GOD-POD Method For The Estimation of Blood Glucose - Principle, Procedure

What is GOD-POD Method for Glucose Estimation?

GOD-POD method is referred to as Glucose oxidase-peroxidase method (Trinder method). It is a enzymatic method which is used for estimation of blood glucose. It is highly specific and it gives true glucose value. This method is based on two step enzymatic reaction and colour development.

In first step glucose oxidase (GOD) catalyzes oxidation of glucose present in sample. In presence of oxygen and water glucose is converted to gluconic acid and hydrogen peroxide (H2O2) is formed. This reaction is specific for glucose so interference is less.

In second step peroxidase (POD) acts on hydrogen peroxide. It breaks down H2O2 and oxidative coupling reaction is carried out with chromogenic mixture. The chromogen is usually phenol and 4-aminoantipyrine and red coloured quinoneimine dye is formed. The intensity of red colour is directly proportional to glucose concentration in sample.

The absorbance of red colour is measured by colorimeter or spectrophotometer. The wavelength is usually taken between 505 nm to 530 nm. By this reading the glucose concentration is calculated and non sugar reducing substances does not interfere like older chemical methods.

Principle of GOD-POD Method

Principle of GOD-POD method is based on two step enzymatic reaction and colour formation. It is used for estimation of blood glucose and it is specific for glucose. In this method glucose is oxidized first and hydrogen peroxide is formed then colour is developed by peroxidase reaction.

In first step glucose oxidase (GOD) catalyzes oxidation of glucose in presence of oxygen and water. The reaction is as follows-
Glucose + O2 + H2O → Gluconic acid + H2O2
Hydrogen peroxide is produced and it is used for next reaction.

In second step peroxidase (POD) acts on hydrogen peroxide. The H2O2 reacts with chromogen mixture (phenol and 4-aminoantipyrine) and red or pink coloured dye is formed. This dye is referred to as quinoneimine. The intensity of colour formed is directly proportional to amount of glucose present in blood sample.

The absorbance of colour is measured by colorimeter or spectrophotometer. The wavelength is usually around 505 nm (it can be measured upto 530 nm). From absorbance the glucose concentration is calculated.

Requirements

Requirements for GOD-POD method are as follows-

Reagents and solutions are-

Buffer– Phosphate buffer is required to maintain pH (7.0 to 7.5) for enzyme activity.
Enzymes– Glucose oxidase (GOD) and Peroxidase (POD) is used.
Chromogenic couplers– 4-aminoantipyrine (4-AAP) and phenol (or chloro-4-phenol) is used for colour development.

Glucose standard– Standard glucose solution of known concentration is used (usually 100 mg/dL or 5.55 mmol/L).
Distilled water– It is used for blank.

Laboratory instruments and materials are-

Spectrophotometer or colorimeter– It is used to measure O.D. of red colour (wavelength 505–530 nm).
Cuvettes or test tubes– Clean dry test tubes or cuvettes (1 cm light path) is used for reading.

Micropipettes and tips– Accurate pipettes are required for adding small volume (usually 10 µL) of sample and standard.
Incubator or water bath– It is used to keep reaction at 37°C (or room temperature as per protocol) for colour development.
Centrifuge– It is required to separate serum or plasma from whole blood.

Specimen requirement are-

Sample type– Clear unhemolyzed serum or plasma is used (CSF and urine can also be tested).
Collection method– Blood for plasma is collected in sodium fluoride and potassium oxalate tube (it prevents glycolysis of glucose before test).

Procedure of GOD-POD method

Procedure of GOD-POD method is as follows-

Three clean dry test tubes are taken and it is labelled as Blank Standard and Test.

In Blank tube 10 µL distilled water is added.
In Standard tube 10 µL glucose standard solution is added (usually 100 mg/dL).
In Test tube 10 µL patient serum or plasma sample is added.
GOD-POD working enzyme reagent is added in all tubes (1.0 mL or 1000 µL).

The contents are mixed properly so reaction is carried out evenly.
The tubes are incubated for colour development. It is done at 37°C for 5–15 minutes (or at room temperature 15–25°C for 15–30 minutes).
Colorimeter or spectrophotometer is set at 505 nm (range 500–530 nm can be used).

Blank is used to set zero absorbance then O.D. of Standard and Test is read and recorded.
Blood glucose is calculated by formula– (Absorbance of Test / Absorbance of Standard) × Concentration of Standard.

Calculation for GOD-POD Method

Calculation for GOD-POD method are as follows-

Measurements are-

O.D. (Absorbance) of Blank is taken.
O.D. of Standard is taken.
O.D. of Test is taken.
(Colorimeter or spectrophotometer is used.)

Primary formula is-

Glucose (mg/dL) = (O.D. of Test / O.D. of Standard) × Concentration of Standard

If Blank reading is not zero then correction is done-

Glucose (mg/dL) = [(O.D. of Test – O.D. of Blank) / (O.D. of Standard – O.D. of Blank)] × Concentration of Standard

Simplified formula (when standard is 100 mg/dL)-

Glucose (mg/dL) = (O.D. of Test / O.D. of Standard) × 100

Unit conversion (optional)-

Glucose (mmol/L) = Glucose (mg/dL) × 0.0555

Uses of GOD-POD method

Uses of GOD-POD method are as follows-

Quantitative estimation of glucose in body fluids– It is used for in vitro estimation of glucose in serum plasma CSF and urine.
Diagnosis and monitoring of diabetes mellitus– It is used for screening and diagnosis of diabetes by detecting hyperglycemia and for monitoring blood glucose.
Glucose tolerance testing– It is used in Oral glucose tolerance test (OGTT) and 2 hour post prandial glucose estimation.

Monitoring during insulin therapy– It is used to check blood glucose in diabetic patient who are on insulin treatment.
Diagnosis of hypoglycemia– It is used to detect low blood glucose (hypoglycemia) in condition like insulinoma adrenal insufficiency or decreased glucose synthesis.
Detection of other endocrine and metabolic disorders– It helps in diagnosis of disorder showing abnormal glucose level like hyperthyroidism hyperpituitarism Cushing syndrome and acromegaly.

Limitations of GOD-POD method

Limitations of GOD-POD method are as follows-

Interference by reducing substances– The first step is specific for glucose but second step (peroxidase reaction) is not fully specific. Reducing agent like ascorbic acid (vitamin C) uric acid and bilirubin can interfere. It competes for hydrogen peroxide or it inhibits colour reaction so glucose value may be shown false low.
Drug and chemical interference– Some drugs and chemicals like L-Dopa L-Cysteine and citric acid can affect the reaction and accuracy is reduced.
Lipemia (turbidity) problem– Lipemic sample contains high fat and it causes light scattering during reading. Due to this glucose value may be shown false high.

Catalase interference– Catalase enzyme can be present due to bacterial contamination or some blood disorder. It breaks hydrogen peroxide before dye reaction and glucose value is shown false low.
Linearity limit– It is linear only upto certain glucose concentration (around 300 mg/dL to 600 mg/dL depends on kit). If glucose is very high the sample is diluted and test is repeated.
Anomeric specificity– Glucose oxidase reacts mainly with β-D-glucose. Mutarotase is added in modern reagent to convert α-glucose to β-glucose but if conversion is affected then reaction rate is altered and reading may change.

In uremic patient error– Very high uric acid and creatinine can affect the test so it may not be fully accurate in uremic patients. In such case Hexokinase method is preferred.

References

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GOD-POD Method For The Estimation of Blood Glucose – Principle, Procedure