UNIT 4 PROTEINS

Structure
4.1 Introduction
4.2 Proteins - An Overview
4.2.1 Classification
4.2.2 Food Sourccs
- 4.2.3 Digestion, Absorption and Transport
4.2.3.1 Digestion
4.2.3.2 Absorption
4.2.3.3 Transpoit of Amino Acids
4.2.4 Functions
4.3 Methods of Delermination of Proteins and Amino Acid Content in Foods
4.4 Improvement of Quality of Protein in the Diet
4.5 Methods of Estimating and Assessii~gProtein Requirements at Different Stages
01Life Cycle
4.6 Nutritional Requirements and Recommended Allowances for Proteins and
Amino Acids
4.7 Rotein Deficiency
4.8 Let Us Suin Up
4.9 Glossary
4.10 Answers to Check Your Progress Exercises
-- -

4.1 INTRODUCTION
The term protein (Greek: proleuo- to occupy first place) was first suggested by
Berzelius to describe the complex organic nitrogenous substailces found in animal
and plant tissues. Proteins form three-fourths of the animal body on a moisture-free
basis. They are essential for life processes. All the basic functions of life depend on
proteins. Indeed, no form of life exists without proteins. They are found in every cell,
make up the contractile elements and enzymes that catalyze the release of energy
for the maintenance of life and there is no physiological function in which they do
not participate. Now in this unit let us get familiarized with the chemical nature,
digestion, absorption, utilization and other nutritional aspects of proteins.
Objectives
After studying this unit, you will be able to:
discuss the classification and functions of proteins,
identify the food sources of proteins,
explain the processes of digestion and absorption of proteins,
recognize the methods of estimating the protein quality, as well as, requirements
at various stages of life, and
describe the symptoms of protein deficiency.

4.2 PROTEINS - AN OVERVIEW

We just read that proteins are essential for maintaining and sustaining life. What are
proteins or what constitutes proteins that make it so important for us?
Proteins contain carbon, hydrogen, nitrogen, sulphur and some also contain phosphorus.
In the Nutritional Biochemistry Course, Unit 2, ybu may recall studying that proteins
are chains of amino acids held together by peptide bonds, When a peptide chain is 99
Advance Nutrition extended by more and more amino acids, until a chain length of one hundred to
several thousand amino acid residues is reached, it is classified as a 'protein'. The
nitrogen content of proteins varies from about 14 to 20 percent. The average value
of 16 percent is used conunonly for converting nitrogen content of food sh~ffsor
tissues into proteins by multiplying with the factor 6.25 (100116) (i.e. Crude protein
= Nitrogen x 6.25).

Let us now study and find out how proteins are classified.

4.2.1 Classification
Proteins vary widely in. their properties. You may recall reading about the properties
of proteins in the Nutritional Biochemistry Course, in Unit 2. We suggest you look
up this unit once again now as the information about proteins and their properties
given there will supplement your understanding of the functions and other aspects of
proteins discussed here in this unit.
Let us consider the two very familiar proteins. One of them is the egg white protein,
which is very sensitive. It denatures on heating, dissolves easily in water and is quite
reactive, while the other one is keratin of nails and hoofs, is wholly insoluble, tough
and chemically inert and resistant. Hence, it is not easy to classify proteins. We
simply distinguish proteins which are insoluble and fibrous and function as structural
material (scleroproteins) and globular proteins, those represented by egg white or
serum proteins, which are soluble in water or salt solution and consist of spherical
molecules.
Besides classifying proteins on the basis of soluble and insoluble, proteins have
been further classified based on the following attributes:
Classification based on chemical nature
I. Classification based on chemical properties
Classification based on amino acid content
Let us get to learn about proteins based on this classification.

A) Classification based on chemical nature

Frequently proteins are classified based on the chemical nature of amino acids (such
as solubility and prosthetic group), as simple, conjugated and derived proteins. Let
us get to know each of these.

I) Simple proteins: Simple proteins are those which contain only amino acids or
their derivatives and no prosthetic group. They yield only amino acids or their
derivatives on hydrolysis. Let us see which are these and where are they
found.
1) Albumins: Proteins such as egg albumin and serum albumin are sol~~ble
in
water and coagulable by heat.
2) Globulins: These proteins are insoluble in pure water, but soluble in neutral
salt solutions. For example, serum globulin, tuberin (potato), arachin and
conarachin (peanuts).
3) Glutelins: These are insoluble in all neutral solvents but soluble in very
dilute acids and alkalis. e.g. glutenin of wheat.
4) ProZnrnins: Proteins soluble in 70-80% alcohol. e.g. gliadin and zein.
5) Fibrous proteins: These proteins are characteristic of the skeletal structures
of animals and also of the external protective tissues, such as the skin, hair,
.etc. e.g., collagen, elastin and keratin.
 6) Histones: Soluble in water and insoluble in very dilute ainmo~iia.On Proteins
hydrolysis, they yield several amino acids, ainong which the basic ones
predominate. The important proteins of this group are the thymus, histones
and the globin of haemoglobin.
Let's now move on to the next category of proteins i.e. conjugated proteins and
its types. . ,

11) Conjzigated proteins: Conjugated proteins contain some non-protein substances.

Most proteins occur in cells in coinbination with prosthetic groups and hence are
important for the nutritionist. These include:
1) Glycoproteins: Most of the naturally occurring coiljugated proteins are
glycoproteins. Sugar molecules are covalently bound to them, especially
those secreted from the cell. They range in size from a nlolecular weight
of 15,000 to more than one million. The carbohydrate component varies
from 1 to 85%. Glycoproteins with more than 80% of their molecules as
carbohydrates are called 'p~+oteoglycans.'
Lipoproteins: These are the multiconzporzcnt complexes of lipicls and
proteins that fomz distinct moleculnl- aggregates. They contain polar and
neutral lipids, cholesterol or cholesterol esters in addition to protein. The
proteins and lipids are held together by non covalent bonds. Lipids are
primarily hydrophobic and cannot be easily transported through a11 aqueous
environment as blood. The lipoprotein combination renders the lipid molecule
hydrophilic and is transported in the blood to tissues which can use or store
the lipids.
3) Nucleoproteins: Nucleoproteins are conlbinations of nucleic acids land
sirnple proteins, wlzich usz~allyconsists of a large rzunzber of basic
aniino acids. N~cleop~oteins have very coinplex structures and nunlerous
functional activities. All living cells contain nucleo~~roteins.
Some cells, such
as viruses, are composed of nucleoprotein.
4) Other conjugated proteins: The phospho proteins and the metallo proteins
are loose (as with phosphate carrying protein) or tight (as with the phosphate
in casein or the iron in ferritin) associations of proteins with phosphate
groups or such ions as zinc, copper and iron.
What are derived proteins? Let us find out next.
111) Derived proteins: Derived proteins are the derivatives of the protein moleczile,
apparently fonned thro~tglzh,ydrolyti changes in the molecule. These are
either primary or seco1zdar-y protein derivatives. Let us get to know them.
I) Primary Protein Derivatives: These are the derivatives of the prorein
molecule formed by hydrolysis involving slight nlteralions. Examples
include:
a) Proteins: These are the insoluble products which result from the incipient
action of very dilute acids or enzymes. e.g. casein (curdled milk), fibrin
(coagulated fibrinogen).
I
b) Metaproteins: Proteins resulting from the action of acids and alksllis
whereby the n~oleculeis sufficiently altered to form proteins soluble in
weak acids and alkalis, but insoluble in neutral solvents.
c) Coagulated Proteins: Insoluble proteins which result from the action of
heat on protein solutions or the action of alcohol o n the protein. e.g.
cooked egg albumin or egg albumin precipitated by alcohol.
1 2) Secondary Protein Derivatives: These are the products of further hydrolytic
i cleavage of the protein molecule, Examples include:

182
Advance Nutrition a) Proteoses: Soluble in water, not coagulable by heat, precipitated by saturating
their solutions with anunonium.

b) Peptorzes: Soluble in water, not coagulable by heat and not precipitated by

saturating their solutions with ainmonium sulphate. These represent a hirther
stage of cleavage than the proteoses.

c ) Peptides: These are the compounds containing two or more anlino acids.
An anhydride of two amino acids is called a 'dipeptirle', one having three
amino acids, a 'tripepticle' and containing several amino acids, a
'polypeptide'. Peptides result from further hydrolytic cleavage of the
peptones.

Next, let us review the classification based on chenlical properties.

B) Classification based on chemical properties

Depending on their chemical propelties and optical activity, the amino acids in proteins
are classified under the followillg heads:

1) Morzo anzino mono carboxylic acids: Examples include glycine, alanine, valine,
leucine, isoleucine, serine and threonine.
2) Mono amino dicurboxylic acids: Examples include aspartic acid and glutamic
acid.
3) Diamino nzorzo carboxylic acids: Arginine 'and lysine.
4) Sulpfitlr containing amino acids: Cysteine, cystine and methionine
5) Aromatic and heterocyclic amino acids: Phenylalanine, tyrosine, histidine,
tryptophan , proline and hydroxyproline.
The next classification is based on the amino acid content. Let us get to learn about
this ~Iassification.

C) Classification based on amino acid content

Nutritionally, amino acids are classified on the basis of the body's ability to synthesize
them - as essential (indispensable and not synthesized in the body) and norz-essential
(dispensable and that can be synthesized in the body) amino acids. Indispensable
amino acids must be a part of the diet while dispensable amino acids need not be
present in food. These definitions, however, become blurred at the metabolic level.
Lysine and threorzine are perhaps the only metabolically indispensable amino acids
because they are not transaminated to any nutritionally significant extent. This is a
crucial point because lysine and threonine are the first and second limiting amino
acids in cereal protcins. Lysine is the first limiting amino acid in human milk. Glutarnic
acid and probably serine are the only truly dispeilsable amino acids since these are
the only amino acids which can be synthesized by the reductive amination of the
appropriate keto acid. You may recall studying about these reactions in the Nutritional
Biochemistry Course, in Unit 7. There is a third group of amino acids which are
'conditionally essential', and are characterized by two features. Firstly, their synthesis
uses olher amino acids as carbon precursors and is confined to specific organs. This
is an i~nportantmetabolic distinction from the dispensable amino acids. For' some
conditionally essential amino acids, e.g., tyrosine, the precursor is a dispensable amino
acid; while for others such as cysteine both an essential amino acid, lnethionine as
sulphur donor and a non essential amino acid, serine are required. At the metabolic
level, the organism's ability to synthesize a conditionally essential amino acid is
constrained by the availability of a suitable amino acid precursor. Secondly, the
maximuin rate at which their synthesis proceeds may be limited and potentially
restricted by ~Ievelo~mental or pathophysiological factors. Thus, very low birth weight
infants are unable to ~ynthesizecysteine and proline and lack the ability to synthesize
glycine. These factors are important because human milk proteins have low glycine Proteins
content. Amino acids are classified based on the amino acid content as given in the
Table 4.1.
Table 4.1: Classification of amino acids

Essential (indispensable) Conditionally-essential Non-essential (dispensable)

Methionine Tyrosine Glutamic acid
Tryptophan Cystine Alanine
Valine Aspaltic acid Proline, Hydroxyproline
Isoleucine Serine Glycine ,

Leucine
Phenylalanine
Lysine
Threonine
Arginine
Histidinc (only for infants)

We now know that protei~lsare composed of amino acids and that proteins differ in
their amino acid make-up. Proteins lacking in one or more of the essential arnino
acids, cannot be utilized to meet the protein requirements of the body hence they are
not good quality proteins. The nutritive value of a protein will b e high if the amino
acid make-up is very similar to that of the body proteiils and will be low if it lacks
partially or completely any one of the 10 essential amino acids (refer to Table 4.1)
or if its amino acid composition is very much different from that of the body
proteins. Based on their nutritive value or amino acids make-up, proteins are therefore
classified as:

I) Complete proteins - e.g., egg p oteins. These proteins promote growth and
provide all the essential amino acids.
11) Partially complete proteins - e.g., wheat proteins. These promote moderate
growth and partially lack one or more essential amino acids.
111) Incomplete proteins - e.g., gelatin or zein. They do not promote growth and
completely lack one or more essential arnino acids.
After understanding how proteins are classified, let's move on to sources of protein
in our next sub-section.

