separated by density. When the density in the solvent equals the
density of the particle, the denominator of the equation equals zero and therefore

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Biochemistry velocity equals zero - the particle reaches its equilibrium density in the solvent
this is called equilibrium density gradient centrifugation or isopycnic banding.

(a) Before centrifugation

CsCl and sample uniformly
distributed
CsCl DNA
Notes (b) After centrifugation
CsCl redistributes giving
density gradient
Nucleic acid species form
bands at ''equal density''
levels
Fig.28.5: Before and After Centrifugation

28.8 ISOPYCNIC CENTRIFUGATION

In isopycnic technique, the density gradient column encompasses the whole
range of densities of the sample particles. The sample is uniformly mixed with
the gradient material. Each particle will sediment only to the position in the
centrifuge tube at which the gradient density is equal to its own density, and there
it will remain. The isopycnic technique, therefore, separate particles into
separate zones solely on the basis of their density differences, independent of
time. In many density gradient experiments, particles of both the rate zonal and
the isopycnic principles may enter into the final separations. For example, the
gradient may be of such a density range that one component sediments to its
density in the tube and remains there, while another component sediments to the
bottom of the tube. The self generating gradient technique often requires long
hours of centrifugation.

Low density

Medium density

High density

Fig. 28.6: Isopycnic separation with self-generating gradient - the sample is evenly
distributed throughout the centrifuge tube

Isopycnically banding DNA, for example, takes 36 to 48 hours in a self-

generating cesium chloride gradient. It is important to note that the run time

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cannot be shortened by increasing the rotor speed; this only results in changing Biochemistry
the position of the zones in the tube since the gradient material will redistribute
farther down the tube under greater centrifugal force.

Main band DNA

Concentration of DNA

Notes

Satellite DNA

1.69 1.701
Density (g/cm3)

Fig. 28.7: Isopycnic banding of DNA

INTEXT QUESTIONS 28.2

1. The relative centrifugal force, F = ...................
2. What are the two types of rotors found in high-powered centrifuges?
3. Write the type of centrifugation based on the given characteristics:
z separate particles into separate zones solely on the basis of their
density differences, independent of time ...................
z particles with same size but different shapes separate ...................
z separate macromolecules that differ only slightly in size or density
...................
4. True/False.
z Moving boundary centrifugation is also called differential
centrifugation
5. Equilibrium density gradient centrifugation is also called as ...................

28.9 SAFETY MEASURES

28.9.1 Safety while using centrifuge
1. The work surface must be level and firm. Do not use the centrifuge on an
uneven or slanted work surface.

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Biochemistry 2. Balance the tubes in the rotor! If you want to run a tube with 10 ml of liquid,
put another tube with 10 ml of water in the opposing hole on the rotor. If
the liquid has a higher or lower density than water, you must balance the
tubes by mass, not volume.
3. Do not open the lid while the rotor is moving. Even though many centrifuges
have a “safety shutoff” if the lid is opened, the only thing this does is stop
Notes powering the rotor. The rotor will still spin due to its own inertia for a while
until friction slows and eventually stops it.
4. If you see it wobbling or shaking, turn it off or pull the plug. A little vibration
is normal, but excessive amounts can mean danger. FIRST, double check
that you have correctly balanced the tubes. If the answer is yes and the
wobbling still happens, contact the manufacturer or dealer and get the unit
serviced. Do NOT continue to run a centrifuge that wobbles visibly when
the rotor is spinning.
5. Wear a face shield and / or safety goggles if you have to work anywhere
near a centrifuge that’s in use.
6. Do not bump, jar, or move the centrifuge while the rotor is spinning. Make
sure you don’t have the cord dangling from a table edge where someone
could catch their foot in it and pull down the centrifuge.

28.9.2 Precautions – working with bio-hazardous materials

The following procedures for centrifugation shall be used when working with
bio-hazardous materials:

z Examine tubes and bottles for cracks or stress marks before using them.
Discard any centrifuge tubes that have cracks in them.
z When working with bio-hazardous materials, wipe outside of tubes with
disinfectant prior to removal from the biological safety cabinet and before
placing in safety cups or rotors.
z Place all tubes in safety buckets or sealed rotors when centrifuging
infectious materials.
z Inspect the “O” ring seal of the safety bucket and the inside of safety buckets
or rotors. Open safety buckets or rotors in a biological safety cabinet.
z If any spills or leakage are apparent in the centrifuge rotor should be cleaned
with a mild detergent, rinsed thoroughly with distilled water, and allowed
to air dry completely (while in bio-safety cabinet).
z Clean the rotor and centrifuge well after each use.

