Introduction
- Glucose is the main carbohydrate present in blood.
- It is the major source of energy for body tissues.
- Brain cells depend mainly on glucose for continuous energy supply.
- Blood glucose estimation is one of the most important routine biochemical laboratory tests.
- It is used for diagnosis and monitoring of diabetes mellitus.
- It also helps in evaluation of hypoglycemia and endocrine disorders.
- Accurate estimation is necessary because glucose level changes quickly after sample collection.
- Delay in sample processing can lower glucose value due to glycolysis by blood cells.
- In routine laboratories, glucose estimation is commonly done by the Glucose Oxidase–Peroxidase method (GOD-POD method).
Principle
The estimation of glucose is based on the Glucose Oxidase–Peroxidase enzymatic principle (Trinder’s method).
Step 1: Oxidation of Glucose
Glucose present in the sample is oxidized by the enzyme glucose oxidase in the presence of oxygen and water.
Glucose+O2+H2O→Gluconic acid+H2O2
Step 2: Color Formation
The hydrogen peroxide produced reacts with phenol and 4-aminoantipyrine in the presence of peroxidase enzyme to form a red colored quinoneimine compound.
H2O2+Phenol+4-AAP→Red quinoneimine dye+H2O
Important Concept
- The intensity of red color formed is directly proportional to glucose concentration.
- Absorbance is measured at 500 nm.
- Higher absorbance indicates higher glucose concentration.
Specimen
The following specimens can be used for glucose estimation:
Serum
- Fresh non-hemolyzed serum is preferred.
- Serum should be separated quickly from clot to avoid glycolysis.
Plasma
- Fluoride plasma is ideal because fluoride inhibits glycolysis.
- Heparinized or EDTA plasma may also be used.
Urine
- Urine can also be used after dilution when required.
Important Precautions
- Avoid hemolysis.
- Analyze sample immediately or refrigerate.
- Delay in testing lowers glucose value because cells consume glucose.
Stability
- Fluoride sample stable for 2 days at room temperature.
- Stable for 7 days at 4–8°C.
- Serum without preservative stable for 8 hours at room temperature.
Reagents
The glucose reagent contains:
Reagent Composition
- Phosphate buffer
- Glucose oxidase
- Peroxidase
- Phenol
- 4-aminoantipyrine
Standard
- Glucose standard of known concentration provided in kit
Reagent Features
- Ready to use liquid reagent
- Stable at 2–8°C
Materials Required
The following materials are required for glucose estimation:
- Test tubes
- Micropipette
- Tips
- Photometer / Colorimeter / Semi-auto analyzer
- Cuvette (1 cm path length)
- Distilled water
- Timer
- Centrifuge
- Glucose reagent kit
- Standard glucose solution
Procedure
| Components | Blank | Standard | Test |
|---|---|---|---|
| Glucose reagent | 1000 µL | 1000 µL | 1000 µL |
| Distilled water | 10 µL | — | — |
| Glucose standard | — | 10 µL | — |
| Sample | — | — | 10 µL |
Incubation
- Mix all three tubes properly.
- Incubate at 37°C for 5–10 minutes.
Reading
- Measure absorbance of standard and test against blank.
- Read absorbance at 500 nm wavelength.
Calculation
The glucose concentration is calculated by:
Glucose (mg/dL) = Absorbance of Test / Absorbance of Standard × Concentration of Standard
Example
If:
- Test absorbance = 0.45
- Standard absorbance = 0.50
- Standard concentration = 100 mg/dL
Normal Reference Values
Fasting Blood Glucose
- Adults: 74–100 mg/dL
Postprandial Blood Glucose (2 hours after meal)
- Less than 120 mg/dL
Urine Glucose
- 1–15 mg/dL
Age Variation
- Newborn values are lower
- Elderly values may be slightly higher
Clinical Significance
Increased Blood Glucose (Hyperglycemia)
Occurs in:
- Diabetes mellitus
- Stress conditions
- Cushing syndrome
- Hyperthyroidism
- Acute myocardial infarction
- Pancreatitis
- Intravenous glucose infusion
Decreased Blood Glucose (Hypoglycemia)
Occurs in:
- Excess insulin administration
- Insulinoma
- Liver disease
- Starvation
- Adrenal insufficiency
- Inborn errors of carbohydrate metabolism
Importance in Clinical Practice
- Screening of diabetes
- Monitoring diabetic treatment
- Detecting hypoglycemic episodes
- ICU patient monitoring
- Endocrine evaluation
