CLINICAL CHEMISTRY I

MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

OBJECTIVES o After the sample collection, you stand the sample
• Know the specimen handling and storage samples for for about 5-10 minutes and allow the sample to
Glucose testing. clot then centrifuge it. Next is to separate the
• Discuss the different glucose methodologies. serum from the plasma.
• Describe the different laboratory testing for glucose • In separated, nonhemolyzed sterile serum, stable as
analysis. long as 8 hours at 25 degrees Celsius and up to 72
SPECIMEN CONSIDERATIONS hours at 4 degrees Celsius.
POSSIBLE SAMPLES STORAGE SAMPLES
• Whole blood → 11% lower than serum or plasma • Refrigerated (2-8 degree Celsius)
• Plasma o For preservation, so that you can still use the
• Serum → Unhemolyzed Venous plasma/serum sample when needed.
o We don’t accept hemolyzed sample in glucose o Serum or plasma: stable up to 48 hours
determination especially for plasma and serum, o Whole blood: 2mg Na Fluoride per mL of whole
not only in clinical chemistry sections but all blood (48hrs)
sections in the laboratory. GLUCOSE METHODOLOGIES
• Cerebrospinal Fluid (CSF) • Chemical Method
• Pleural Fluid • Enzymatic Method
• Urine CHEMICAL METHOD
• Most of the diagnostic laboratory and hospitals are A. OXIDATION REDUCTION METHOD
using the plasma or/and the serum in glucose 1. ALKALINE COPPER REDUCTION METHOD
determination. a. Folin Wu Method
SEPARATION OF LIQUID PORTION b. Nelson Somogyi Method
• Serum can be separated within 30 minutes. c. Neocuproine Method
o When you extract sample, you transfer that to a d. Benedict’s Method (Modification of Folin Wu)
test tube and allow the sample to stabilize or to Principle: Reduction of cupric ions to cuprous ions
stay at room temperature for 5-10 minutes. Then, forming cuprous oxide in hot alkaline solution by glucose.
centrifuge. o Cupric ions = Copper II cation
o It is important to separate the serum from the o Cuprous ions = Copper I cation
sample because the WBCs or RBCs tend to use
the glucose. When these cells use the glucose
present in the sample, there will be glycolysis
leading to false decrease of glucose.
A. FOLIN WU METHOD
• Serum w/o bacterial contamination & w/o leukocytosis
(high WBC) = acceptable up to 90 minutes delay only. • The glucose reduces cupric ions present in the
alkaline copper reagent to cuprous ions or the copper
• Plasma must be separated from the cellular fraction
sulfate is converted to cuprous oxide which reduces
(even with Na Fluoride)
the phosphomolybdic acid to phosphomolybdous acid
o Sodium fluoride is present in the glucose
which is the blue color when optical density is
determination tube.
measured at 420 nanometers
o Sodium fluoride inhibits the glycolytic enzyme
called the enolase which also acts as an • This is part of the alkaline copper reduction method
anticoagulant. which is under the general classification of the
o Acetate also inhibits the glycolytic enzymes of the chemical method.
glyceraldehyde 3-phosphate dehydrogenase. • Cuprous Ions + Phosphomolybdate →
o Centrifugation must be done upon receiving the Phosphomolybdic Acid or Phosphomolybdenum Blue
sample. B. NELSON SOMONGI METHOD
• Glycolysis decreases serum glucose by • The sugars with reducing property arising out of the
approximately 5% to 7% in 1 hour (5 to 10 mg/L) in presence of the potential aldehyde or keto group are
normal uncentrifuged coagulated blood at room called the reducing sugars.
temperature. o Examples of the reducing sugars are glucose,
galactose, lactose, and maltose