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28.9.3 Maintenance of Centrifuge Biochemistry

z Quality control
z Initial installation
z Initial calibration should be performed only by a qualified service technician.

28.9.3.1 Daily maintenance

z Wipe the inside of the bowl with disinfectant solution and rinse thoroughly. Notes
z The centrifuge must not be used if the interior is hot, if unusual vibrations
or noises occur, or if deterioration (corrosion of parts) is detected. A
qualified service technician should be contacted.
z Most vibrations are due to improper balancing and can be corrected by re-
balancing the buckets and tubes.

28.9.3.2 Monthly maintenance

z Clean the centrifuge housing, rotor chamber, rotors and rotor accessories
with a neutral cleaning agent.
z Clean plastic and non-metal parts with a fresh solution of 5% sodium
hypochlorite (bleach) mixed 1:10 with water (one part bleach plus nine parts
water).

28.9.3.3 Annual maintenance

z The centrifuge must be serviced annually by a qualified service technician
who must ensure that the unit operates safely and properly.
z The service should include cleaning condenser coils, fans, screens and
filters, checking the centrifuge brushes, bearings, timer, temperature and
speed, and checking for electrical integrity.
z The service technician must issue an inspection certificate indicating
compliance with safety and proper operation. The most recent inspection
certificate must be displayed close to the centrifuge.

28.9.3.4 Rotor safety

z It is an unavoidable fact that rotors age and become fatigued with repeated
use. For the highest speed centrifuges, ultracentrifuges, rotors must be
“derated” (their maximum rpm reduced gradually over time) and then
eventually retired after a period of continuous use. This is due to the high
stress on the titanium or aluminum from the ultra high gravitational forces,
which will eventually lead to failure. Further, equipment that lasts 10–15
years can have multiple users, and rotors are often passed down over the
life of the centrifuge. It is therefore important to manage all the rotors;

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Biochemistry modern centrifuges have many built-in features that assist in this process,
even for multiple user environments:
1. Rotor-life management allows end users to manage ultracentrifuge
usage by rotor serial number, total number of hour’s used or total
number of cycles. This enables the centrifuge to alert the end user
when it is time to derate or retire a rotor. Furthermore, once a serial
number is denoted as derated, the centrifuge will not allow the rotor
Notes to be used in excess of the new reduced speed.
2. Automatic rotor ID prevents the running of non specified rotors in
a given centrifuge. Rotor ID is achieved in a variety of ways, such
as a magnet configuration on the bottom of the rotor, which is read
by a sensor in the bottom of the chamber. Sometimes rotors are
identified by inertia readings taken during acceleration, which compare
the amount of energy being used to turn the rotor by the motor to the
on-board database, which is programmed with the correct amount of
energy required for all rotors usable in that unit.
3. Over speed protection and rotor ID are closely related. During an
inertia check, the on-board system confirms that the programmed
speed does not exceed the maximum rpm for the specified rotor. In
the case of ultracentrifugation, speed disks on the bottom of the rotor
are read by an optical eye, which limits the maximum speed by the
number of black segments on the disk.
4. Rotor inspection/clinics can be conducted in the laboratory by most
service technicians. Trained service technicians can inspect and
educate laboratory staff on warning signs to look for that indicates
imminent rotor failure.

INTEXT QUESTIONS 28.3

1. What is the meaning of the word “derated”?
2. True/False.
z The work surface must not be level and firm.
z Automatic rotor ID prevents the running of non specified rotors in a
given centrifuge.
3. Match the following.
1. Daily maintenance (a) Qualified service technician
2. Monthly maintenance (b) Derated
3. Annual maintenance (c) Disinfectant solution
4. Rotor safety (d) Nueral cleaning agent
4. ................. and ................. are closely related in rotor safety

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Biochemistry

WHAT YOU HAVE LEARNT

z Centrifuge plays a vital role in the separation of biomolecules
z The techniques are very simple but has its role in advanced studies
z Different types of centrifuges are available for different purposes
Notes

ANSWERS TO INTEXT QUESTIONS

28.1
1. Sedimentation
2. z Inertial force
z Reaction force
3. Svedberg equation
4. 10 -13 ; Unit – seconds
5. Settling is the falling of suspended particles through the liquid, whereas
sedimentation is the termination of the settling process.