4.2.2 Food Sources

The important sources of proteins in the diets of low-income groups are cereals and
legumes. Meat, fish, eggs and milk are important sources of proteins of high biological
value. Oilseeds, oilseed meals and soy are rich potential sources of proteins. The
protein content of some important foods is given in Table 4.2.

'Iltble 4.2: Protein content of some important foods

Foods Protein (g 1100g) Foods Protein (g 1100 g)

Cereals 6 - 14 Milk 53.5 - 4.0
Legumes 18 - 24 Milk powder (full cream) 26 - 27
Soybean 43 Milk powder (skimmed) 35 - 38
Nuts and oilseeds 18 - 40 Fish 18 - 20
Oilseed meals 45 - 55 Meat and liver 18 - 22
Egg, hen 12 - 13

r
Advance Nutrition We shall continue with our discussions on the metabolism and function of proteins.
However, you must first attempt the check your progress exercise given below
before procceeding further to recapitualate the learnt concepts.

Check Your Progress Exercise 1

1) Fill in the blanks
a) The nitrogen coiltent of rood-stuffs are converted into proteiils by
multiplying the factor ......................
b) A11 example of conjugated proteins is ...................................
C) Glycoproteiils with nlnre than eighty percent of their inolecules as
carbohydrates are called ......................................
d) An example of complete proteiils is .................... protein.
e) Zein is an example of ................................ protein.
2) Match the following: &
A B
1. Siinple proteins a) Lipoprotein cornbinatioil
2. Tixly indispensable aminoacids b) Soyabeans
3. Proteins of high biologic value c) Zeiil
4. Richest plant source of protein d) Albulnin
5. An anhydride of two amino acids e) Serine
6. Renders the lipid lnolecule hydrophilic f) No11 vegetarian foods
7. Incomplete protein g) dipeptide

After having sound knowledge of what proteins are coinposed of, its types and food
sources, let us now study about their physiological aspect, i.e. digestion, absorption
and transpol-t in the body.

4.2.3 Digestion, Absorptioil and Tral~sport

Under this sub-section, we shall learn how proteins that we consuine are digested,
absorbed and transported to various body tissues. You may recall studying about this
aspect in the Nutritional Biochemistry Course in Unit 5, sub-section 5.4.2. We suggest
you look up this unit now, This will facilitate your understanding on this topic. A brief
review on the process of digestion, the enzymes involved, their location and target
amino acids is present next for your recapitulation.

4.2.3.1 Digestion I

The daily protein intake (of about 50-100 g) and the protein of enzymes, sloughed
(bled or drop off ) epithelial cells and mucins, which are found in the gut is almost
completely digested and absorbed. This is a vely efficient process and ensures a
continuous supply of amino acids to the whole body's amino acid pool. The puipose
of protein digestion is to liberate the amill0 acids of the consunled proteins. This is
accomplished through a series of enzymes which have specific target linkages as
their point of action as shown in the Table 4.3.
 Table 4.3 : Enzymes and their target linkages Proteins

Enzyine Location Target Linkages

Pepsin Stomach Peptide bonds involving the
aromatic amino acids
Trypsin Small intestine Peptide bonds involving arginine
and lysine
Chymotrypsin Small iiilestine Peptide bonds involving
tyrosine,tryptophan,
phenylalanine, methionine and
leucine
Elasrase pcptidase A Small intestine Pepticie bonds involving alanine,
serine and glycinc
Cxboxy Small inlestjne Peptide bonds involving valine,
peptidase A pepticlase A leucine, isoleucine
and alanine
Carboxy Sinall intestine Peptide bonds involving lysi~le
peptidase B and pcptidase B arginine.
Ei~dopeptidase Cells of brush Di and tripeptides tlial enter the
Aminopeptidase aminopeptidnse blush border of
Dipeptidase the nbso~ptivedipeptidase cells.

The protein hydru1nse.s. called as peptidrl.r.es, fall into two categories. Those that
attack internal peptide bonds and liberate large peptide fragments for s~~bsequent
altack by other enzymes are called the '~~izdopeytidnse,~' and those that attack the
terminal peplide bonds and liberate single ai~li~lo acids from the protein structure are
c:illed 'exopepti~la,se,s'.T11c exopeptidnses are further subdivided according lo whether
they attack at the carboxy end of the amino acid chain (carboxy peptidases) or the
amino end of the chain (amino peptidases). The initial attack on the intact protein is
catalyzed by the intestinal epithelial cells. The l~ydrolysisof proteins in the gastro-
intestinal tract is completed by pinteases secreted in the gaskic juice and pancreatic
juice and also by proteases in thc intestinal mucosa.
In Tablc 4.3, you would have noticed that thc digestion of protein starts only in the
ston~ach.11.1contrast to carbohydrate and lipid digestion, which is initiated in the
mouth with the salivary amylase and the lingual lipase, protein digestion does not
begins until the protein reaches the stomach and the food is acidified with the gastric
hydrochloric acid (HC1). Let us get to know about gastric digestion in greater details.
Gastric digestion
If gastric HC1 production is low and not adequate to inaintain thc pH of the stomach
contents between 2 and 3, protein digestion in the stomach may be negligible. This
will happen in aclzlor/lydria (a condition characterized by the failure o l the intragastric
pH to fall to less than 4.0), achylia gastricn (absence of gastric juice, partial or
complete) or periziciuus anaemia. The HCl serves several functions i n gastric
digestion. It acidifies the ingested food, killing potential pathogenic organisms. However,
not all pathogens are killed. Some are acid-resistant or are so plentiful in the food that
the amount of gastric acidification is insufficient to kill all of the pathogens. HC1 also
serves to denature the food proteins, thus making them more vulnerable to attack by
the proteolytic enzymes present in the gastric juice namely, pepsin and endopcptillase
(it hydrolyzes peptide bonds in the interior of the protein molecule).

Pepsin has a strong clotting action on milk. This factor is important in the digestion
of milk proteins in infants. The optimum pH of pepsin action is about 2.0. Actually
pepsin is.not a single enzyme. It consists of pepsin A which attacks peptide bonds
involving phenylalanine or tyrosine and several other enzymes which have specific
Advance Nutrition attack points. The pepsins are released into the gastric cavity as pepsinogen. When
the food entering the stomach stimulates HC1 release and the pH of the gastric
contents fall below 2, the pepsinogen looses a 44-amino acid sequence. The activation
of the pepsins from pepsinogen occurs by one of the two processes-autuactivation
and autocatalysis. Let's understand what these are. The first one is called
autoactivatiorz and occurs when the pH drops below 5. At low pH, the bond
between the 44'" and 4Sh amino acid residue falls apart and the 44*-amino acid
residue (from the arnino tenninus) is liberated. The liberated residue acts as an
inhibitor of pepsin by binding to the catalytic site until pH value of 2 is achieved. The
inhibition is relieved when this fragment is degraded. Degradation occurs at pH 2 or
by the action of pepsin. Thus, pepsin hydrolyzes mainly peptide bonds containing
phenylalanine, tyrosine or tryptophan and leucine and other acidic amino acids and
since food remains in the stomach for a limited time, dietaty proteins are hydrolyzed
mainly into a mixture of polypeptides as highlighted herewith.
Pepsin
Dietary proteins rPolypeptides
Let us get to know about the autocatalysis process next. 'Autocatalysis' occurs
when already active pepsin attacks the precursor pepsinogen. This is a self-repeating
process and serves to ensure ongoing catalysis of the resident protein. The cleavage
of the 44-amino acid residue, in addition to providing activated pepsin, also serves as
a signal peptide for cholecystokinin release in the duodenum. This sets the stage for
the subsequent pancreatic phase of protein digestion.
Let us get to know about protein digestion in the intestine next.
Proteolysis in the intestines: The main digestion of polypeptides produced in the
stomach takes place in the intestines. Cholecystokinin stimulates both the exocrine
pancreas and the intestinal mucosal epithelial cells to release its digestive enzymes.
The proteases involved in the digestion are trypsin, chymotrypsin and
curboxypeptidase (as highlighted in the Table 4.3) secreted in pancreatic juice and
arnino peptidases present in the intestinal mucosa. The intestinal cells release an
enzyme, enteropeptidase or enterokinase, which serves to activate the psotease
trypsin, released as trypsinogen by the exocrine pancreas. This trypsin not only acts
on food proteins, it also acts on other preproteases released by the exocrine pancreas,
thus activating them. Thus trypsin acts as an en(toprotease on chymotrypsinogen
by releasing chymotrypsin, on proelastase by releasing elastase and on
procarboxypeptidase by releasing carboxypeptidase. Trypsin, chymotrypsin and
elcrstase are all endoproteases, each having specificity for particular peptide bonds.
Trypsin and chymotrypsin act at pH 7.4 to 8.0. Tlypsin hydrolyses mainly peptide
linkages containing tyrosine or phenylalanine.
Trypsin and Chymotvypsirz
Proteins and polypeptides ~Peptides+ Amino acids
Each of the three proteases i.e, trypsin, chymobypsin and elastase have serine as a
part of their catalytic site so that any comporlnd that ties up the serine will inhibit the
activity of these proteases. One such inhibitor, diisopropylphosphofluoridate reacts
with serine and stops protein digestion. Through the action of pepsin, trypsin,
chymottypsin and elastase, nuinerous oligopeptides are produced which are then
attacked by the amino and carboxypeptidases of the pancreatic juice and those 011
the brush border of the absorptive cells. Caboxypeptidase A hydrolyzes the end
group in peptides containing aromatic or aliphatic amino acid, thus releasing flee
amino acids as shown herewith. Carboxypeptidase B hydrolyzes peptides containing
arginine and lysine residues. The intestinal mucosa contains a group of amino peptides
which complete the hydrolysis of peptides to amino acids. The intestinal mucosa also
contains tripeptidase, dipeptidase,etc., which hydrolyze tri and dipeptides as highlighted
in the reaction given herewith:
 Peptides
Carhoxy peptidnsr

Atnilzn peplid(~ses
,Amino acids Proteins

Dipeptirlase
Dipeptides )Amino acids
T~.ipepticlc~.~e
Tripeptides )Amino acids

Thus, the ultimate products of digestion of proteins, namely, amino acids are liberated
from these chains one by one and are absorbed and appear in the poi-tal biood.
With this, we come to an end on our discussion on digestion. In the Iollowing sub-
section, we will look at how amino acids fornled are absorbed in our body.
4.2.3.2 Absorption
Although single amino acids are liberated in the intestinal contents, there is insufficient
power in the enzymes of the pancreatic juice to render all of the amino acids singly
for absorption. The brush border of the absorptive cell, therefore not only absorbs the
single amino acid but also the di- and tripeplides. In the process of absorbing these
small peptides, it Ilydrolyzes them to their constituent anlino acids. There are specific
transport systems for each group of funciionally similar amino acids and peptides.
The site of absorption is the 'small intestine'. The process of absorption requires
energy. It is observed that L-isomers (natural isomers) of amino acids are more
rapidly absorbed than D-amino acids and are hence biologically more impoi-tant.
What are L and D isomers of amino acids? We have already talked about this in the
Nutritional Biochemistry Course, in Unit 2. Do look up sub-section 2.6.2 now. These
isomers are transported by an active carries system againsl a concentration gradient.
Similarly, neutral amino acids are more rapidly absorbed than the basic amino acids
and in general, amino acids conlpete with or~eanother for absorption. In several
instances, the carrier is a shared one, that is, the carrier ti-ansports more tha~lone
amino acid. Vitamin B, is essential for antino acid absolption Let us now learn how
this transport mechanism works.

