Estimation of blood glucose by Folin-Wu Method (Reduction of Cupric to Cuprous Salt )

Reduction of Cupric to Cuprous Salt (Folin-Wu Method)

This is one of the oldest methods for the estimation of blood sugar. However, it is still very much in use in countries where enzyme preparations are not easy to obtain. 

Reduction of Cupric to Cuprous Salt (Folin-Wu Method)

Principle

 The method is based on three stages:

(i) Precipitation of blood proteins with copper tungstate.

(ii) Reduction of cupric sulphate to cuprous oxide.

(iii) Colorimetric measurement of the subsequent blue-green colour produced on the addition of molybdate reagent to the cuprous oxide.

Reagents

Sodium tungstate (10 %) Dissolve 10 g per 100 ml distilled water. 

Sulphuric acid, 2/3 N Add 2 ml of conc.sulphuric acid to about 50 ml of distilled water and dilute up to 100 ml with distilled water. 

Alkaline copper tartrate reagent Dissolve 40 g sodium carbonate and 7.5 g tartaric acid in about 400 ml of distilled water. Separately dissolve 4.5 g copper sulphate in about 100 ml of distilled water. Mix both the solutions together and make the volume up to1 litre with distilled water. Filter before use.

Phosphomolybdic acid reagent Dissolve35 g molybdic acid and 5 g sodium tungstate in 200 ml of 10 % sodium hydroxide. Add it to 200 ml of distilled water in one litre beaker. Boil for 45 minutes to remove ammonia, cool and add slowly 125 ml of 89 % phosphoric acid (specific gravity 1.75). Make up the volume to 500 ml with distilled water.

Stock glucose standard solution (1 g/dl) Dissolve 1 g of glucose in 100 ml of saturated benzoic acid (0.3%).

Working glucose standard solution (10mg/dl) Dilute the stock standard 1:100 with saturated benzoic acid to give 10 mg/ dl glucose solution (Iml glucose stock Standard + 99 ml benzoic acid).

Technique

• Place 1.0 ml of blood in 7.0 ml of distilled water and mix.

• Add 1.0 ml of 10 % sodium tungstate. Mix.

• Add 1.0 ml of 2/3N H2SO4. Mix byinverting several times. Allow it to stand for5 minutes at room temperature.

• Centrifuge or filter using Whatman No. 1 filter paper.

• Set up 3 Folin-Wu tubes as follows:

	Blank	Standerd	Test
Distilled water	1.0ml
Working standard		1.0ml
Filtrate /supernatant			1.0ml
Alkaline Copper tartrate	1.0ml	1.0ml	1.0ml

• Place the tubes in boiling water bath for 10minutes.

• Cool and add 1.0ml phosphomolybdic acid reagent to each tube.

• Shake the tubes to get rid of the air bubbles; and add distilled water up to 12.5 ml mark.

• Mix and read the absorbance (Ab) immediately at 680 nm or using a red filter.Set the zero using the blank.

Calculations

Note

1.0 ml of blood was diluted 1:10 for protein precipitation. 1.0 ml of the diluted blood was then used for the test. Therefore, the actual volume of blood used for the test is 0.1 ml. Also, 1.0 ml of the working standard (10 mg/dl) contains 0.1 mg of glucose.

Alternatively

the concentration of glucose in the test sample can be obtained by the use of a calibration curve as in the oxidase-peroxidase method,

Causes of Abnormal Glucose Levels
Severe hyperglycaemia	Mild/transient hyperglycaemia	Severe hypoglycaemia
1. Diabetes mellitus	1. Severe liver disease	1. Insulinoma
2. Adrenal cortical hyperactivity (Cushing's syndrome)	2. Pancreas disorder	2. Hypopituitarism
3. Acromegaly	3. Pituitary disorder	3. Ectopic insulin production from tumours
4. Obesity	4. Steroid therapy	4. Adrenal cortical insufficiency
	5. Shock 6. Convulsions
	7. Acute stress reaction (Physical or emotional)