28.2
1. F = Mr2
2. Fixed angle rotors and Swing-out rotors
3. z Isopycnic centrifugation
z Rate zonal centrifugation
z Density gradient centrifugation
4. True
5. Isopycnic banding

28.3
1. The meaning of the word “derated” is the rotor’s maximum rpm reduces
gradually over time.
2. z False z True
3. 1. (c) 2. (d) 3. (a) 4. (b)
4. Over speed protection and rotor ID

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 MODULE Primary and Secondary Standards

Biochemistry

29
Notes
PRIMARY AND SECONDARY
STANDARDS

29.1 INTRODUCTION
Often in our life we come across the word ‘standard’. Whether it is the standard
of bus service or behaviour or education, we use this term very commonly in
our life. So why are we obsessed with standard? Is standard necessary in all
spheres of our life? If the answer is yes then should we not follow a standard
in our laboratory too? If yes, then next question arises how to prepare a standard
in our lab.

OBJECTIVES
After reading this lesson, you will be able to:
z define standard
z explain its uses
z classify standard with proper example
z explain various properties of standards

29.2 STANDARDS
In Biochemistry, the word standard means a material containing a substance of
our interest with a known concentration. We can express this with definite
numbers with proper units. By using this standard we can find out the
concentration of that substance in a new material. Therefore, primary standard
serves the purpose of being the primary calibrator or primary reference material.

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1. Functions Biochemistry

Therefore, standard has the following uses in Biochemistry lab

(a) To provide a reference using which we can determine unknown concentration
(b) To standardize volumetric solutions
(c) Preparation of secondary standard
(d) To calibrate an instrument. Notes

2. Types
Standards can be divided into two types:
1. Primary standard
2. Secondary standard

29.2.1. Primary standard

From the name itself it is obvious that this is a standard which comes first. That’s
why the name is primary.
A primary standard is a chemical or reagent which has certain properties such
as
(a) It is extremely pure,
(b) Highly stable
(c) It is anhydrous
(d) It is less hygroscopic
(e) Has very high molecular weight
(f) Can be weighed easily
(g) Should be ready to use and available
(h) Should be preferably non toxic
(i) Should not be expensive

Having said that lets us understand each point one by one.

z A primary standard material should be extremely pure which means that
it should be a chemical of high grade of purity, preferably 99.98%. In a
chemistry lab you will come across chemicals of different grade of purity.
If you check the label you will notice a number with percentage termed as
purity. So when a chemical has purity of 99.98% or more it is a suitable
material to be considered for primary standard.

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Biochemistry Usually those chemicals that exceed the requirement of American

chemical society (ACS) are extremely pure and can be used for making
primary standard. It is an analytical reagent of extreme purity which is
specially manufactured for the purpose of being used as primary standard.
z It should be highly stable which means it usually does not react easily when
kept in its pure form. Or in other words it should have very low reactivity.
This is important because if a reagent reacts easily with atmospheric oxygen
Notes or water or changes its property over time then it is unreliable. We can never
use such unstable and unreliable chemicals as standard.
z It should be anhydrous which means that it does not contain any water
molecule in its molecular structure. For example, in a chemistry laboratory
you will come across same chemical with different number of water
molecules attached with it. Let us see the example of Epsom salt. The
chemical name is magnesium sulphate. So we write the formula is MgSO4.
But the chemical Epsom salt which is found in grocery or drug store is a
chemical with formula MgSO4.7H2O. Therefore if you want to prepare a
primary standard of magnesium sulphate you should purchase an anhydrous
MgSO4 preferably an analytical reagent grade chemical and with purity
greater than 99.98%. Remember such reagents are usually available with
common vendors.
z Just being anhydrous is not sufficient. The chemical preferably should be
less hygroscopic that is on opening the container it should not absorb water
molecules from atmosphere. Why water should not enter into chemical?
This will be clearer in the following point where it is explained how
presence of water molecule can affect the simple calculation of standard
concentration making the entire standardization procedure unreliable.
z Has very high molecular weight compared to its other similar forms. If we
take the same example of Epsom salt you can understand this statement.
Let us take 1 gram of MgSO4 fit for making a primary standard and we
call it salt A. Now take 1 gram of MgSO4.7H2O fit for common uses and
name it salt B. Now you compare the actual weight of magnesium sulphate
to make a standard solution for both chemicals. The molecular weight of
MgSO4 is 108 but for MgSO4.7H2O it is 234.
In case of 1 molecule of salt A the weight of actual MgSO4 will be 108
atomic mass unit. But In case of 1 molecule of salt B the weight of actual
MgSO4 will be 108 out of its total weight of 234 atomic mass unit.
108 gram salt A (MgSO4) will give 108 gram of MgSO4
So 1 gram MgSO4 salt will give = 108/108 = 1 gram of MgSO4
But 234 gram salt B (MgSO4.7H2O) will give 108 gram of MgSO4
So 1 gram MgSO4.7H2O salt will give = 108/234 = 0.461 gram of MgSO4