SECOND SEMESTER 1
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

• This method is one of the classical and widely used
methods for the quantitative determination of the
reducing sugars.
• The reducing sugars when heated with the alkaline
copper tartrate reduce copper from the cupric state
and cuprous oxide is formed.
• Cuprous Ions + Arsenomolybdate → Arsenomolybdic
Acid or Arsenomolybdenum Blue
• When the cuprous oxide is heated with the
arsenomolybdic acid (or arsenomolybdate) there will
be the reduction of molybdic acid to molybdenum
which has the color blue.
• Cuprous ions with the reaction of the
arsenomolybdate form the arsenomolybdenum blue
• color varies from green to dark red or brick red or rusty
or the arsenomolybdic acid.
brown depending on the amount and type of sugar
o The blue color that is developed from this whole
• Fehling’s test has the principle that the aldolases are
reaction is compared with the standards in
easily oxidized to yield carboxylic acid.
colorimetry at 620 nanometers.
C. NEOCUPROINE METHOD (2,9 DIMETHYL 1,10
PHENANTROLINE HYDROCHLORIDE)
• The copper in the oxidation state reacts with
neocuproine forming a complex.
• This complex is extracted into a chloroform-methanol
mixture giving a yellow or yellow-orange solution.
• Cuprous Ions + Neocuproine → Cuprous-
Neocuproine Complex (Yellow or Yellow-Orange)
D. BENEDICT’S METHOD (MODIFICATION OF
FOLIN-WU)
• Used for detection and quantitation of reducing
substances in body fluids like blood and urine.
• Used citrate or tartrate as a stabilizing agent.
• This benedict’s test is used to test for simple • The cupric ion complex with tartrate iron is reduced to
carbohydrates. It identifies reducing sugars example cuprous oxide forming the product of a brick red color.
of which we have the monosaccharides and some • What's the difference between Fehling’s test and
disaccharides which have three ketone or aldehyde benedict’s test?
functional groups. o Reducing sugars and aldehydes are chemical
• These benedict’s solutions okay can be used to test compounds that can get oxidized by reducing
for the presence of glucose in the urine. some other components. The concept can be
• Benedict’s test has a principle that when the reducing used to identify the presence of them in a
sugars are mixed with benedict's reagent and heated compound mixture.
a reduction reaction causes the benedict agent to ▪ For identification of this compound the
change its color. Benedict's and the Fehling’s test can be
used. This test uses specific reagents known
as the Benedict solution and the Fehling’s
test solution respectively
▪ Benedict solution contains the Copper (II)
Citrate.
▪ Fehling’s solution contains Copper (II)
Tartrate.

SECOND SEMESTER 2
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

2. ALKALINE FERRIC REDUCTION METHOD • The horseradish peroxidase is used to catalyze the
HAGEDORN JENSEN second reaction and the H2O2 is used to oxidize a dye
• Has the principle of involving the reduction of yellow compound.
ferricyanide the colorless ferrocyanide by glucose. • two commonly used chromogens are used in this type
• it includes or uses the principle of inverse of method:
colorimetry method. o 3-methyl-2-benzothiazolinone hydrazone
B. CONDENSATION METHOD o N,N-dimethylaniline
ORTHO-TOLUIDINE (DUBOWSKI METHOD) • The shift in the absorbance can be monitored
• Uses the principle of condensation of glucose with a spectrophotometrically and is proportional to the
primary aromatic amine in glacial acetic acid, forming amount of glucose present in the specimen.
an equilibrium mixture of glycosylamine and the • This copper reaction is so this couple reaction is also
corresponding Schiff base. known as the Trinder reaction
• Glucose + Aromatic amines (applied with Glacial HAC • Two different reactions from the B-D-glucose
and heat) → glycosylamine + Schiff's base producing the gluconic acid and the H2O2 and from
• This method shows that having this type of procedure, that we will have the Trinder reaction in which the
the glucose in the protein-free filtrate (3% TCA) reacts H2O2 using the enzyme peroxidase will form the
to ortho-toluene in hand acidic solution will yield a oxidase chromogen and one of its products is the
green-colored compound with the maximum water.
absorbance at 630 nanometers. • Take note:
• This produces the product solution having the color o Increased levels of uric acid, bilirubin, and
green in the color compound. ascorbic acid can cause falsely decreased values
ENZYMATIC METHOD as a result of these substances being oxidized by
This acts on glucose but not on other sugars and not peroxidase, which then prevents the oxidation
another reducing substance: and detection of the chromogen.
1. Glucose Oxidase Method o Strong oxidizing substances, such as bleach, can
A. Colorimetric Glucose Oxidase Method (Saifer cause falsely increased values.
Gerstenfield Method) B. POLAROGRAPHIC GLUCOSE OXIDASE
B. Polarographic Glucose Oxidase • It measures the rate of oxygen consumption which is
2. Hexokinase Method proportional to glucose concentration.
3. Glucose Dehydrogenase Method • Glucose oxidase in the reagent catalyzes the
4. Dextrostics (Cellular Strip) oxidation of glucose by oxygen under first-order
5. Interstitial Glucose Measuring Device conditions, formingH2O2.
1. GLUCOSE OXIDASE METHOD • The enzymatic conversion of glucose is quantitated
A. COLORIMETRIC GLUCOSE OXIDASE METHOD OR by the consumption of oxygen on an oxygen-
SAIFER GERNSTENFIELD METHOD sensing electrode.
o From that, H2O2 is prevented from re-forming
oxygen by adding molybdate, iodide, catalase,
• Glucose + O2 + H2O2 (catalyzed with glucose and ethanol.
oxidase) → gluconic acid + H2O2
• It states that the glucose oxidase is a more specific
enzyme reacting with only the B-D-glucose and this • Glucose + O2 + H2O2 (catalyzed by glucose oxidase)
glucose oxidase converts this B-D-glucose to → gluconic acid + H2O2
gluconic acid and the oxygen is consumed and H2O2 • Oxygen consumption can be used to perform the
is being produced in this type of oxidase method. direct measurement of oxygen by the polar
polarographic technique.
• Oxygen depletion is measured and is proportional to
• H2O2 + reduced chromagen (catalyzed by the amount of glucose present.
peroxidase) → oxidized chromagen + H2O2 • Polarographic glucose analyzers measure the rate of
oxygen consumption because glucose is oxidized by