4.2.3.3 Transport of Amino Acids

More than one transport or cmier sysletn functions in the absol-ption of amino acids.
The active carrier system for neutral amino acids shares a common membrane
carrier. Neutral amino acids and those with t l ~ eshori or polar side chains (serine,
tllreonine and alanine) are transported by the shared carrier system. The basic amino
acids, that is, lysine, xginine and hislidine share a carrier system with cystine. The
mechanism of transport and uptake are depicted in Figure 4.1.

Figure 4.1: Carrier-mediated sodium-dependent amino add transport

Advance Nutrition In Figure 4.1, you may have noticed that the amino acid transport is dependent on
the Na+ ion. The dependence of amino acid transport on Na+ ion suggests a direct
interaction between the carrier and Na+ ion. This is similar to that observed in the
absorption of glucose, as you may recall studying in the last unit. The amino acid
associates with the carrier and Na+ ion in the microvilli and the complex travels to
the inner side of the membrane where it dissociates, releasing the amino acid and Na
ion into the cytoplasm as illustrated in Figure 4.1. Thus the amino acid leaves the
absorptive cell with sodium. The carrier, Na ion is recirculated back to the lumen for
reuse. As sodium enters the cell, potassium is pumped out via a Na+ Kt ATPase
system. As sodium leaves the cell, potassiuin flows back in and the electrolyte
balance is maintained. The Na+ ion is then actively trailsported out of the cell.

So we have looked at the digestion, absorption and Lransport of proteins and amino
acids. What are the functions of proteins in our body? Let us study and find out next.

4.2.4 Functions
Each of the various proteins in the body serves a specific function in the maintenance
of life. Any loss in body protein, in reality, means a loss in cellular function. In
contrast to lipids and carbohydrates, proteins have no true body reserve. Humans
when deprived of or insufficiently supplied with protein, compensate for this dietary
deficiency by catabolizing some, but not all, of their tissue functionality. Cells, tissues,
organs and whole systems cannot exist without proteins serving their various functions.
So let us get to know the varied functions of proteins in our body. We begin our
discussion on funqtions, first with the body-building function.

I) Body-building functions ofproteins: The primary functions of proteins, as you

might be aware, is tissue growth and maintenance. Protein contains amino acids
- the building blocks - hat our bodies use to build and maintain muscles, bone,
skin, blood and other organs. Thus, proteins play an important role in growth and
body-building. For the constant growth of human beings from birth to adulthood,
a regular supply of dietary protein is required. Now this does not mean that the
body does not require proteins once the growth ceases. During adulthood, worn
out cells, body tissues need contiiluous replacement. Proteins are required for
maintenance of our body. Proteins, therefore, are crucial and required through
out our life span for growth, body-building and maintenance.

2) Protein as nn energy source: Proteins contribute to the body's energy need.

If diet does not furnish enough calories from carbohydrates and fats, proteins
are catabolized to give energy. One gram protein yields 4 Kcal. But, what is
important for us is to understand is that this is not the major fi~nction of
proteins. This only happens when, as mentioned earlier, the diet does not supply
enough energy-giving nutrients.
Proteins as enzymes: From conception to death, living cells use oxygen and
metabolize fuel. Cells synthesize new products, degrade others, and generally
are in a state of metabolic flux. For these processes to occur, catalysts are
needed lo enhance each of the many thousands of reactions occulring in the
cell. These catalysts called 'erzzyrnes' are proteins as you have already studied
in the Nuttitional Biochemistiy Course in Unit 4. Enzymes make up the largest
and the most specialized class of proteins. Each enzyme is unique and catalyzes
a specific kind of reaction. In the cell, enzymes are found in cellular compartments
(cytoplasm, nucleus, mitochondria, etc.), as well as, the membranes within and
around the cell wall. The location of an enzyme is one of its characteristics and
dictates, in part, its role in metabolism. Many enzymes are complex proteins;
they consist of a protein component and a prosthetic group. The protein part is
called a p o e n ~ m eand the prosthetic group, 'coenzyme' as illustrated in Figure
 4.2. A detailed discussion on enzymes is presented in the Nutritional Biochemistry Proteins
Course, Unit 4. We suggest you read this unit now for a better understanding
of the functions of proteins as enzymes.
Substrate

uApoenzyme
Cofactor
(nonprotein portion) u .
Holoenzyme
@rotein portion) (whole enzyme)
Coenzpne + apoenzyme -+(holo-) enzyme

Figure 4.2: Holoenzyme

Enzymes consist of specific sequences of amino acids. The catalytic function of an

enzyme is intimately related to its ainino acid sequence. Enzymes nust possess a
shape that will compleinent the reactive molecular shape of the substrate in the same
way as a key fits into a lock. This is coinmonly r e f e ~ ~ etod as 'lock and key
mechanism'. This shape is a function of the enzyme protein's primLuy, secondary,
tertiary and quatelllary structure. Jn the same way, substrates should also have
specific shapes in order to be catalyzed by their respective enzymes.

Enzyme -I-Substrate Enzyme - Substrate

This is the reason why only D-sugars or L-amino acids can be metabolized by
rnamnalian cells. These stereoisomers coilrorm to the shape required by the enzynle
which serves as its catalyst. Wllile enzymes show absoliite specificity, the specificity
generally applies to the entire molecule. If howcvel; the substrate is large and complex,
the structural requirements are less stringent in that ccnly that part of the substr~te
involved in the enzyme-substrate complex sl~oulclhave the appropriate molecular
arrangement. The portion of the substrate not involvecl in the reaction need not be
the appropriate conformation.
Some enzymes are specific for only one substrate; others may catalyze several
related reactions. While some are specific for a particular substrate, others are
specific for certain bonds. This is called 'group specificity'. For example, glycosidases
act on glycosides, pepsin and trypsin act on peptide bonds and esterases act on ester
linkages. Within this group, certain enzymes exhibit greater specificity. Chymotrypsin
preferentially acts on peptide bonds in which the carboxyl group is a part of the
aromatic ainino acids (phenylalanine, tyrosine or tryptophan). Enzymes such as
carboxypeptidase catalyzk the hydrolysis of the carboxy-terminal or amino-telminal
amino acid of a polypeptide chain. This bond specificity, rather than molecular
specificity, is useful to the animal in that it reduces the number of enzymes needed
within the organism. Incidentally, the above enzymes are very useful to the protein
chemist in his 1 her determination of the amino acid sequence of a given protein.
Cells synthesize enzymes in much the same fashion as they synthesize other proteins;
yet enzymes are relatively short lived. Cells must continually synthesize their enzymes
if they are to survive. So, we have looked at the functions of proteins as enzymes.
Proteins also function as carriers in the body. Let us get to know about this function
next.
B-

Advance Nutrition 4) Proteins as carriers: A large variety of compounds are carried in the blood
between tissues and organs of the body. Some of the compounds require specific
protein for their transport. Not only is this specific protein necessary for the
transport of coinpounds insoluble in blood, but it is also necessary to protect
these compounds from further reactions that take place during the transport
process. Some of the membrane proteins are carriers and some are both carriers
and enzymes. Both intracellular and extracellular carriers have been identified.
The plasma proteins wlzich can have a carrier finction are the albumin and the
a- and P-globulins. The best studied of the plasma carriers are those associated
with the transport of lipid (called Ilpoproteins), since these lipoproteins (carriers plus
lipids), when levels are elevated, appex to be related to the development of a variety
of diseases. These lipoproteins comprise of about 3% of the plasma proteins. They
are the loose associations of such lipids as phospholipids, triacylglycerols and cholesterols
and represent an example of how proteins function as carriers. The lipids they carry
are either from the diet or are synthesized de liovo in tissues, such as the liver. The
P-globulin proteins carry these lipids to such sites as muscle or adipose tissue, wl~ere
they are either used or stored. The release of the lipid from the protein carrier is a
complicated process. In adipose tissue, the lipoprotein is attached to a membrane
receptor site-an enzyme, lipoprotein lipase cleaves the lipid from the protein. The lipid
is then picked up by another protein called a lipid binding protein and is cmied to the
interior of the cell for storage. The P-globulin protein carrier, once free of its lipid,
returns to the liver or intestinal mucosa and is recycled.

The plasma lipids, phospholipids, acylglycerols, cholesterol, cholesterol esters and free
fatty acids are usually transported as loosely associated lipid-protein complexes. At
least three different proteins have been identified. Albumin usually transports the free
fatty acids, whereas the a and P-globulins transport the phospholipids, acylglycerols
and cholesterols. The different lipoprotein complexes can be separated and identified
011 the basis of their antigenicity, their electrophoretic mobility and their density. The
low density or P-lipoproteins contain the P-peptide, cholesterol and some phospl~olipids.
The majority of phospholipids are carried as a-lipoproteins. With age, the lipid content
of the plasma tends to rise and the rise is reflected almost entirely as an increase
in P-lipoproteins. As the density of the lipoproteins decreases, the molecular weight
and complexity of the lipid it carries decreases. The a-lipoproteins carry mainly (up
to 60%) acylglycerols. These glyceroles are usually those synthesized in the body
rather than coming from the diet. The dietary acylglycerols are usually c a ~ i e das
chylomicrons. These particles are the largest and least dense of the lipid-protein
complexes. Read Unit 7 in the Nutritional Biochemistry Course for more information.

In addition to serving as carriers of lipids, some of the globulins in the plasma can
combine with iron and copper, as well as, with other divalent- cations. These
colnbinations are called 'metalloproteins'. The globulins serve to transport these
cations from the gut into the tissues where they are used. The monovalent cations,
sodium and potassium, do not need carriers but most other minerals do.