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Therefore if you by mistake make a standard out of salt B, you will actually Biochemistry
be taking 0.461 gram of MgSO4 and calculating it as 1 gram. So with this
faulty standard estimation of MgSO4 in other unknown solution will give
less result than the actual concentration
Therefore it is important that primary standards must be anhydrous and of
high molecular weight.
z It can be weighed easily because it is so pure that its weight is in fact a Notes
true representative of number of moles present in its actual weight.
z One of the uses of primary standard is to standardize a volumetric solution.
That means they are used for standardization of titration of solutions. It can
be used for titration of acids as well as bases. Let us see how a primary
standard is used for titration.
„ In a biochemistry laboratory the most common standards for acid
titration we use basic chemical such as sodium carbonate (Na2CO3),
Tris which is also known as trisaminomethane [(CH2OH)3CNH2] etc.
you should know that Tris is a very commonly used chemical in
Biochemistry and Molecular biology lab.
„ For base titration we use potassium hydrogen phthalate [(KHP):
KHC8H4O4] etc.
„ Another type of titration is redox titration which is very common in
biochemistry lab. For this we frequently use potassium dichromate
(K2Cr2O7) very often as primary standard. Sodium oxalate (Na2C2O4)
is also used for this purpose.
z The primary standard is used for calibration of secondary standard or for
method validation against a definitive method and it corresponds to the true
value of the substance analyzed.

29.2.2 Secondary standard

From the name itself it is obvious that this is a standard which comes second.
That’s why the name is secondary.
A secondary standard is used by standard laboratories such as companies
involved in preparation of reagents, kits or laboratories responsible for
producing quality control material for other labs. They use primary standard as
the primary calibrator or primary reference material. Secondary standard in turn
is used for the purpose of calibration of control material in smaller lab for
analysis of unknown concentration of a substance. So basically, secondary
standard serves the purpose of external quality control for smaller labs. This
makes it essential that the secondary standard must first be standardized against
the primary standard.

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Biochemistry z There are other points one has to remember. For preparing the secondary
standard solution one must use aquous solution of high grade purity. It must
be deionized if aquous solvent used is water. Without pure solvent, the
standard solution prepared will be worthless (These points are also
applicable for preparing primary standard solution).
z Similarly before using high grade chemicals one should also be vigilant and
Notes check for date of manufacture, expiry date, date of receipt of chemical,
whether the conditions for its transport was followed or not, if the seal is
not tampered with, its purity, standard reference material used etc.
A secondary standard is a chemical or reagent which has certain properties
such as
(a) It has less purity than primary standard
(b) Less stable and more reactive than primary standard
(c) But its solution remains stable for a long time
(d) Titrated against primary standard
Usually a chemical fit for being a standard chemical yet does not meet the
requirements of a primary standard
z The best example is anhydrous sodium hydroxide (NaOH). It is extremely
hygroscopic. As soon as the bottle is opened NaOH starts absorbing
moisture from atmosphere and within no time it becomes moist. You can
experiment it in your lab. Take the NaOH bottle near an analytical balance.
Place a Petridis and make its weight as zero (by using the tare button). Now
open the container and place little NaOH crystal on it and quickly note the
weight. Now keep the glass windows of the analytical balance open for few
minutes and notice the gradual increase in its weight in terms of mg units.
This is because the NaOH crystals absorb water molecule from air.
(Remember to use a glass container such as a Petridis to weigh the chemical.
Because, NaOH is a corrosive for metal panel of balance).
z Another example is potassium permanganate (KMnO4) very often as
secondary standard. It is a good oxidizing agent or in other words it is
reactive so less stable. More often due to its reactivity, its own oxidized
product manganese oxide (MnO2) contaminates the content. That’s why it
is unsuitable for being a primary standard. But it can be used very well as
a secondary standard.
z Next question is why is secondary standard called still a standard? This is
so because secondary standard is used as a calibrator by smaller laboratories
involved in actual analysis of unknown samples.

