QUANTITATIVE ESTIMATION

OF GLUCOSE
BY
GLUCOSE OXIDASE METHOD
Structure of Blood Glucose
 Synergistic Effect of
Insulin and Glucagon
to Regulate Blood Glucose Level
 Methods to Determine
Blood Glucose

• Colourimetric chemical methods.

• Colourimetric biochemical/enzymatic methods.
 CHEMICAL METHODS

• OXIDATION-REDUCTION — oxidation
reduction of copper or iron ions.

• CONDENSATION— with o-toludine or anthrone

 Disadvantages of
Chemical Methods

Non specifically glucose is oxidized –

so any other reducing bio-molecules
present in the blood an interfere.
 Biochemical/ Enzymatic Methods

• Hexokinase method
• Glucose Dehydrogenase method
• Glucose Oxidase/Peroxidase method
 Reaction Schemes of
Biochemical/Enzymatic Methods
BY HK —
• Glucose +ATP +HK  ADP + G6P
• G6P+NAD + G6PDH 6 P-gluconolactone +NADH+H
BY DH —
• Glucose +NAD+ GDH  Gluconolactone +NADH+H
BY GOD/POD —
• Glucose + O2 + H2O+ GOD  Gluconic acid +H2O2
• H2O2+ POD  H2O + oxidised chromogen
 Advantages of
Glucose Oxidase Method
• Estimation from any complex mixture.

• Most specific for β-D glucose.

• Most concise polarographic estimation —

dis -appearence of oxygen by coupling with
pre-chromogenic substance.
 Glucose Oxidase/Peroxidase Method
• Saifer–Gerstenfeld method —o-dianisidine is
used as oxygen acceptor to transform to red
chromogen (λmax 520nm)

• Trinder’s method (GOD/POD) method —

4 amino antipyrin/4- amino phenazone is used
as accept oxygen to form red colour quinione
imine dye (λmax 505nm)
 Importance of Trinder’s Method

The chromogen used here 4 AAP and

phenol is more specific to couple with
molecular oxygen and to produce red
coloured Quinoneimine dye measured
spectrophotometrically at 505nm
wavelength.
Trinder’s Method
(GOD/POD) method
 Introduction
• The enzymatic clinical as well as diagnostic
method to determine blood glucose which is
first oxidized by the enzyme GOD to
Gluconic acid and H2O2.
• Then this reaction is coupled with another
enzymatic reaction by Peroxidase where
H2O2 is oxidatively coupled to give
chromogenic product—can be quantitatively
determined.
• The final product formation is proportional to
the amount of initial blood glucose.
 Trinder’s Method
(GOD/POD method) Reaction

• Glucose + O2 + H2O -- GOD  Gluconic

acid +H2O2
• H2O2 +Phenol + 4-AAP -- POD 
Quinoneimine dye (measured at 505nm)
 Principle of the Method
• By Glucose Oxidase (GOD) first
glucose is converted to Gluconic acid
and H2O2

• Then this H2O2 is converted to H2Oand

O2 which is in conjunction with 4 AAP
and Phenol gives red colouration of
quinoneimine dye—
spectrophotometrically measured.
Chemical Structural Reaction Scheme
for GOD/POD Method
Measurement Device
Spectrophotometer
 Principle of Spectrophotometer
• Obey Lambert-Beer’ s law through
polaroscopic device
O.D or absorbance A = € X C X l
• Absorbance or Optical Density is measured
at particular wavelength of light where there is
absorption maxima λmax.
• A= -log I/I0where I0 is the intensity measured
with no sample and I is the intensity measured
with the sample.
• Light is passed through the sample kept in the
cuvette —absorbance is measured.
 Commercially Available Kit
for Blood Glucose Estimation (GOD/POD) Method
 Components of the Kit
Reagents—
• Reagent I (10 vials)
• Reagent II (1 big amber bottle)
• Reagent III (Standard– 1 bottle of low
standard—100 mg/dl and 1 bottle of high
standard—500 mg/dl)
Reconstitution bottle—(1 amber bottle to mix
reagent I and reagent II)
Instruction manual.
 Kit storage and viability

• The kit must be stored at 2-8°C till expiry

date mentioned in the kit.
• If opened Reagent I is mixed with reagent
II—at 2-8°C—stored for ¾ weeks.
• If the reagents become unclear—should not
be used.
Sample (Blood) Collection
 Storage of Blood Sample

• 2 ml of collected blood sample can be

stored at 2-8°C .
• It must be mixed with anticoagulants—
EDTA and sodium fluoride mixture.
 Reagents Required
• REAGENT I—Phosphate buffer, GOD, POD,
4 AAP, stabilizer.

• REAGENT II—Glucose diluents.

• REAGENT III—Glucose standard.

 Reagent I
• Phosphate buffer—pH 7.0; 100mM.
• GOD—150,000 U/L
• POD—16,00 U/L
• 4-AAP—0.28mM
• Stabilizer
 Reagent II
• Glucose diluents –Phenol (10mM)
supplied in amber bottle as it is light
sensitive.
 Reagent III
• Glucose standards —100 mg/dl, 300
mg/dl and 500 mg/dl.
 Methods Used
• Preparation of Glucose standard
(reagent III)—100mg/dl, 300 mg/dl, 500
mg/dl.