Many hormones and vitamins require transport or carrier proteins to take them from
their point of origin to their active site. In addition, there are inlracellular transpoi-t
proteins such as the lipid binding proteins that are responsible for the transport of
mateiials between the various cellular compartments. Lastly, there are transport
proteins which carry single molecules. The classic example is haemoglobin, the red
cell protein, responsible for the transport of oxygen from the lungs to every oxygen-
using cell in the body.
From carrier function, we move on to the regulatory function of proteins.
5) Proteins as regulators of water balance: As substrates and solutes are
transferred or exchanged across membranes, water has a tendency to follow to
maintain equal osmotic pressure on each side of the membrane. If oslnotic
 pressure is not maintained, the individual cells either shrink from lack of internal Proteins
water or burst from too much. You may recall studying about this phenomenon
in the Food Microbiology and Safety Course, in Unit 3. The balance of water
between intracellular and extracellular compartmeilts is closely regulated.
Water balance across the capillary membrane is carefully controlled. A close balance
is maintained between the osmotic pressure of the plasma, the interstitial fluids and
the cells and the hydrostatic pressure exerted by the pumping action of the heart. The
total osmotic pressure of the plasma and of the intra- and extracellular fluids is the
result of its content of inorganic electrolytes, its organic solutes and its proteins. The
concentrations of the electrolytes and organic solutes in plasma, interstitial fluid and
cells are substantially the same so that the contribution to the osmotic pressure by
these substances is practically equal. However, since there are more proteins in
plaslna than in the cells, plasma exerts an osmotic pressure on the tissue fluids. The
result of this inequity of solutes is the drawing of fluids from the tissue spaces and
from the cells into the blood. Opposing this force is the hydrostatic pressure, exerted
by the pumping action of the heart, which moves fluids from the blood into the tissue
spaces and into the cells. The hydrostatic pressure is greater on the arterial side of
the capillary loop than on the venous side. There is some kind of interplay between
these four kinds of pressure- blood osl?iotic pressure, tissue osmotic pressure,
blood hydrosmtic pressure and tissue h~~drostlztic pressure. This interplay results
in the filtration of solutes and metabolites and thc transfer of oxygen from the arterial
blood into the tissues and cells it supplies and on the venous side, a resoiptio~lfrom
the tissue space of C 0 2 , metabolites and solutes back into thc blood supply,
:-
Albumin plays a more significant role in maintaini:ig the osmotic pressure than the
,other blood proteins because of its size and abundance. It is a small lnolecule and
has a greater number of particles per unit volun~ethan the other larger serum
proteins. With fewer proteins in the serum, water leaks out into the interstitial space
and accumulates. You lnay be familiar with this condition commonly referred to as
'oedema'. The oedema of protein deficiency may also be Lhe result of the body's
inability to regulate the protein hormone, particulnrly, Anti-Diuretic Hormone (ADH).
This hormone plays a role in controlling water balance. The effect of protein is on
the distribution of water amongst the various body coinparlments than on the total
body water.

So now you have understood what important role, proteins cnn play in regulating the
water balance. Proteins also function as biological buffers. What do we mean by this
and how proteins function as biological buffers is discussed next.

6) Proteins as biological bug'ers: Proteins have the ability to accept or donate

hydrogen ions and by doing so they serve as biological buffers you may recall
studying under the properties of proteins in sub-section 2.6.2 in Unit 2 in the
Nutritional Biochemistry Course. In blood, there are three important buffering
systems - plasma proteins, haemaglobirz and carbonic acid bicarbonate.
The equilibrium reactions for .each of these buffering systems is as follows:
H Protein w H+ + Protein-

The first of these buffering systems, the plasma proteins, functions as a weak acid1
<, -t<
salt buffer when the free carboxyl groups on the protein dissociate, or as a weak
baselsalt buffer when the free amino groups dissociate. Although the buffering ability
of the plasma protein is extremely important in maintaining blood pH, it is not as
important as the other two systems,
The second buffering system, carbonic acid-bicarbonate, is extremely effective because
there are reactions which follow this equilibrium which will regulate either acids or 111
Advance Nutrition bases. The I-12C03level in plasma never goes too high because it is in equilibrium
with C02 (H2CO3-+ C02 + H20), which is expired by the lungs. In blood, this
equilibrium proceeds very quickly because of the presence of carbonic anhydrase, an
enzyme found in red blood cells which catalyze it. If the carbonic acid-bicarbonate
reaction goes in the opposite direction, the concentration of the HCOl so foilned will
be regulated by the kidneys.
The third important buffering system in blood results from haemoglobin. Haemoglobin
has six times the buffering power of the plasma proteins. It functions well as a buffer
because it is present in large amounts, it contains 38 histidine residues (Histidine
residues are good buffers because they can dissociate to H+ and the irnidazole group)
and because haemoglobin exists in blood in two forms, reduced haemoglobin and oxy
haelnoglobin. It is thus a weaker acid and a better buffer.
Next let us look at the function of proteins as st~vcturalelements.
7) Proteins as structural elements and structural units: The liver cell membrane
analysis shows that this membrane contains 50-60% protein, 35% lipids and 5%
carbohydrates. The carbohydrate present is joined pii~narilyto the protein forming
glycoproteins, con~poundswhich constitute the receptor sites of several hormones.
The protein portion of the nlembrane is so oriented that its hydrophilic aspects
are also in close proximity to the intracellula and extracell~ilarfluids. The
protein molecules are interspersed within the lipids and lend both structural
stability and fluidity to the membrane. Memhrane function depends on how the
proteins are placed in the nlembrane and on the fluidity, which results from the
combination of proteins in a lipid mixture. If the lipid is more saturated, a more
rigid crystalline structure will fornl. Many lipids being fluid and less rigid, allow
the proteins to change their shape in response to ionic changes and hence these
proteins function as enzymes, carriers, binding or receptor sites or entry ports
for a large variety of materials binding, entering or leaving the cell. Thus proteins
scrve as the structural and functional units of the cell membrane.
Proteins are also important intracellular stnictural units. Muscle is composed of 20%
protein, 75% water and 5% inorganic mate ial, glycogen and other organic compounds.
The major proteins in ~nuscleare inyosin - a large globular protein, and actin - a
smaller globular protein. These two proteins, plus the filamentous tropomyosin and
troponin a e the moleculas coinponents of the muscles. The muscle proteins are
characte~izedby their elasticity, which contributes to the coiltractile power of the
tissue.

The most important structural function of protein is related Lo skin and connective
tissue. The skin is composed of epithelial tissue which covers not only the exterior
of the body but also lines the gastrointestinal tract, respiratory tract and the urinary
tract. One of the major protein found in the skin is 'melanin'. Melanin is a tyrosine
derivative and. provides the pigmentation or characteristic colour to the skin. Persoils
unable to form this pigment are albinos and the disease is called 'nlbinism.' Keratin
is the protein which fol-ms hair, nails, hooves, feathers or horns. Each of these
structures is slightly different but all contain keratin. This protein is insoluble in
water and is resistant to most digestive enzymes. It has a high percentage of
cystine.

Connective tissue is that tissue which holds various cells and tissues together. It
includes bones and teeth also, since they start with a matrix protein into which various
amounts of minerals are deposited. Collagen and elastilz are the two distinct types
of proteins in the connective tissue. It contains proline, hydroxyproline and glycine.
These proteins are not easily degradable and are inert metabolically. Even in protein
deficient states, the body will synthesize collagen and elastin and these proteins will
not be catabolized for needed amino acids. However, this protein can be degraded
to a limlted degree by boiling in acid. It is then coiiverted to gelatin. The collagen of

r
bone, skin, cartilage and ligaments differ in chemical composition from that of Proteins
the white fibrous tissue which holds individual cells together within muscle, liver and
other organs. Elastin and chondroalbumoid are two other proteins in the connective
tissue. They are present in small anlounts and serve as a part of the structural
protein.

Proteins as structural elements have been studied above. Interestingly proteins also
surround the joints and function as lubricants. Let's look at this lub~icantfunction of
proteins next.

8) Proteirzs as lubricants: Tbe inucous of the respiratory tract, oral cavity, vaginal
tract and the rectal cavity reduces the irritation which might be caused by
materials moving through these passages. This inucous is a mucoprotein, a
conjugated protein which contains hexosamine. Proteins as lubricants also surround
the joints and facilitate their movement. The absence of these lubricants or
substantial decrease in their fluidity through depositioil of nzinerals, incakesskeletal
movement difficult and painfill.
Last but not the least, proteins also have an important role to play in the immune
system. Let us find out how.

9) Proteins in the immune syster?~:Proteins sucb as y-globulin serve to protect the

body against foreign cells. The iinmunoglobulins produced by ly~nphocylesare
the large polypeptides having more than one basic n~onoinericunit. These proteins
differ in their ainina acid structure, which in turn, affect thcir secondary, tertiary
and quaternary structures. Just as the amino acid sequence of an enzyme
determines substrate specificity, the ainino acid sequence of the imlllunoproteiil
assures antigen-antibody specificily. The initiation of synthesis of particular
iitununoglobulins by the lyinphocytes requires the binding of WL antigen (a foreign
protein) to the cell surface at particular locations called 'antigen receptors'. The
immunoglobuliilsynthesized binds with the foreign protein iinmobilizing it. Thus,
the complex antigen-antibody is formed ahout which we have already studied in
great details in the Applied Physiology Course in Unit 3. Do look up this unit
now to understand this process.
With this, we come to an end of our study on lhe functions of proteins.
Check Your Progress Exercise 2
1) Write a note on the enzymes that are ii~volvedin protein digestion.
.....................................................................................................................
....................................................................................................................
.....................................................................................................................
2) Bring out the role of HC1 in gastric digestion.
.....................................................................................................................
....................................................................................................................
....................................................................................................................
.....................................................................................................................
3) List the functions of protein.
.....................................................................................................................
.....................................................................................................................
................................)..............................*..............................*..............a,.
Advance Nutrition
4) Which protein plays an important role in maintaining the osmotic pressure?
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
............................................................

.....................................................................................................................
5) Give examples of proteins as carriers.
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................
.....................................................................................................................

With a clear understanding on the functions of proteins, next we shall review the
methods of determination of proteins and amino acid content in foods.

4.3 METHODS OF DETERMINATION OF PROTEINS

AND AMINO ACID CONTENT IN FOODS
The methods for protein quality evaluation are grouped under the following headings:
I) Analytical methods for the determination of nitrogen and amino acids in foods
11) Chemical and microbiological assays of protein quality
111) Evaluation of protein quality in experimental animals
IV) Clinical methods for evaluation of protein quality
In this section we shall review both the qualitative and quantitative evaluation of
protein and amino acid content in foods. We shall begin with the quantitative/analytical
method for the determination of nitrogen and amino acid in foods.
Quantitntive/Annlytical Method for Esti~nntionof Nitrogen and Protein
You may'recall reading earlier that proteins contain nitrogen along with carbon,
hydrogen and some other substances. Nitrogen (N) in food not only comes from
amino acids in protein but also exists in additional forms that may or may not be used
as a part of the total nitrogen economy of humans and animals. Thus, determination
of nitrogen is commonly used to determine the protein content of a sample. Total
nitrogen, which is determined by the Kjeldahl procedure, is conve ted to crude protein
by a factor of 6.25, as mentioned earlier. Thus, the conventional measure of "protein"
or "cntde protein" in foods is N x 6.25, and it is recommended that this one factor
be used in nutritional studies in which whole diets contain more than one source of ,
nitrogen.

The Kjeldahl's method is commonly used in estimation of protein content of foods and
has been extensively used for protein estimation of various foodstuffs. Although over
a period of time many other methods have emerged for determination of organic
nitrogen, this method still remains an old favourite because it is reliable and has very
well standardized procedures.