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In our text we have used the term calibration. So what is calibration? This is the Biochemistry
process by which we compare the measurements by a standard or an instrument
(primary) with another standard or an instrument (secondary). By doing so, we
try to eliminate any variation or difference in measurement by the secondary
standard or an instrument. The other term for calibration is standardization. Have
you noticed how the shopkeeper puts a standard weight on his balance to his right
and adds potatoes or tomatoes etc to the left? In this case the weight is the
standard and the vegetables are being standardized with equal measurement of Notes
for their weight. So if there is a mistake in the weight he uses (let us think it
is less than what is written on it) on his right side we may be cheated and get
less vegetable than we are paying for
z Calibration using titration: take the example of vitamin C estimation from
lemon. For this the standard is ascorbic acid which is available in your
laboratory. Take ascorbic acid and prepare a standard solution of known
concentration (for example 40mg/dl). Next prepare a diluted solution of
indicator called 2 Di -chloro indophenol (2- DCIP) and add ascorbic acid
drop by drop till it is completely decolourized. Let us say we used 20 ml.

Fig. 29.1: Original colour of 2 DCIP indicator

100 ml ascorbic acid standard solution has 40 mg of ascorbic acid in it
1 ml of standard solution has = 40/100 = 0.4 mg ascorbic acid
Now 20 ml of standard solution has = 0.4 ×20 = 8 mg ascorbic acid
This says that our 2- DCIP requires 8 mg of pure ascorbic acid for its complete
neutralization or titration.

Fig. 29.2: Lemon

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Biochemistry Next let us use our lemon. Weigh the lemon. Let us say it weighs 4 g. add lemon
juice drop by drop. Let us say it took several drops of lemon juice for complete
neutralization of 2- DCIP. Now weigh lemon and now it weighs 2 g. So it proves
that 2g of lemon juice present in 4 g of lemon has 8 mg of ascorbic acid. Why
8 mg of ascorbic acid? Simple. This is so because; 8 mg of ascorbic acid is
required to completely neutralization 2 DCIP. So from this we came to know
that
Notes
4 g of lemon has 8 mg of ascorbic acid

So 100 g of lemon would contain = 50 mg of ascorbic acid

So concentration of ascorbic acid in our variety of lemon is 50 mg/100 g that

is equal to 50 mg%.

z The secondary standard is used for calibration of control materials in a lab

using a reference method and it is closer to the true value of the substance.
So it is used for external quality control programme.
z Whereas in a clinical laboratory unknown samples are analyzed against
control materials using a routine/ field method
Let us compare the situation in biochemistry laboratory dealing with human
samples. Here, we often use the term primary and secondary calibrator instead
of calling it primary and secondary standard. A primary calibrator is a preparation
with a stated purity (> 99.98%). The reference measurement procedure is
calibrated with primary calibrator. Using this reference measurement procedure
the Institute for Reference Materials and Measurements (IRMM) determines
the value of secondary reference materials.

Even the companies, which sell their kits and reagents to us, use these secondary
reference materials. So their calibrator products are basically secondary
reference materials. We use these secondary calibrators as reference in our
laboratories. The primary calibrator is used only by institutes like IRMM which
are involved in quality assurance at National level.

Preparing a primary standard out of a pure chemical is relatively easier

(Remember? It is one of the criteria to be a primary standard.). For biological
analytes, we can not prepare the secondary standard by dissolving the analyte
in water. It is so because in our blood they are dissolved in plasma and not in
water. For example, for plasma proteins, there is a human reference serum
material which comes with a certificate of concentrations present for 12 serum
proteins from IRMM.

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Biochemistry
Primary standard At the National level

Reference measurement procedure/Definitive method

At the Company level

Secondary standard (prepared and supplied by company)
Notes
Company measurement procedure/ Reference method

Product calibrator = Secondary standard supplied by company

(external quality control)

At the End-User level

Routine procedure in lab/ Field method
(standardization against Product calibrator)

Value assignment/Internal quality control/analysis of sample etc.