• Preparation of working reagents—

mixing of reagent I and II
 Preparation of Glucose
Standards
• Either by diluting supplied standard glucose
reagent (Reagent III) with distilled water the
series of standards can be prepared
following the formula:
V1 X S1 = V2 X S2
• Or supplied glucose standards are used as
usual.
 Table to Prepare
Standard Glucose Solutions
Standard Stock Distilled Total
conc. glucose water volume
solution
added
(500mg/dl)
100 mg/dl 2 ml 8 ml 10 ml
200 mg/dl 4 ml 6 ml 10 ml
300 mg/dl 6 ml 4 ml 10 ml
400 mg/dl 8 ml 2 ml 10 ml
500 mg/dl 10 ml 0 ml 10 ml
 Preparation of
Working Reagents

• Within 50ml of Reagent II , 1 vial of Reagent I

is mixed within the supplied Reconstitution
Bottle.
 Parameters Involved

• Method—end point detection

• Wavelength range—500-550 nm

• Absorption maxima—505nm

• Time taken—7-10 min

• Temperature—RT/37°C

• Blank—with water.
Procedure
Observation
 Results 1.
Standard Curve for Glucose –
O.D vs. series of glucose concentration
Example O.D Data
for Standard Curve
Example—Standard Curve
 Results 2.
Calculation Formula with One
Standard Glucose Concentration
 Example - from
Calculation with Formula
 Comments

• Interpretation of the method used

• Interpretation of the result/data-clinical

significance and investigations
Interpretation of the Method Used
• In case of glucose standard curve —it has to
be followed that the linearity must be
maintained within the standard curve.
• O.D value must not exceeds than 2 because
2= -log I/I0= 100= total absorbance by the
sample that means no transmittance—gives
faulty and saturated results concomitantly.
• Otherwise sample must be diluted and the
result must be multiplied by dilution factor.
 Interpretation of the Result/Data

• The sample concentration should not exceeds

either the linearity range or any particular
standard concentration take or from the
standard curve.
• Then sample should be diluted and the result
should be multiplied by the dilution factor.
 Clinical significance
• Plasma glucose: Fasting: 70 –110 mg/dl
• Post-prandial: 80 – 140 mg/dl
• Random: 60 – 140 mg/dl.
Exceeding the normal ranges are called
hyperglycemic condition.
Lowering to the normal ranges are called
hypoglycemia.
 Performance

• Linearity Limit: 500mg/dl.

• If linearity by the standards are not

maintained then the system will be
checked once again according to the
precession.
 Precision
• Quality Control: The patient results
obtained for each batch can be validated by
using normal and abnormal control sera with
assayed values for glucose.
• Biostatistical analysis: It is recommended
to perform the test with each of the sample
thrice. To calculate standard deviations of
the data to have corrected level (glucose
here).
 Quality Control
• With the commercially available sera the
machine should be standardized within-
the-day and between-the day.
• The standard reference value may be
different in different laboratory—but the
condition maintained during the
experiments should be kept constant.
 Bio-Statistical Analysis

The data must be given with standard

deviation and percent of coefficient of
variation.
Errors May Involved in the Method
Causes DESCRIPTION
Human error Pipetting error, mislabeling,
wrong sample reading,
Unclean cuvette.
Material Expired kit reagents and
improper reagent
preparation
Substances/chemical Increased uric acid/bilirubin
can be reacted by
Peroxidase, Other
aldohexose sometime may
react
 Limitations
• Glucose value > 500 mg/dl should be diluted .
• At high plasma levels uric acid, glutathione
and bilirubin may interfere with the assay
• Reagents are sensitive to light and
temperature
• Ascorbic acid will decrease glucose values
• Haemolytic and lipemic samples may result in
false elevated results.
• Catalase enzyme may inhibit other enzymes.
 Advantages
•This is rapid

•Simple enzymatic Colourimetric method.

•The stability of the glucose oxidase reagent

is excellent.

•Low cost per test.

•Specific and sensitive.

 Precaution
• Dry and clean glassware should be used.
• Gloves should be used.
• The working reagent should be prepared
fresh.
• Incubation period should be kept constant
for all the samples.
 Specificity and Sensitivity
• According to Sonowane et al., 1976 High
degree of specificity than any other
method.

• The test is sensitive from 100-500 mg/dl

range
 Therapies
According to the data obtained it is to be
determined whether the patient is
HYPOGLYCEMIC and
HYPERGLYCEMIC.

Next accordingly therapies are done.

Symptoms for Hyperglycemia
Symptoms for Hypoglycemia
Proper Diet Against Hypoglycemia
 Application

• Quantitative determination of D-glucose in

foodstuffs such as baking agents, diet
beer and dietetic foods, as well as in
pharmaceuticals, cosmetics and other
biological samples.

• Used in blood glucose quantification

Glucometer
 Conclusion

• A method that is used regularly in all

clinical laboratory.
• Routine method to check up blood
glucose.
 Case Study
Interpretation of the Data