SECOND SEMESTER 3
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

the first-order conditions using glucose oxidase • It provides results in close agreement with the
reagent. hexokinase procedures. Mutarotose is added to
shorten the time.
Glucose Dehydrogenase Method Reaction:
or

• The H2O2 that has been one of the products in the first
reaction must be eliminated in a side reaction to
prevent the reaction from reversing.
DEXTROSTICS (CELLULAR STRIPS)
• The catalase is used to catalyze the oxidation of
• These cellular strips are touched to a drop of blood
ethanol by the H2O2 and form acetaldehyde and the
and then inserted into the meter which gives a digital
water.
reading of the blood sugar with a unit of mmol/L
• Another option is the use of molybdate to catalyze
• An enzyme-impregnated strip used with a small
the oxidation of iodide to iodine by the H2O2.
portable electronic colour-measuring device for
HEXOKINASE METHOD
convenient estimation of the blood sugar levels by
• More accurate than the glucose oxidation method
diabetics.
because the coupling reaction using the glucose 6-
• One of the Point-Of-Care Devices (POC).
phosphate hydrogenase is highly specific. Therefore,
• It should not be used to diagnose diabetes or
it has less interference than the couple glucose
hypoglycemic disorders.
oxidase procedure
• Hexokinase, in the presence of ATP, converts
glucose into glucose 6-phopshate. Now, this glucose
6-phosphate and the co-factor NADP are converted
to 6-phosphogluconate and NADPH by glucose 6-
phopshate dehydrogenase at 340 nanometers
maximum absorbance measuring the rate of
appearance of NADPH.
• Plasma collected using heparin, EDTA, flouride,
oxalate or citrate may be used for this test.
• Other samples can also be used: Urine, CSF, and INTERSTITIAL GLUCOSE MESURING DEVICE
serous fluids. • Used for continuous monitoring of glucose levels in
• Most Specific Glucose Method; REFERENCE people with diabetes.
METHOD • Uses electrochemical methods to automatically and
Hexokinase Method Reaction: frequently measure glucose levels in the interstitial
fluid of dermis or subcutaneous fat tissue.
• The result of this test provides an idea of the glucose
patterns over the hours or days of the patient.
Take Note: Rate of appearance of NADPH can be
monitored spectrophotometrically and is proportional to
the glucose present in the sample. Gross hemolysis and
extremely elevated bilirubin may cause a false decrease
in results.
GLUCOSE DEHYDROGENASE METHOD
• When glucose is measured using a glucose
dehydrogenase method, glucose is reduced to
produce a chromophore that is measured
spectrophotometrically. the amount of NADH
generated is proportional to the glucose
concentration.

