Glucose

Specimen

Whole blood, plasma or serum. The container should contain a potassium oxalate-sodium fluoride mixture (3 parts potassium oxalate and one part sodium fluoride-add 3 mg/ml blood). Potassium oxalate prevents clotting by precipitating calcium and sodium fluoride (prevent glycolysis within the cells by inhibiting the enolase enzyme of the glycolytic pathway) for collecting whole blood and plasma. If serum is to be used, it should be separated immediately after clotting to avoid getting falsely low values of glucose concentration

Methods

1. Folin-Wu method
2. Ortho toluidine method
3. Glucose oxidase method

Folin-Wu Method

Principle

A protein-free filtrate is heated with an alkaline cupric tartrate solution. Glucose present in the specimen reduces cupric ions to cuprous ions which precipitate as insoluble cuprous oxide. The amount of cuprous oxide formed is measured by reducing phosphomolybdate to molybdenum blue.

Reagents

1. King’s isotonic diluent: used instead of water to minimize the interference of non-glucose-reducing substances inside the red cells.
2. Sodium tungstate (10 g/dL)
3. Schaffer–Hartman alkaline copper tararate
4. Phosphomolybdic acid 5. Glucose standard solution (100 mg%)

Procedure

Preparation of protein-free filtrate from blood.

• In a test tube, take 7 ml of distilled water,
• 1 ml blood sample, 1 ml 10% sodium tungstate solution and 1 ml
  2/3N H2SO4 solution (dropwise and with shaking).
• Thus, the dilution of the blood sample is 1 in 10.
• Let it stand for 10 minutes, then filter and collect the filtrate in a dry beaker.

	Test	Standard	Blank
Alkaline copper reagent, ml	2.0	2.0	2.0
PFF	2.0	2.0	2.0
Boiling water bath for 8 min. and	immediately cool
Phosphomolybdin acid, ml	2.0	2.0	2.0

Read the OD at 420–490 nm or blue filter.

Calculation

Glucose concentration in mg/dl = absorbance of T/absorbance of S × 100

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Orthotoluidine Method

Principle

Glucose condenses with orthotoluidine in glacial acetic acid at 100°C to form N- N-glucosamine, which is blue-green. Glucose concentration is proportional to the intensity of the colour

Reagents

1. Orthotouidine reagent
2. 10% trichloroacetic acid
3. Glucose standard solution (100 mg%)

Procedure

Preparation of protein-free filtrate (PFF) from blood.

• In a dry test tube, take 3 ml of distilled water,
• 0.5 ml blood and 1.5 ml 10% TCA. ( Dilution of blood ≡ 1 in 10).
• Mix, keep for 10 minutes, and filter in a dry test tube to obtain a clear solution of PFF

	Test	Standard	Blank
PFF, ml	1.0	–	–
Glucose standard, ml	–	1.0	–
3% TCA, ml	–	–	1.0
Orthotolouidine reagent, ml	5.0	5.0	5.0

Mix and keep the tubes in a boiling water bath for 10 min, cool and read the OD using a colourimeter with the wavelength 630–690 nm red filter.

Calculation

Glucose concentration in mg/dl = absorbance of T/absorbance of S × 100

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Glucose Oxidase (GOD/POD) Method

Principle

The enzyme Glucose oxidase converts glucose to gluconic acid and hydrogen peroxide. Hydrogen peroxide then splits to form water and nascent oxygen. The nascent oxygen then combines with a chromogen to form a pink colour. Glucose oxidase enzyme specifically acts on glucose. So, this method gives the true value of glucose levels.

Reagents

1. Phosphate buffer, pH 7.0.
2. Enzyme reagent: Containing GOD, POD, 4-aminoantipyrine and phenol in phosphate buffer.
3. Glucose standard: 100 mg%

Procedure

	Test	Standard	Blank
Serum, ml	0.020	–	–
Glucose standard, ml	–	0.020	–
Enzyme reagent, ml	2.0	2.0	2.0

Mix and incubate at 37°C for 15 minutes. Take OD at 530 nm and calculate the result as above.

Calculation

Plasma glucose (mg/dl) = absorbance of T/absorbance of S × 100

Normal Range

1. RBS – 110 – 140 mg/dl
2. FBS – 70 – 110 mg/dl
3. PPBS – 110 – 140 mg/dl

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Clinical significance

Hyperglycaemia

1. Diabetes mellitus
2. Hyperthyroidism
3. Hyperpituitarism
4. Increased adrenocortical activity

Hypoglycaemia

1. Decrease in Blood glucose above normal. •
2. Overdose of insulin for diabetic treatment.
3. Hypothyroidism,
4. Hypopituitarism
5. Hypoadrenalism
6. Insulinoma