We shall not go into the details related to the method of this process here in this unit,
since it has already been covered in the Nutritional Biochemistry Practical Course
(MFNL-002) in Practical 5. We suggest you look up this Practical now. Next, we Proteins
shall review the methods for protein quality evaluation.
Qualitative Estimation of Protein/Amino Acid Content in Foods
The protein and amino acid content of foods helps to interpret nutritional differences
among foods in terms of their amino acid make-up. This has been obtained only
through experiments on animals or human beings. The methods available to determine
protein quality are based on several Rarameters. They are categorized as methods
based on:

I) Growth and body, weight clzanges: The methods under this category include-
Protein efficiency ratio (PER)
Net protein ratio (NPR)
e Gross protein value
Rat repletion method
Nitrogen growth index
Slope ratio method
11) Carcass nitrogen analysis: This method includes-
Nitrogen retention method
Net protein utilization (NPU)
Rat repletion metllods
111) Nitrogen balance includes-
Nitrogen baln~lce
Digestibility coefficient, Biological value and net utilization of dietary proteins
Nitrogen balance index
Egg replacement method
IV) Regeneration of blood and liver constituents includes-
Liver protein regeneration
Blood protein regeneration
Regeneration of liver enzymes
V) Availability of amino acids includes-
Chemical methods
Enzymic methods
Microbiological methods
Animal assays
VI) Miscellaneous par'ameters include-
Plasma amino acid levels
Microbiological methods
VII) Chemical scoring i n c l u d e
Chemical score
Essential amino acid index
Simplified cl~emicalscore
Advance Nutrition Only a few of these methods measure the overall nutritive value of a protein - protein
efficiency ratio (PER), net protein utilization (NPU) and biological value (BV). PER,
NPR and NPU are widely used as the methods for the evaluation of dietary proteins
and amino acids. You would realize that no single method gives a,complete evaluation
of protein quality and hence a combination of method is preferred.

Let us get to know these methods. We shall start off with PER i.e Protein Efficiency
Ratio.
Protein Efficiency Ratio (PER)
PER method was developed by Osborne, Mendel and Ferry in 1919 and is based
on the growth of young rats. The diets usually contain 10% of dietary protein to be
tested and are complete in all other dietary essentials. Groups of albino rats (21 days
old) are fed for a period of 4 weeks on different diets. Records of the gain in body
weight and protein intake of the rats are maintained. The protein efficiency ratio
(PER) is calculated using the following fonnula:
Gain in body weight (g)
PER = = gain in weight per g of protein consumed
Protein intake (g)
PER, therefore, is a measurement of the eJt'cient utilization of the proteins in the
bod)].You would be interested to lu~owthat the egg protein has the highest protein
efficiency ratio. Hence, egg protein is the standard to which all other forms of
proteins are measured. Next, let us learn about digestibility coefficient.
Digestibility Coefficient
You have earlier learnt that dietary proteins are hydrolyzed to amino acids during
digestion. The digestion begins in the stomach by the action of pepsin and later
completed in the intestines by ttypsin, erepsin and other enzymes. Proteins differ in
their digestibility. Egg and milk proteins are easily digested and converted into amino
acids while pulse proteins are slowly digested to amino acids. Amino acids are
absorbed into the blood stream and the unhydrolyzed portion of the protein is wasted
in the faeces. Thus, digestibility coefficient of protein refers to the percentage of the
ingested protein absorbed ilzto the blood stream after the process of digestion
is complete.

When an animal is fed on nitrogen (N)-free diet, certain amount of nitrogen is

excreted in the faeces. This is derived mainly from the digestive juices. This is called
'endogenous faecal N.' When a protein food is given, the N found in faeces
consists both of endogenous N and food N lost in digestion. To find out N lost in
digestion, endogenous faecal N should also be determined. For the determination of
the digestibility coefficient, therefore, the data on food nitrogen intake (I,), total faecal
nitrogen excreted (F,) and endogenous faecal nitrogen (F,) are required. The digestibility
coefficient can thus be calculated using the formula:
N intake - (N in faeces - endogenous faecal N)
Digestibility coefficient = 100 x
N intake
100 x I, - (Fn - F,) I
i
-
I"
I

where, Fn - F, is the food nitrogen lost in digestion.

The digestibility coefficients of proteins are influenced by several factors, such as, the ,
presence of indigestible carbohydrates like cellulose and hemi-cellulose and the presence
of proteolytic enzyme inhibitors, I

In section 4.3 above, we read about the term "protein quality". A term which is
closely and syiionymously used with protein quality is "Biological Value" of proteinwe ,
know that higher the biological value, the better is the protein quality. So let us read Proteins
about this further.
Biological Value
A method for determining the biological value of proteins was developed by Mitchell
in 1925. It measures the quantity of dietary proteins utilized by the animal for
meeting its protein needs for maintenn~zceand growth. Groups of albino rats (28
days old) are fed successively on the following diets for a period of 10 days: (1)
protein-free diet, and (2) diet containing 10 percent protein to be tested. Urine and
faeces are collected by keeping them .in metabolism cages. Records of food intake
are maintained. The diet, urine and faeces are analyzed for nitrogen. Biological value
is then calculated using the following formula:
(Nitrogen digested - Nitrogen losl in metabolism) x 100
Biological value =
Nitrogen digested
Let us determine the expression for numerator and deno~ninator.

Nitrogen digested = N intake (I,,)- N in faeces (F,,) on the protein diet - N in

faeces (F,) on protein free diet, i.e.
Nitrogen digested = In - (Fn- F,)
Nitrogen lost in metabolism = N in urine (U, ) on protein diet - N in urine (U, ) on
an protein free diet = Un- U,
Hence, Biological value can be expressed as:

Thus, BV is . a measurement of protein quality expressing the rate of efficiency

with which the protein is used for growtlz. On a scale with 100 representing top
efficiency, Table 4.4 preseiits the biological values of proteins in several foods.

'Pable 4.4: Biological values of proteins in several foods

Food Items BV
Whole egg 93.7
Milk 84.5
Fish 76.0
Beef 74.3
Soybeans 72.8
Rice, polished 64.0
Wheat, whole 64.0
Corn 60.0
Beans, dry 58.0
Source: Food and Agriculture Organization of the United Nations. The Ainino Acid Content of Foods
and Biological Data on Proteins. Nutritional Sludy #24. Roine (1970).

Net Protein Utilization (NPU)

Mitchell (1922) introduced the term 'Net Utilization of Dietary Protein' which is a
product of digestibility coefficient and biological value divided by 100, as
shown herewith.
Advance Nutrition Digestibility coefficient x Biological Value
NPU =
100
A direct method of estimating Net Protein Utilization (NPU) was developed by
Miller and Berzder (1955). Groups of albino rats 28 days old were used. One group
was fed on a non-protein diet while the other groups were fed on the test diets
containing different proteins at 10 percent level for a period of 10 days. The food
intakes of the animals were measured. The animals were killed at the end of 10 days
and the body nitrogen was determined by Kjeldahl method on a sample of dried and
powdered carcass. The NPU is calculated according to the formula:
Body N of the test group - Body N of the non-protein group +
N consumed by non-protein group
NPU = xl00
N consumed by test group

The main advantage of determining NPU of a food or diet is that it helps in the
calculation of the net available protein of the diet. Studies have shown that there is
a good correlation between the values for PER, NPU and cl~emicalscore.
Let us now understand what is a Net Protein Ratio or NPR, and how is it determined.
Net Protein Ratio (NPR)
This method was introduced by Bender and Doell (1957) and is a modification of
the PER method. In this method, an allowance is made for the protein requirements
for maintenance. The method consists of feeding a group of weanling rats on a diet
containing 10 percent of the test protein and another comparable control group on a
non-protein diet for a period of 10 days. The NPR is calculated by adding the loss
in weight of the control gronp to the gain in weight of the test group and dividing the
total weight (g) by the quantity of protein consumed by the test group according to
the following formula:

Gain in weight (g) of the test group + loss in weight (g) of the
non-protein group
NPR=
Protein intake (g)
NPR values have been reported to correlate closely with NPU values.
Chemical Score
Since egg proteins contain all essential amino acids in adequate a~nountsand possess
the highest nutritive value among dietary proteins, Block and Mitchell (1946) assigned
a chemical score of 100 to egg proteins. Since the nutritive value of the proteins
depend on the essential amino acid most limiting in the dietary proteins, they evolved
a chemical score (based on the most limiting essential amino acid) which can sellre
as an index of the nutritive value of the proteins. The chemical score is tlze 'ratio
betweerz the content of the most limiting amino acid in tlze test protein to the
content uf the same anzino acid in the reference protein (egg protein) exl~ressed
as a percentage'. The chemical score formula is given herewith. Chemical score of
milk proteins is 65 and those of gelatin and zein are 0.

mg of amino acid in l g of test protein

Chemical Score (CS) = x 100
mg of amino acid in 1 g of reference protein

Lastly, nutrient-to-energy ratio has attracted wide interest as indices of dietxy quality.
The protein-energy ratio as a measure of dietary quality is explained next.
 Protein-Energy Ratio (NDP Cal%) Proteins

Platt and his colleagues (1961) are largely responsible for the introduction of the ratio
of protein energy to total energy (PE ratio) as a us.efu1 measure of dietary quality in
human nutrition. To take into account both the quality and concentration of the
protein, they introduced the concept of the net dietary protein calories as a percentage
of total calories (NDP Cal%). The NDP Cal%, is the net dietary protein value
expressed as percent of total calories. The NPU or the chemical score could be
utilized in calculating the NDP Cal% as both of them are indicative of protein value.
The formula used is as under:
Protein calories in the diet
NDP Cal% = x NPU x 100
Total Calories

Protein calories in the diet

NDP Cal% = x Chemical Score x 100
Total caloiies
For adults, diet with an NDP Cal% of 5% would be adequate to maintain health. In
infants, children, adolescent and pregnant women, growth is supported only by a diet
providing an NDP Cal% of 8% or above.

Now that we have studied and understood the various inethods of determining proteins,
it is important that we practically assess the proteinlamino acid quality of our diet or
perhaps of other population groups. To help you in this task, we have included various
activities in the Practical Manual (MFNL-004) accompanying this course. So go
ahead c a q out these activities.

Let us now proceed further and find out the ways by wl~icl~
we can improve protein
quality of our diets.

4.4 IMPROVEMENT OF QUALITY OF PROTEIN IN THE

DIET
Since the net protein utilization (NPU) values of inilk or egg proteins are higher than
those of proteins of average diets consumed in different countries, a correction has
to be made for this variation in the NPU of dietary proteins, This is shown
herewith:
Safe level of intake of egg or milk proteins x
NPU of egg or milk proteins
Dietay protein requirements (g) =
NPU of dietary proteins
For example, if the NPU of the dietary proteins is 45 and egg protein is 90, the
coi~ectionfactor will be 2. In other words, the amount of this dietary protein needed