SECOND SEMESTER 4
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

LABORATORY TESTING FOR GLUCOSE FASTING PLASMA GLUCOSE
There are 7 laboratory tests for glucose, and these are: ● Formerly fasting blood sugar (FBS)
• Random plasma glucose (Random Blood Sugar) ● It needs fasting (nothing by mouth)
• Fasting plasma glucose (Fasting Blood Sugar) ● Sometimes in the laboratory they will accept if the
• Tolerance test patient drank a small amount of water provided that
• HbA1c (Glycosylated immunoglobulin test) the attending physician of the patient has been
• Fructosamine informed.
• Urine Microalbumin ● Specimens collected after 8.10 hours fasting (new
• Ketone testing guidelines)
RANDOM PLASMA GLUCOSE
• Formerly known as the random blood glucose test.
● If you have the result of more than 7.0 mmol/L or 126
• Specimens are collected anytime of the day (does not
mg/dL, it is more likely diagnosed as diabetes mellitus
requires fasting)
NORMAL VALUES FOR SERUM OR PLASMA, OR
• Usually done in glucometer (applies the same
WHOLE BLOOD
principles with the dextrostics)
Serum 70-110 0.05551 3.9 -
• Has no normal values (reference values) as the
Glucose, or mg/dL 6.1
glucose is measured randomly.
fasting plasma mmol/L
White 60-100 3.3 -
blood mg/dL 5.6
mmol/L
ORAL GLUCOSE TOLERANCE TEST (OGTT)
● This is multiple blood sugar test
● It is recommended to measure glucose concentration
in urine but in practice, the serum is enough as a
• The principle of glucometer works with the sensors or sample of choice.
amperometry. ● The purpose of this test is to determine well your body
• The first step to measure the glucose in the blood is metabolizes glucose over a required period of time.
to convert glucose concentration into voltage or ● Before the oral glucose test is given to a patient, a
amount or current signal. This is possible with a fasting sample will be extracted.
special sensor strip for amperometry. 1. Fasting sample (1st sample) + urine
• The sensor uses the platinum or silver electrode to 2. 75 grams glucose load is orally taken within
form part of the electric circuit where hydrogen 15 minutes.
peroxide is electrolyzed. Hydrogen peroxide is ▪ The patient also needs to vomit it out or else
produced as a result of the oxidation of glucose on the the procedure needs to be repeated.
glucose oxide membrane and the current flowing 3. 1-hr sample (2nd sample) + urine
through the circuit provides the measurement of the 4. 2-hr sample (3rd sample) + urine
concentration of hydrogen peroxide, giving now the ● NOTE: There will be three samples extracted from the
concentration of glucose. patient.
● The OGTT is used to diagnose diabetes mellitus.
● Also used to Diagnosis of GDM (Gestational diabetes
Mellitus) for pregnant women
o Fasting >5.1 mmol/L (>92 mg/dL)
o 1h: >10.0 mmol/L (>180 mg/dL)
o 2h: >153 mg/dL (>8.5 mmol/L)
GLYCATED/GLYCOSYLATED HEMOGLOBIN
● HbA1c (Aka glycated Hemoglobin)
o “Hemoglobin A that is irreversibly glycosylated at
one or both N-terminal valines of the B-chains of
the tetrameric hemoglobin molecule”

SECOND SEMESTER 5
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

(International Federation of Chemistry Working
Group on HBA1c)
● HbA1c is the largest subfraction of normal HBA
involved in diabetic and non-diabetic individuals
● It represents the weighted average of glucose levels
with the youngest erythrocytes contributing to a
greater extent than the older ones.
● Principle
o A red blood cell lives for approximately 120 days
o Hemoglobin is contained in the red blood cell
o Glucose (sugar) enters the bloodstream and the
red blood cell
o Glucose naturally binds to hemoglobin
o This binding creates glycated hemoglobin
(HbA1c)

• A reliable test in monitoring the long term glucose

concentration. it should be done or performed in 3-6
months in individuals with diabetes mellitus in order
CRITERIA FOR THE DIAGNOSIS OF DIABETES AND
to properly monitor the glycemic control of the
PREDIABETES INDIVIDUALS
affected individual.
● Now the preferred test to assess glycemic control
● Widely used marker of chronic hyperglycemia
(reflecting average blood glucose levels over a 2- to
3- month period of time)
METHOD
● Affinity Chromatography
● HPLC (High-performance liquid chromatography)
● Electrophoresis
● Spectrophotometry
● 2-step Non-enzymatic Method
● Among the methods that have been mentioned,
Affinity chromatography and spectrophotometry are Definition: mg = milligram, dL = deciliter
the commonly used method. ● For all three tests, within the prediabetes range, the
● Specimen requirement for HBA1c is? higher the test result, the greater the risk of diabetes.
o Whole Blood ● The prediabetic values are impaired glucose results
● Anticoagulant of Choice: or values which are characterized by glucose
o EDTA concentrations between the normal and diabetic
values.
● Patients who are diagnosed of diabetes mellitus are
consistent with increased glucose results in HbA1c.