Flow chart summarizing the use of primary and secondary standards

in a clinical laboratory set up

INTEXT QUESTIONS 29.1

1. Standard can be of ................... types.
2. A primary standard should be ................... pure.
3. Between primary and secondary standards, ................... standard is more
reactive.
4. We can calibrate a ................... or ................... using primary standard.
5. Secondary standard can be prepared by ................... using primary standard.

WHAT HAVE YOU LEARNT

z A standard also called a calibrator is a material containing a substance of
our interest with a known concentration, which can be expressed with
definite numbers and units. It is of 2 types primary and secondary. Primary
standard is purer, more stable, less reactive, anhydrous and less hygroscopic

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Biochemistry compared to secondary standard. Secondary standard is titrated or calibrated

against primary standard and used as a reference material in all labs across
country for the purpose of analysis. Preparation of standard solution should
use pure, de-ionized aqueous solvent to keep the quality of standard intact.
In a clinical lab set up, primary standard acts as a primary reference material.
Secondary standard is used as an external quality control for calibration of
internal quality control in labs. Unknown sample is analyzed after both
Notes external and internal quality check is complete and satisfactory.

TERMINAL QUESTIONS
1. What are the criteria of a primary standard?
2. What are the criteria of a secondary standard?
3. What are the differences between primary and secondary standards?
4. What are the uses of primary standard?
5. Why should the primary standard be of high molecular weight?
6. Why can’t you use NaOH as a primary standard?
7. Why can’t you use KMnO4 as a primary standard?

ANSWERS TO INTEXT QUESTIONS

29.1
1. Two
2. >99.8%
3. Secondary
4. Secondary standard or an instrument
5. Titration

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Radioactive Isotopes, their Application in Biomedical Research MODULE
Biochemistry

30
Notes
RADIOACTIVE ISOTOPES

30.1 INTRODUCTION
In the previous chapter we have seen about radioactivity and its types. In this
topic we are going to see how different radioactive radiations can be measured.
Based on its ability to ionize and excite molecules radioactivity can be detected
and measured.

OBJECTIVES
After reading this lesson, you will be able to:

z describe the measurement of Radioactivity

z describe about tracer technique

z explain clinical and biochemical uses of radioactive substances

z describe health effects of radiations

30.2 MEASUREMENT OF RADIOACTIVITY

The 3 commonly used methods are

1. Ionisation of gases

2. Excitation of solids and liquids

3. Ability to expose photographic emulsions i.e.autoradiography

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Biochemistry
1. Ionization of gases
Principle: Radiation causes ionization of gaseous particles in its path. When this
takes place between electrodes in a closed chamber, an electric pulse is
generated. The magnitude of it depends upon applied potential and number of
radiation particles entering the chamber.

Notes Instrumentation: Ionization chamber consists of a gas chamber with insulated

base. It consists of an anode wire and metallic cathode. The circuit is closed by
connecting to a battery and Resistor. The electric pulse produced is measured
by amperometer.

Types

30.2.1 Ionization Chambers or simple ionization

In this type only one ion pair is produced per collision

Disadvantages:

z Current production is low

z Sensitive devices are needed for detection

30.2.2 Proportional Counters or gas amplification

At high voltage ionized electrons moves with high speed. This causes secondary
and tertiary ionization. Finally, large number of electrons reaches the anode. This
is known as Townsend avalanche effect.

Advantages
z Current production is high

Disadvantages
z Constant voltage is needed as it affects ionization and amplification

Uses
z To detect alpha emitting isotopes

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30.2.3 Geiger-Mueller Counters Biochemistry

All the isotopes including beta emitters induce complete ionisation. Current flow
is independent of primary ions. Maximal gas amplification is seen. The output
pulse is same in considerable voltage range. Number of times the pulse produced
will give the radioactivity. Dead time is the time taken by the ion pairs to reach
their respective electrodes. This is usually 100-200µS.
Notes
Advantages
z Can detect beta radiation

Disadvantages
z Ionising particles entering into the tube during the dead time cannot be
detected.
z Some ions escape from the electrodes and gives continuous discharge

Uses
z Routine check of radioactive laboratory for contamination
z Qualitative analysis of radioactivity.
z Quick screening of gels & chromatographic fractions for labeled components