1
I
to satisfy the requirements of a given population group will be twice as high as that
, of egg or milk protein.
The next thing that might come to your mind is regarding the protein quality of the
I
Indian diets. Average Indian diets as coilsulned in different parts of the country
I consists chiefly of vegetable source of protein. The amount of animal protein depends
I on the diet habits, with only milk providing a source of animal protein in vegetarian
diets to varying amounts of meat and flesh in the non .vegetarian diets. The question
of whether the protein quality of our predomintantly vegetarian diets is adeq~ateand
what are the ways in which the quality of protein in our diets can be imprhved has
been addressed through a number of studies.
Habitually, Indian diets are cereal-based diets, limiting in lysine, sm essential amino
acid critical for growth and development in children. The term limiting is used to 110
Advance Nutrition describe that indispensable anzirzo acid which is present in the lowest quantity in
the food, in cornpal-ison with the same amino acid in a reference protein suclz
as egg or milk, the quantity af the amino acid expressed in terms of per g nitrogen.
Further, the diet is predominantly vegetable-based, and foods of animal origin do not
usually find a place because of their high cost. A large percentage of people are
vegetarian and their diets include pulses, vegetables, cereals and grain products.
These plant foods tend to have too little of one or more indispensable amino acids
(i.e. lysine, threonine, tryptophan or methionine, particularly in legumes). In other
words, individual plant foods such as cereal alone or pulses alone tend to be relatively
deficient in one or more essential arnino acids and thus exclusive consumption of
single plant foods such as chiefly rice or wheat would result in deficiency of an
essential amino acid and if this consumption pattern is continued over a long period
of time, it can result in protein deficiency. However, a combination of plant foods,
such as cereal-pulse-vegetable based diets are fully capable of meeting protein needs,
when consumed in amounts that satisfy energy needs. Fortunately, for us, the amino
acid deficient in cereals, namely lysine is present in ample quantities in pulses and
green leafy vegetables. Similarly, the essential amino acid methionine which is relatively
low in pulse, is present in larger quantities in cereals. Thus, the different sources of
vegetable proteins complement each other in terms of the amino acids they provide.
Therefore, if we ensure that diet at every meal is a combination of cereals, pulses,
and vegetabIes with nuts and milk contributing wherever one can afford, it will take
care of the protein requirements. In this context, when we coilsider the Indian cuisine
we notice, North and West Indian meals consist of chapatis or rotis and rice as
staples, eaten with a wide variety of side dishes like dals, curries, yoghurt, chutney
and achars. South Indian dishes are mostly rice-based, sambhar, rasam and vegetables
being important side dishes. The pulsesAegun~es,included in the diet, with their high
content of lysine but low content of methionine, complement the grains (cereals),
which are more than adequate in methionine and cystiene but limiting in lysine. Such
cereal-pulse combination diets when consumed help the body receive all the
indispensable amino acids. Hence, there is no need to worry about protein if we are
eating a varied vegetarian diet! It is easy to get protein from lentils, dal, beans, curd,
rice, soy milk, and cereals when eaten in combillation so that their amino acid
patterns become complementary. The iilclusion of pulses in cereal-millet-based diets
is critical not only in iilcreasiilg the protein content, but also in improving the nutritional
quality of the protein.

So, then, that brings us to the second question i.e, can we improve the nutritive value
of protein? Yes, as discussed above, the nutritive value of a protein can be improved
in two ways : (1) by nzutual supplenzentation, that is, by blending two or inore
proteins so that the excess of essential amino acids present in one protein makes up
the deficiencies of the same amino acids in another protein and (2) by s~pplenzentation
of the dietary proteins with limiting esse~ztialamino acids. Let us understand
these methods by way of few examples.
1) Mutual sztpplernentation
a) Improvement o f cereal proteins: Cereals, in generaI, are limiting in lysiile and
threonine while legumes, milk, meat and fish are good sources of the anlino
acids. Hence, the proteins of legumes, inilk, meat and fish supplement effectively
cereal proteins. '
b) Soyabean and sesame proteins: Soyabean proteins are good sources of lysine
but are deficient in methionine while sesame proteins are good sources of
methionine but are deficient in lysine. Hence, the proteins of sesame supplement
effectively those of soyabean.
c) I~nprovementof cereal diets with legunze and inilk proteins: The proteins of
poor diets based on different cereals are limiting in lysine and threonine and their
quaIity can be improved effectively by incorporation of legumes, soyabean or
milk proteins in the diet.
So it is clear that mutual supplementation is the answer to improve the nutritive value
of proteins. Another way to do so would be to supplement with individual amino acids
as discussed next.

2) Supplementation vvitlz irzdividucrl arnirzo acids

a) Improvement of cereal diets by saipplenientation tvitlz Iysirze and tlzreoraine:
Cereal diets supplemented with lysine alone or a mixture of lysine and tl-xeonine
nlarkedly increases the PER of cereal proteins or protcins of poor cereal diets.
b) Illzprovement of soybean and cowk milk protein witlz ntethionine: The proteins
of soybean and cow's milk are deficient in methionine. Supplementation with
methionine increases the PER of the diet from 2.0 to 2.9 for soy bean and 3.0
to 4.0 for milk proteins.
c) Improvement of sesame orzd szln.flower seed proteins with lysi~ze:
Supplementation with the limiting amino acid lysine increases the PER of sesame
proteins from 1.7 to 2.9 and sunflower seed proteins from 1.2 to 1.8.
The discussion so far focused on methods to determine the protein quality and also
on methods one could adopt to improve the quality of protein in a particular diet. Next
let us move on to protein requirements - how they are estimated and assessed?

4.5 METHODS OF ESTMATING AND ASSESSING

PROTEIN REQUIREMENTS AT DIFFERENT
STAGES OF LIFE CYCLE
In this section, we are going to deal with the methods that are used to estimate
protein requirements, as well as, the factors which affect it. Let's read and find out
first what we mean by protein requirement and its significance.
Human protein and amino acid requirements have beerr studied for well over 100
years using a variety of techniques. Nutrition scientists have collected data on the
quantity of protein foods consumed in health, growth and weight gain of varions
populations. The assumption was made that whatever healthy people ale was probably
what kept them, healthy'and should, therefore, be used as a standard of comparison
for other diets. These standarcls with respect to protein were invariably high for
populations having an abundance of meat, milk, poulhy and fish in their diets. Voit and
Atwater around the torn of the 20"' century, found intakes of 118 and 125 g protein/
day, respectively for an adult woman and man.

As nutrition developed as a science, inore accurate methods for assessing nutrient

needs were developed. Among these methods were those for assessing the intakes
and excretion of nitrogen compounds. The Kjeldahl method, about which,we learnt
above, and other methods for determining the nitrogellous end products of metabolism
were devised. These methods made possible the development of the concepts which
today's scientists use to delermine the nutrient requjrements of humans, as well as,
other species.
In protein nutrition, it was realized that the body consists of two pools of protein: one
which has a short half life and which must be constantly renewed and one which is
slowly broken down and rebuilt. If one assumes that over a short period of time, the
pool having the long half life contributes almost nothing to the nitrogenous metabolic
end products and then a measure of the arnount of nitrogen excreted will reflect only
the turnover of the short lived proteins. These proteins have to be replaced by
proteins newly synthesized from the amino acids provided by the diet. Hence, the
term protein requirement means that 'amount of protein which must be consunzed
to provide the amino acids for the synthesis of those body proteins irreversibly
categorized in the course of the bodyS metabolism'. The intake of nitrogen from
Advance Nutrition protein must be sufficient to balance that excreted; this basic concept is called
nitrogen balance. This concept is useful in understanding the minimal need for
protein in the diet.

Protein requirement is greatly influenced by many factors such as age, environmental

temperature, energy intake, gender, micronutrient intake, infection, activity, previous
diet, trauma, pregnancy and lactation. Let us take up each of these factors in detail.

1) Age: Protein in excess of maintenance needs is required, when a new tissue is

being fornled. Certain age periods, when growth is rapid, require inore dietary
protein than other periods. Age differences in protein tuimover, as well as,
protein synthesis explain some of the effects of age on protein needs. Premature
infants (those born before their 10 lunar month gestation time) growing at a very
rapid rate require between 2.5 to 5 g proteinkglday if they are to survive.
Studies of full term infants have indicated that a protein intake of 2.0 to 2.5 gkgfday
resulted in a satisfactory weight gain and that further increases in protein intake did
not measurably improve growth. Older infants and children, whose growth rate is not
as rapid as the premature or new born infant, require considerably less protein (1.25
g/kg/day). As growth rate increases during adolescence, the protein needs increase.
Again, this can be related to the demands for dietary amino acids .to support the
growth process. As the human completes his growth, the need for protein decreases
until it arrives at a level which is called the 'rnaintena~zcelevel'. It is at this level
that the concept of body protein replacement by dietary protein applies. During the
growth period, it is very difficult to separate the requirements for maintenance from
those of growth. The impulse for growth is so strong that it will occur in many
instances at the expense of the maintenance of body tissues. For example,
malnourished children will continue to grow taller even though their muscles, as well
as, other tissues show evidence of wastage due to dietary protein deficiency.

Growth carries with it not only a total nitrogen requirement but also a particular amino
acid requirement. Maintenance, on the other hand, appears to have only a total
protein requirement. The adult can make a number of short-term adjustments in his
protein metabolism that can compensate for possible inequities of imbalances in
amino acid intake as long as the total protein requirement is met. The young growing
animal is not that flexible. The essential amino acid requirements are age dependent.
Although histidine can be synthesized in sufficient quantity by the adult to meet
maintenance needs, yet it is no1 synthesized in great enough amounts to support
growth or tissue repair. Thus, histidine is an essential amino acid ,for the infiazt,
growing child and injured adult. This is due to the nature of the growth and repair
processes.

Let us move on to the next factor and find out what environment influences have to
do with our protein requirement.

2) Environmental temperature: As environmental tenlperatures rise or fall above

or below the range of thermic neutrality, animals begin to increase their caloric
expenditure to maintain their body temperature. 111eilvironments that are too
warm, vasodilation (widening of blood vessels) occurs along with sweating and
increased respiration. All of these mechanisms are designed to cool the body
and all require an increase in the basal energy requireinent expressed as per unit
of body surface area. In cool environments, vasoconstriction and shivering occurs
in an effoit to wann the body and prevent undue heat loss. Again, an increase
in basal energy requirement is observed. Smtzts (1934) found that nitrogen
requirements were related to basal energy requirements. Through the study of
a large number of species, he concluded that 2 mg nitrogen were required for
every basal kilocalorie required when the energy requirement was expressed on
a surface area basis. Thus, any increase in basal energy needs due to a change
in environmental temperature will be because of the relationship between protein
 and energy. An increase in energy needs would be accompanied by an increase Proteins
in the protein requirement for maintenance. In addition, profuse sweating as
occurs in very warm environtnents carries with it a nitrogen loss which must be
accounted for in the deternlination of minimal protein needs.
The next factor that require attention in case of subjects selected for estimating
protein needs is information regarding the previous diet i.e., the kind of diet consuined
by subjects in the past.

3) Previous diet: The effects of previous diet in the determination of protein

requirements may be rather profound. If, for example, the subjects selected for
studies on protein needs have been poorly nourished prior to the jnitiation of the
study, their retention of the protein during the study will be greater than would
be observed in subjects who have been well nourished piior to the initiation of
the study. In other words, malno~uishedsubjects have a higher protein requirenlent
than well-nourished subjects. This of course raises the issue of whether there
are body protein reserves. Voit, Wilson, Cuthbertson, Fisher (1890) and others
observed that animals fed on' a protein-free diet exhibit a lag before their nitrogen ,
excretion level is minimized. During this phase, the animal is ~netabolizinghis
protein reserve. Other investigators maintain that every protein in the body has
a function and if some of these proteins are lost, there is a loss in body function.
Support for this concept is seen in the reduced ability of protein depleted animals
to fight infection or respond to the metabolic effects of trauma.
Whether one believes that there is such an entity as a protein reserve inay depend
upon whether one perceives a difference between personal opinions on how nutrient
requirements should be defined, Some nutritionists believe in stating the absolute
minimum requirement to sustain life and then adding on increments for each body
function above mere survival. This is known as the 'particulate approach'. Other
nutlitionists believe that one cannot separate and quantify the individual requirements
of each function beyond survival. They advocate a protein intake sufficient for
optimal function of the animal. This is known as the 'integrative approach.' The
particulate and integrative approaches each have their ineiits when argued intellectually.
However, since humans do not merely exist, inally huinan nutritionists tend to take
the integrative approach to human nullition requirements in their determination of
protein needs.
Next, we move on to another impoi-tant factor that determines our protein needs, that
is, physical activity.
4) Physical activity: Research on protein needs for n ~ ~ ~ s c uwork
l a r had its beginning
in 1863 when Von Leiberg postulated that inuscle protein was destroyed with
each contraction of the muscle. On this basis, he recornnlended that heavy
muscular work required a heavy prolein diet. This theory has been amply
disproved, yet even today many believe that a protein rich diet will contribute
to athletic prowess. Today, we know that muscle contraction does not result in
destruction of the muscle. It however, requires energy in the form of ATP,
glucose and fatty acids and does result in the breakdown of creatine pl~osphate
to creatine which is then converted to creatinine, a nitrogenous waste product
excreted in the urine.
As the energy requirement is increased to support the increase in muscular activity,
so too is the protein requirement in much the same inanner as described above for
the effects of temperature. In a number of studies, the athletic performance of
subject's could not be directly related to the quantity of protein consumed above that
determined to be the requirements, their lnuscular deficiencies were reduced unless
a vigorous training programme was included as a part of the experiment's protocol,
Since most of the studies were of short duration and since muscle protein has a
relatively long half-life, the lack of any demonstrable effect of protein intake on
muscle performance is not surprising.
Advance Nutrition Other factors such as sex, pregnancy, lactation and trauma affecting the protein
requirements have been studied. As can be anticipated, males due to their greater
physical activity and larger body size have a larger protein requirement than females.
Pregnancy, lactation and trauma increase the protein requirements.
So, now you realize that a large number of factors play a role in determination of
protein requiremei~ts.Let us next find out what are the recommended allowances for
proteins for different age-groups.