SECOND SEMESTER 6
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

CORRELATION BETWEEN HEMOGLOBIN A1C AND URINE MICROALBUMIN
PLASMA GLUCOSE LEVELS • Test to detect very small levels of
protein (albumin) in urine
o To detect early diabetic
nephropathy (kidney damage)

KETONE TESTING
• Beta-HBA, acetoacetic acid,
and acetone
o The ratio of beta
hydroxybutyric acid or
hydroxybutyrate to the
acetoacetate or
Take note that there are still special considerations in the
acetoacetic acid is greatly
HbA1c test.
increased in diabetic
● HbA1c is considered as the weighted average of the ketoacidosis due to
glucose concentration or the glucose level in altered rate of state and elevated levels of NADH
erythrocytes. So, any defect or abnormality in the
in hepatic mitochondria
RBC may affect the HbA1c.
o In serum acetone, if the serum acetone is
● What if the patient has hemoglobinopathy?
increased it is indicative of defect in the
o It will cause a decreased value for HbA1c.
metabolism of carbohydrates.
o Hemoglobinopathy
• To detect ketosis in DM type
▪ a genetic defect that results in abnormal
• Take note: the ketone test is recommended when
structure of one of the globin genes of the
plasma glucose values reach the 300 mg/dL
hemoglobin molecule.
• METHODS:
o Conditions associated with shortened red blood
o Electrochemistry
cell survival or lower mean red blood cell age
o Chromatography
such as hemolysis, recovery from acute blood
o Electrophoresis
loss, transfusion, or splenectomy will lower the
o Colorimetric methods
HbA1c test or HbA1c value/level as a result of the
o Gerhardt’s
reduced exposure to plasma glucose.
▪ Used of ferric chloride reacted with
FRUCTOSAMINE
acetoacetic acid to produce a red color
• Fructosamine is also called as the glycosylated or the
o Sodium Nitroprusside
glycated albumin. It is a reflection of the short-term
▪ Reacts with acetoacetic acid in alkaline pH to
glucose control.
form a purple color
• Most widely used to
▪ Urine reagent strip test and Acetest tablets
assess short-term (3-6
o 3-hydroxybutyrate dehydrogenase
week) glycemic control
▪ To detect either 3-b-hydroxybutyric acid or
• Most useful if the acetoacetic acid
HBA1c is unreliable 2-Hour Postprandial Glucose
due to
• 2 hours following regular meal
hemoglobinopathy or
o For example: after taking or ended lunch at
hemolysis
exactly 12 o’clock, the 2-hour postprandial
• Not ideal: serum glucose test will be performed after two hours
albumin level is <3 g/dL or when serum albumin 2-Hour Postprandial Glucose (more standardized)
turnover is accelerated (cirrhosis)
• 75 grams glucose load
• Blood collection after 2 hours

SECOND SEMESTER 7
CLINICAL CHEMISTRY I
MLS 414 | LABORATORY | MIDTERM

CARBOHYDRATES: LABORATORY APPLICATION

o Has almost the same procedure with the oral level. Excessive glucose is stored as an inactive
glucose tolerance test but in this case the blood glycogen mainly in the liver and little in the muscle.
collection is done after 2 hours. • Basically, this product insert shows or follows the
o 200 mg/dL – DM principle of enzymatic colorimetric determination
▪ If the result of the sample after 2 hours glucose according to the following reactions as being
postprandial glucose reaches the 200 mg/dL, showed in the picture.
more likely the patient is a candidate of the • Take note: in every reagent make sure that you have
diabetes mellitus read the reagent storage and stability.
DIAGNOTIC CRITERIA FOR DIABETES MELLITUS • The sealed reagents are stable up to their expiry date
• Random Plasma Glucose stated on the label, when stored at 2-8°C and
o >200 mg/dL (11.1 mmol/L) protected from light in order to prevent deterioration
• Fasting Plasma Glucose or early deterioration of the sample/reagent.
o >126 mg/dL (7.0 mmol/L) • Once opened, the reagent is stable up to 4 weeks
• 2-hour Plasma Glucose OGTT only at 2-8°C if contamination is to be avoided.
o >200 mg/dL (11.1 mmol/L) • Take note of the procedure notes, the laboratory
• HbA1c procedure for the semi-automated analyzer, it should
o >6.5% have a blank when performing spectrophotometry
method, the calibrator, as well as the sample or
control.
• Take note: you cannot run the sample not unless you
have run already the control. That is to follow the
quality assurance or the quality control in the lab.

• In this type of manufacturer, it states that glucose is

the major carbohydrate present in the blood and
serves as a primary source of energy. It is usually
obtained from ingested starch and sugar. The glucose
concentration is normally maintained at a constant

SECOND SEMESTER 8