Fig. 30.1: Key Component in a Simple Ionization Chamber

30.3 EXCITATION OF SOLIDS AND LIQUID

Principle: Radioactive isotopes can excite a compound called fluor. This emits
photons of light which can be detected and quantified using a photomultiplier
tube. This is called scintillation counting.
Types: There are 2 types of scintillation counting
1. Solid scintillation counting and
2. Liquid scintillation counting

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Biochemistry 30.3.1 Solid Scintillation counting

Instrumentation: In solid scintillation counting the fluorescing substance is
held in a light tight aluminium chamber. Radiations can penetrate through the
aluminum and can cause excitation of solid crystals to emit photons of light. This
fluorescence can be detected and quantified using a photomultiplier tube.
In photomultiplier tube, the electric pulse that results from the conversion of
Notes light energy to electrical energy. This is directly proportional to the radioactive
event.
Crystal used for alpha isotopes: Zinc sulfide
Crystal used for beta isotopes: Anthracene
Crystal used for gamma isotopes: Sodium iodide

Advantages
z Gamma isotopes have weak penetrating power hence it rarely collides with
molecules to cause excitation. In solid scintillation the atoms are densely
packed in h crystal so the probability of collision is more

Disadvantages
z Cannot detect weak beta emitters like 3H, 14 C& 35S as they are not able
to pass through the wall of the counter.

Fig. 30.2: Solid scintillation counter

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30.3.2 Liquid scintillation counting Biochemistry

In liquid scintillation counting the sample is mixed with a scintillation cock tail
containing solvent and one or more fluors

Instrumentation
In liquid scintillation counting, the radioactivity cause fluorescence of solvent.
This emitted light in turn causes fluorescence of another compound called Notes
primary fluor. This emitted light in turn causes fluorescence of another
compound called secondary fluor. This wavelength can be detected efficiently
by the photomultiplier tube.

Advantages
z Can detect weak beta emitters like 3H, 14 C& 35S

z More than one isotope can be detected at a time

z Counting efficiency is high

Disadvantages
z Costly
z Quenching is the interference given by several substances in detecting actual
fluorescence

30.4 ABILITY TO EXPOSE PHOTOGRAPHIC FILMS

(AUTORADIOGRAPHY)
Principle: Ionising radiations acts upon photographic emulsions to produce a
latent image
Instrumentation: A radiation source and photographic emulsion consisting of
silver halides embedded in solid phase such as gelatin are needed. Radiation
converts silver halide to metallic silver which forms a latent image. This can be
developed as a blackening of the film and can be stored as a permanent image.
Uses: Distribution of radioactivity in biological specimens.

30.5 TRACER TECHNIQUE

It is technique in which one or more atoms of a chemical compound have been
replaced by a radio isotope to trace the metabolic pathways. A radioactive tracer
can also be used to track the distribution of a substance within a natural system
such as a cell or tissue. Radioactive tracers form the basis of a variety of imaging
systems, such as, PET scans, SPECT scans and technetium scans.

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Biochemistry Principle
It is based on the principle that a stable isotope is replaced by a radioisotope.
The radioisotope is capable of emitting radiations that can be detected and
analysed. It is powerful than chemical reactions as they can be detected even in
lower concentration as seen inside a cell. In general, isotopes of hydrogen,
carbon, phosphorus, sulphur, and iodine have been used extensively to trace the
Notes path of biochemical reactions.

Uses

1. Tracing Metabolic pathways

When a labeled chemical compound undergoes reaction inside the cell ,one or
more of the products will contain the radioactive label. Analysis of it provides
detailed information on the mechanism of the chemical reaction.

2. Distribution of the compound

Radioactive compound provides a means to construct an image showing the way
in which gets distributed in the organism

Application
1. In metabolism research, Tritium(3H) and 14C-labeled glucose are commonly
used to measure rates of glucose uptake, fatty acid synthesis, and other
metabolic processes.
2. In medicine, tracers are applied in a number of tests, such as 99mTc in
autoradiography and nuclear medicine, including single photon emission
computed tomography (SPECT), positron emission tomography (PET) and
scintigraphy.
3. The urea breath test for helicobacter pylori commonly used a dose of 14C

labeled urea to detect H. pylori infection

30.6 CLINICAL AND BIOMEDICAL USES OF

RADIOACTIVE SUBSTANCES
The Radioactive substances can be used to study various metabolic pathways.
They are also used to diagnose and treat various diseases (eg cancer). Medical
uses of few radioactive substances are given in the table below.