4.6 NUTRITIONAL REQUIREMENTS AND

RECOMMENDED ALLOWANCES FOR PROTEINS
AND AMINO ACIDS
The FAOIWHO Committee (1973) expressed the protein requirements in terms of
egg or milk proteins. The committee defined safe level of protein intake as 'the
amount necessary to meet the physiological needs and maintain the health of
nearly all the individuals in a specified agehex group.'
The cormnittee followed three procedures in arriving at the protein requirements:
1) Amino acid requirements,
2) The factorial method, in which the obligatory nitrogen losses and N required for
growth, pregnancy and lactation are estimated, and
3) Measurements of minimum protein intake required for satisfactory growth and
N balance in infants and children and N equilibrium in adults.
Let us briefly discuss each of these and try to understand these better, starting off
with amino acid requirements:
1) Amino acid requirements
Data regarding the essential amino acid requirements of infants, children and adults
are given in terms of egg protein and cow's milk protein (gtkglday) required to meet
the amino acid needs. These requirements are given in the Table 4.5.
Table 4.5: Essential amino acid requirements
S.No. Age-Group Egg Protein (g) Cow's milk (g) Protein
1. Infants 1.6 2.0
2. Children (10-12 yeas) 0.9 0.9
3. Adults 0.18 0.28
a) Women 0.18 0.28
b) Men 0.26 0.43

The Committee suggested a Reference Anlino Acid Pattern in 1973. Since adequate
experimental evidence for the suitability of the pattern was not available, the Committee
adopted egg and milk proteins as reference proteins and expressed protein requirements
in terms of egg or milk proteins. The Committee assumed that the proteins of milk
or eggs are utilized to the same extent in children and gave a protein score of 100
to egg and milk proteins.
2) Factorial method
The nitrogen requirements have been calculated by a factorial method suggested by
different expert groups, described as follows:
R=U+F+S+G
where
R = N requirements,
U = loss of endogenous N in urine,
I
F = loss of endogenous N in faeces, Proteins ; 1
S = loss of N through skin, i.e., sweat and integumental losses, and
I
G = N required for growth.
Let us look at these different components of the factorial method.
1
1
a) Obligatory N losses: The Committee estimated the total obligatory nitrogen
losses through faeces, urine, skin and other miscellaneous routes in adult men
as 2.0 mg Ntbasal Kcal. The obligatory N losses in adult men on a protein-free
diet is given in Table 4.6,
Table 4.6: Obligatory nitrogen losses (mg)
-- -

( Route N per kg of Body Weigltt N per Kcal of Basal Energy 1

I Urine 37
I Faeces

+
I ski.
Miscellaneous

The Committee used the same figure of 2.0 mgN per basal Kcal Ibr the total
obligatory losses in women, infants and children.

b) N requirenzents for growth: The nitrogen requirements for growth of infants

and children are given in the Table 4.7.
Table 4.7: Nitrogen requirelnents for growth
Nitrogen for Growth ( mgN/kg/day)
--
Infants
0- 3 months
3- 6 months
6- 9 months
9- 12 months
Children
1 year
2 year
3 year
4 - 6 year
7 - 9 year
10 - 12 years

c) N requirements in Pregnancy and Lactotion: The nitrogen accretion in the

pregnant woman (assuming that [he foetus weighs 3.3 kg at term), estimated by
the Committee, is given in Table 4.8.
Table 4.8: Nitrogen accretion during various stages of pregnancy
Stage of Gestation N Accretion /day
(md
I ' First quarter 80
Second quarter 400
Third quarter 740

I Fourth quarter 860

After having a thorough knowledge of the factorial method of estimating N

requirements, surely you would be now in a position to calculate protein requirements.
Advance Nutrition The nutritional requirements of protein for Indians in different age groups and
physiological stages as suggested by the ICMR is given in Table 4.9.
Table 4.9: Nutritional requirements of protein

Group Particulars Body Weight(kg) Protein g/d

Man Sedentay work 60 60

Moderate work
Heavy work
Woman Sedentary work
Moderate work 50 50
Heavy work
Pregnant woman 50 +15
Lactation
0 - 6 months 50 +25
6 - 2 months +18
Infants 0 - 6 months 5.4 2.05kg
6 - 12 months 8.6 1.65Ikg
Children 1 - 3 years 12.2 22
4 - 6 years 19.0 30
7 - 9 years 26.9 41
Boys 10 - 12 yews 35.4 54
Girls 10 - 12 years 31.5 57
Boys 13 - 15 years 47.8 70
Girls 13 - 15 ycars 46.7 65
Boys 16 - 18 years 57.1 78
Girls 16 - 18 years 49.9 63

. Sollrce: ICMR (1988).

Next, let us have a look at the consequences of protein deficiency in our diets.

4.7 PROTEIN DEFICIENCY

One of the most common nutritional disorders in the world today is the deficiency of
protein. Both adults and children are affected, as the populations in the less developed
nations of the world exceed their food supply. Due to the ubiquitous nature of protein
and its role in bodily function, protein deficiency is characterized by a number of
symptoms. In many situations, not only is protein lacking in the cliet but also calories.
For this reason, it is difficult to segregate synlptoms due solely to protein deficiency
froin those of energy deficit. In children, one may observe the different syinptoins
and visualize thein all as parts of a continuum called protei?z-energy or protein-
calorie lnalnutrition (PEM or PCM) rather than distinctly different nutritional disorders.
'Kwashiorkor' was the term used to describe a disease first observed in the Gold
Coast of Africa by Dr. Cicely Williams in 1935 and at first was regarded as a dietay
state where only protein was deficient, not energy. Mamsmus, on the other hand,
was regarded as a dietary state where both protein and energy are deficient. Now
it has become apparent that the syinptoms of any one of the twin diseases may
intermingle with the other so that a clear-cut diagnosis is impossible. A detailed
discussion o n PEM is presented in the Public Nutrition Coursc, Unit 3. We suggest
you look up the unit now as you go about reading regarding PEM here in this unit.
Here a very brief ove~viewof the disorder has been presented.
A. Kwashiorkor
The tenn Kwashiorkor means tlie 'disease the first child gets when the second baby
is born', that is, 'the sickness of the deposed child'. Thus, the disease could be cured
by milk.
Kwashiorkor usually affects the young child after he is weaned. The child is usually
between 1 and 3 years old and is weaned because the mother has given birth to
another child or is pregnant and cannot support both children. If the child has no
teeth, he is given the gruel. This may be a fruit, vegetable or cereal product mixed
with water and hence n0t.a good protein source. Can you think of a few reasons
which could linlit or decrease the protein intake? Let us see what these are. Cultural
food practices or taboos may further limit the kinds and amount of protein given to
the child. Apart from this, concurrent infections, parasites, seasonal food shortages
and poor distrib~rtionof food amongst the family members may also contribute to the
development of kwashiorkor. The deficiency develops not only because of inadequate
intake but also because at this age the growth demands for protein and energy are
high.
The symptoms of kwashiorkor are as ~follows:
a Growth .failure: This is manifested by decreased body length and low body
weight in spite of retention of water in the body (oedema) and presence of
subcutaneous fat in some children. This growth retardation is primarily due to
the general quantitative lack of proteins.
0 Mentcil clzanges: Several workers have stressed on the constant finding of
mental changes described as apathy and peevishness. In advanced cases,
children tend to live in an inert listless condition and show no interest in the
surroundings.
Oedeina: Oedeina occurs at first in the feet and lower legs and then may
irivolve the hands, the thighs and face. The oedema is mainly due to lowered
serum albumin and probably also due to high sodium and low potassium levels
in serum. There is also some evidence that the t ~ o ~ m adiuretic-antidiuretic
l
honnonal control of urine secretioil gets disturbed.
Mzlscle wasting: Muscle wasting is a constant feature of kwashiorkor and a
reduction in the circumference of the upper arm is usually evident. It is less
affected by oedema than in the forearm or leg:
o Moorzface: The :fi111, well-rounded face, known as moon-face, is often present
in kwashiorkor.
Liver clzanges: Liver is slightly enlarged and fatty infiltration of liver is usually
present.
Gastrointestinal tract: Loss of appetite and voiniting are common. Diarrhoea
is present in most cases.
Skin and hair c/zunges: The characteristic skin changes of kwashiorkor are
known as the 'crazy pavement' dermatosis. This is 111ost~narlcedon the buttocks,
back of thighs aild axille. These lesions consist of dark hyperl7igrnental brownish
black areas of skin.
Anaemia: Anaemia is invariably present. It is due to the deficiency of iron and
folic acid. Anaemia may be aggravated by parasitic infection which prevents the
absoiption of nutrients.
Vitainin Deflcierzcy: Signs and syinptoms of vitamin A del'iciency such as
xerophthalmia and keratomalacia are widely prevalent. Angular stoinatitis and
glossitis due to deficiency ol riboflavin may be present.
Biochemical changes: Several biochemical changes have been reported in
children suffering from kwashiorkor.
Serum Albunlin: The serum albumin content is usually low ranging from 0.7 to
2.2 g1100 inl. Seruin albumin level is a good index of the severity of the disease
and the iise in serum albumin level duiing treatnlent is a reliable index of the
rate of recovery.
Advance Nutrition e Enzymes in Ser~unand Digestive Juices: Low levels of choline ester;
allcaline phosphatase, amylase and lipase have been reported in kwashiorkoc
experimental animals fed on protein deficient diet, reduction in lipase, m y 1
and protease activities of pancreas have been reported.
Let us learn about marasmus next.
B. Marasmus
Altl~oughchildren of all ages and adults can suffer from deficiencies of both ener
and protein, the marasmic child is usually less than one year old. In developi
countries, a comnlon cause for inarasmus is the cessation of breast-feeding. M
production by the mother may have stopped because of her poor-health, death
deliberate decision of the mother to bottle-feed her baby. This decision might ha
a socio-econoinic connotation. Can you think of it? Well, it is just that the inotli
may view bottle-feeding as a status symbol or she may be forced to work to ea
a living and may be unable to have her baby with her, or she may not be able
lactate. While under optinla1 conditions of economics and sailitation, it seeins that tl
bottle-fed child may be well fed in emerging nations but this is not always true. Tl
mother nlay not be able to buy the milk formula in sufficient cluantities to adequate
nourish the child, she may over dilute the milk or she may use unsafe water ar
unsanitary conditions to prepare the f o i x ~ ~for
~ l athe child. This plus the insufficiei
nutrient content often precipitously leads to the developnlent of marasmus, a form (
starvation characterized by growth failure with prominent ribs, a characteristic nlonke
like face and 'match stick limbs' with little nluscle or adipose tissue developmen
Tissue wastage but no oedeina is present. Whereas the kwashiorkor child has a poc
appetite, the marasinus child is eager to eat. The child is mentally alei-t but nc
irritable. Anaemia and diarrhoea are present for the same reasons as in kwashiorko
The skin and hair appeal. to be of normal colour.
Protein hormones which regulate and coordinate the use of dietary nutrients aie nc
V o u n d in adequate amounts. Cell hormone receptor sites are affected which furthe
dampens the effectiveness of the hormones produced. Marasmic and lwashiorko
children have decreased blood sugar levels, decreased serum insuli~land growtl
hoirnone levels and in marasmus, decreased thyroid hormone levels. The clinica
features of marasmus are as follows:
N~~tritiol~al
Marasnz~~s: N~~tritionalmarasmus is principally due to the consumptioc
of diets markedly deficient in both proteins and caloiies. It is seen most coinnlonly
in the weaned infants of about 1 year of age in contrast to kwashiorkor, which
occurs inore often among children of the age group 2-4 years. Nutritional
marasmus usually is precipitated by dia~hoealdiseases.
Clinical Features: The two constant features of nutritioilal inarasmus are growth
retardation and severe wasting of rnuscle and s~~bcutaneous fat. ,