Clinical and biomedical uses of radioactive substances.

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Biochemistry
Radioactive isotope Uses
Calcium- 47 Studying cellular function & bone formation
Carbon-14 Metabolism
Cesium-137 Cancer treatment
Chromium-51 RBC studies Notes
Cobalt-57 Diagnosis of pernicious anemia
Cobalt-60 Sterilization of surgical instruments
Copper-67 Treat cancer
Iodine-123,125 Diagnose thyroid disorders
Iodine- 131 Treat thyroid disorders
Phosphorus-32,33 Molecular biology & genetic research
Selenim-75 Protein studies
Sulphur-35 Molecular biology & genetic research
Technetium-99m Organ imaging & blood flow studies
Tritium Drug metabolism
Xenon-133 Lung ventilation & blood flow

30.7 HEALTH EFFECTS OF RADIATIONS

We already know that radioactive radiations are able to ionize & excite the
molecules they encounter. The effects of radiation depend upon the duration and
its type.

30.7.1 Effects
The effects of radiation are
z Generalised effect: Biological effects are due to the ionization process that
causes cell mutation. A given total dose will cause more damage if received
in a shorter time period. A fatal dose is (600 R)
z Acute Somatic Effects: Relatively immediate effects to a person acutely
exposed. Severity depends on dose. Death usually results from damage to
bone marrow or intestinal wall. Acute radio-dermatitis is common in
radiotherapy; chronic cases occur mostly in industry.

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Biochemistry z Critical Organs: Organs generally most susceptible to radiation damage

include: Lymphocytes, bone marrow, gastro-intestinal, gonads, and other
fast-growing cells. The central nervous system is relatively resistant. Many
nuclides concentrate in certain organs rather than being uniformly distributed
over the body, and the organs may be particularly sensitive to radiation
damage, e.g., isotopes of iodine concentrate in the thyroid gland. These
organs are considered “critical” for the specific nuclide.
Notes
30.7.2 Basic Radiation Exposure Control Methods
The three basic ways to decrease the exposure to radiation are
z Decrease Time
z Increase Distance
z Increase Shielding
z Time: Minimize time of exposure to minimize total dose. Rotate employees
to restrict individual dose.
z Distance: Maximize distance to source to maximize attenuation in air. The
effect of distance can be estimated from equations.
z Shielding: Minimize exposure by placing absorbing shield between worker
and source.

INTEXT QUESTIONS 30.1

1. Match the following:
1. Carbon-14 (a) Thyroid disorders
2. Iodine-125 (b) RBC studies
3. Sulfur-35 (c) Treat cancer
4. Copper-67 (d) Metabolism
5. Chromium-51 (e) Molecular biology
2. Fill up:
1. Gamma radiations can be detected .................. counting.
2. Weak beta emitters can be detected by .................. counting.
3. Dead time is seen .................. counters.
4. Fatal dose of radiation is ..................
5. The ability to expose photographic films is called ..................

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WHAT HAVE YOU LEARNT

z Radiations can be measured by methods based upon radiation, excitation
and ability to expose photographic films
z In methods based on radiation Geiger muller counter is important
z In scintillation methods gamma radiations can be detected by solid
Notes
scintillation counting
z In liquid scintillation counting more than one radioactive substance can be
detected at a time
z Autoradiography can be used to study whole animal samples
z Effects of radiation can be minimized by increasing shielding and distance
from the radioactive substance and decreasing the exposure time.

TERMINAL QUESTIONS
1. What are different types of measurement of radioactivity?
2. Write about ionization method of detection of radioactivity.
3. Write short notes on Geiger-muller counters
4. What is scintillation counting. and what are its types
5. Write short notes on solid scintillation counting
6. Write short notes on liquid scintillation counting
7. Writ about autoradiography
8. Write about different radio isotopes used clinically
9. Write about the deleterious effect of radiation
10. What are the different methods to control external radiation?

ANSWERS TO INTEXT QUESTIONS

30.1
1. 1. (d) 2. (a) 3. (e) 4. (c) 5. (b)

30.2
2. 1. Solid Scintillation 2. Liquid Schintillation
3. Geiger - mueller 4. 600R
5. Autoradiography

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