a) Growth retardation: This is usually very severe. Loss of weight is inuch more
marked than decrease in height. The child is usually below 60 percent of the
standard weight.'
b) Wasting of muscle and s u b c ~ ~ t n n e ofat:
~ ~ sThe subject is severely emaciated.
The muscles are wasted. The arms are thin and the skin is loose. Subcutaneous
fat is practically absent.
C) Other changes: The skin is dry and atrophic. The subject shows signs of
dehydration. Eye lesions due to vitamin A deficiency and anaemia inay be
present.
d) Biochelnicnl charzges: There is a slight lowering of senun albumin. Vitamin A
content of serum is low. The i~nporlantdifference in the clinical and biochemical
features between marasmus and kwashiorkor are given in Table 4.10.
 Table 4.10: Clinical and biochemical features of marasamus and kwashiorkor Proteins
I

Features Marasmus Kwashiorkor

Age of maximum incidence 6-18 lnonths 12-48 months
Loss of body weight +++ +Lo+ +
Emaciation (Loss of muscle + + + + +to + +
and subcutaneous fat)
Oedema Absent + to++
Fatty infiltration of liver 0 LO + +++
Skin changes + +++
Seiuin albumin Slightly less Markedly less
Serum enzymes Slightly lowered Markedly lowered
Serum lipids:
Triglycerides Normal No~mal
Choleslerol Normal Lowered
Non-esterified fatty acids Elevaled Elevated
Blood sugar Normal Slightly lowered
Respoilse to adrenaline ExaggclaLed Lowered
Blood urea Normal Lowered
Increase in body weight after Slow Satisfactory
high protein and high calorie
therapy duiing the first 4 weeks

What is the treatmerit qf Kwashiorkor and ~ ~ r u s n z ~ i s ?

The treatment of both kwashiorkor and mwasmic childreil requires care and caution.
As their enzymes for diges~ionand their protein absorption and transport systems are
less active, feeding these children with large quanlities of good quality protein would
be harmful. Well, this might sound strange to you. But il is so. Their diets must
gradually be enriched with these proteins to allow their body sufficient time to develop
the metabolic pathways to handle a better diet. Giving these children solutions of
either predigested proteins or solutions of amino acids may be of benefit initially, but
these solutions too must be used with care. If the ainino acids in excess of iminediate
use are deatninated and if lhe pathway for synthesizing urea is not fully functional,
ammonia can accumulate in the child and become lethal.

We have studied about kwashiorkor and marasinus as conditions typical of protein

energy malnutrition. But, interestingly, a large number of cases show signs and
synlptoms of both maasmus and kwashiorkor. These are the inkmediate forms. Let
us learn about them next.
Marasmic Kwashiorkor
In countries where the incidence of protein-calorie lllalnutrition (PCM) is high, a large
number of cases show signs and syinptoms of marasmus and kwashiorkor. These
intermediate forms are called 'Marasmic-kwashiorkor'. In addition, the inter-relationship
between the two major syndromes is such that the changing circumsta~~ces may
result in a transition from one clinical picture to mother. A child with early kwashiorkor
can develop nutritional marasmus by severe infective diarrhoea and ill-advised prolonged
under-feeding. Conversely, an infant with nutritional marasmus may develop
kwashiorkor if fed on protein deficient carbohydrate rich foods along with adequate
common salt.
With this we end our discussion on the consequences of protein deficiency, as well
as, other varied aspects of protein which may influence human health. In the next
Advance Nutrition unit we shall discuss about lipids in detail. However, you must attempt the check your
exercise below before reading further.
Check Your Progress Exercise 3
1) Choose the correct answers:
i) The NPR method of evaluating protein quality is based on:
a) Growth and body weight changes
b) Carcass nitrogen analysis
I
c ) Nitrogen balance
d) Pattern of liver regeneration
ii) The Chemical score of gelatin is:
a) 100 b) 99.5
c) 50 d) 0
iii) The most availability feature of protein malnutrition is:
a) Weight loss b) Growth failure
c) Oedema d) Night blindness
iv) The response to protein calorie therapy is shown in:
a) Marasmus b) Kwashiorkor
c) PEM d) VAD
v) Moon face is a symptom of:
a) Beri beri b) Kwashiorkor
c) Marasamus d) Scurvy
2) Define the following terms, giving their calculation formula.
a) Digestibility coefficient
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b) NPU
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c ) Chemical Score

3) How can you improve the nutritive value of a protein?

 Proteins
4) List a few factors which affect protein requirements.
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5 ) Give clinical and biochemical features of Kwashiorkor and Marasmus.
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4.8 LETUSSUMUP
This unit covered the in~portantmacronutrient proteins. Proteins, you learnt, are vital
to all body cells, tissues, organs and functions of organ systems. The process of
digestion, absorption and metabolism of protein are complex and involve several
nutritional and non-nutritional factors. Proteins are widely distributed in nature, the
richest vegetarian source being soy beans. Egg protein has the highest biologic value.
Several methods can be elnployed to determine the protein content of foods, the most
popular being those based on growth and body weight changes. Protein quality in the
diet may be improved by mutual supplementation of protein rich foods and cereals.
Nutritional requirements of proteins vary with age and physiological activityhtage.
Deficiency of proteins leads to the twin disorders of marasmus and kwashiorkor and
may be cured by dietary intervention.

4.9 GLOSSARY
Albinism : disease caused in persons unable to form melanin
pigment.
Amino acids ; the building blocks of proteins composed of
carbon, hydrogen, oxygen and nitrogen.
Apoenzyme : protein component of an enzyme.
Autocatalysis : a sdf-repeating and ongoing process of catalysis.
Chemical score : the ratio between the content of the most limiting
amino acid in the test protein to the content of
the same amino acid in egg protein expressed
as a percentage.
Conjugated proteins : proteins which contain some non-protein
substances.
Derived proteins derivatives of the protein molecules formed
ough hydr~lyticchqges in the molecule.
Digestibility coefficien e percentage of the ingested protein absorbed
into the blood stream after the process of
digestion is complete,
Advance Nutrition
Oedema : accumulation of.water in the interstitial space.
Esseiltial amino acids : amino acids that are indispensable and are not
synthesized in the body.
Kwashiorkor : ' disease the first child gets when second baby is
born.
Limiting amino acids : the amino acids present in the least proportion in
a food.
Marasinic kwashiorkor : the intermediate form of disease where signs
and symptoms of marasmus and kwashiorkor
are seen.
Mutual supplementation . : blending two or more proteins so that the excess
of essential amino acids present in one protein
makes up the deficiencies of the same amino
acids in another protein.
Non-essential amino acids : amino acids that are dispensable cud can be
synthesized in the body.
Nucleoproteins : combination of nucleic acids and sinlple proteins
consisting of a large number of basic amino acids.
Protein requirement : amount of protein which must be consumed to
provide the amino acids for the synthesis of those
body proteins irreversibly catabolized in the
course of the body's metabolism.
Proteoglycans : glycoproteins with more than 80% of molecules
as carbohydrates.
Regulators : factors which maintain the body's internal
equilibrium o r balance.
Simple proteins : proteins which contain only amino acids or their
derivatives and no prosthetic group.
Supplements : foods used to enhance the nutrients present in a
person's diet or menu.

4.10 ANSWERS TO CHECK YOUR PROGRESS

EXERCISES
Check Your Progress Exercise 1
1) a) 6.25, b) metallo, c) proteoglycans, d) egg, e) incomplete
2) 1. d; 5. h;
2. e; 6. a;
3. f; 7. c;
4. b;
Check Your Progress Exercise 2
1) The enzymes involved in protein digestion are enumerated in sub-sub section
i
4.2.3.1. Read the section and write the answer on your own.
2) The HCl serves several functions in gastric digestion. It acidifies the ingested
food, killing potential pathogenic organisms. However, not all pathogens are
killed. Some are acid resistant or are so plentiful in the food that,the amount of
gastric acidification is insufficient to kill all of .the pathogens. HCl also serves
to denature the food proteins, thus n~&ing tl~eilimore vulnerable to attack by
 the proteolytic enzyme present in gastric juice namely, pepsin and endopeptidase Proteins
(it hydrolyzes peptide bonds in the interior of the protein molecule).
3) Proteins act as enzymes, carriers for various compounds, act as regulators of
water balance, act as buffers, act as stnlctural elements and structural units,
function as lubricants and are vital to the immune system.
4) Albumin plays an important role in maintaing the osmotic pressure.
5) Examples of proteins as carrier include a albumin, a and P globulin and
haemoglobin.
Check Your Progress Exercise 3
1) i) a
ii) d

iv) a

2) a) The percentage of ingested protein absorbed into the blood stream after the
process of digestion is complete. It is calculated as:

100 x I,, - (F, - F,)

I"
where, I, = N intake
F,, = N in faeces
F, = Endogenous faecal N
b) A prod~zctof digestibility coefficient and biological value divided by 100.
Biological value can be expressed as:
I, - (F, - F,) - (U, - Uv) x 100
BV=
I n - (Fn - Fe)
c) The ratio between the content of the most limniting amino acid in the test
protein to the content of the same amino acid in egg protein expressed as
a percentage.
3) by mutual supplementation, that is, blending two or more proteins so that the
excess of essential amino acids present in one protein makes up the deficiencies
of the same amino acids in another protein, and by supplementation of the
dietary proteins with limiting essential amino acids.
4) Factors which affect protein requirements are age, environmental temperature,
energy intake, gender, micronutrient intake, infection, physical activity, previous
diet, trauma pregnancy and lactation.
5) Clinical and biochemical features of maiasamus and kwashiorkor are given in
Table 4.10. Read the features and answer the question on your own.