Estimation of Blood Glucose level by Folin-Wu method is one of the clinical biochemistry protocol. You can download these protocols from the given link.
What is Folin-Wu tube?
The modification of the Folin and Wu method for quantitative blood sugar determination used in this laboratory consists merely in:
1. Diluting the unknown after the addition of the phosphomolybdate-phosphotungstate sugar reagent and development of color until it is approximately the same shade as the standard, instead of to a constant amount, before a calorimetric comparison is made; and
2. Introducing this variable factor, the amount of dilution, in the final formula for calculating the amount of dextrose in the unknown.
Estimation of Blood Glucose level by Folin-Wu method
To estimate Glucose level in Blood by Folin-Wu method
When glucose or other reducing agents are treated with an alkaline copper solution they reduce the copper with the result insoluble cuprous oxide is formed. The reaction depends on temperature, duration of heating, a degree of alkalinity. The ratio of glucose to cuprous oxide form may be varied after heating far a period. The cuprous oxide form is allowed to react with phosphomolybdate to form molybdenum blue colored complex which can be read colorimetrically using red filter on at 680nm.
- 1% Sugar solution: 1 gram of sugar is added in 100ml saturated benzoic acid.
- Working standard sugar solution:2 mg per 2 ml of the above solution.
- Alkaline copper solution:
- Solution A: Dissolve 2% Na2CO3 in 0.1N NaOH.
- Solution B:5% CuSO4 in 1% sodium potassium tartrate or Rochelle salt
Mix 50ml of solution A with 1 ml of solution B.
- Phosphomolybdic acid: To 35grms of molybdic acid add 5 grams of sodium tungstate, 200 ml of 10% NaOH and 200ml of water is added. It is boiled vigorously for 20-30 minutes so as to remove whole of ammonium present in molybdic acid. The solution is cooled and diluted to about 350ml and 125 ml of 85% phosphoric acid (ortho) is added and makeup to 500ml with distilled water.
- 10% Sodium tungstate
- 2/3 N H2SO4
Deproteination of Blood:
1 ml of Blood is transferred to a boiling tube containing 7 ml of water then 1 ml of 10% sodium tungstate is added mixed well followed by 1 ml of 2/3 N H2SO4 with shaking. It is allowed to stand for 10 minutes, it is then filtered. This filtrate is called tungstic acid blood filtrate and is taken as a Test sample.
2ml of tungstic acid blood filtrate is transferred to folin-wu tube graduated at 25 ml mark and to other similar folin-wu tubes 2 ml of standard glucose solution and 2ml of water as Blank is added. To each of the three tubes, 2 ml of alkaline CuSO4 is added. Now the surface of the mixture is in line with 4ml mark of the Folin-Wu tube. The folin-wu tubes are placed in a boiling water bath exactly for 8 minutes. It is cooled under running water. To each of the tube, 2 ml of the phosphomolybdic acid solution is added. After 1 minute it is diluted with water up to the mark. The solution is transferred to a suitable tube and OD is read at 680nm.
- The normal blood sugar level ranges from 8 to 120 mg / 100ml of blood
- In mild diabetic conditions value if blood glucose from 140-300 mg / 100ml of blood and in severe diabetic conditions value is up to 1200mg / 100ml of blood have been noted.
- Low blood sugar level values are formed in insulin administration, Addison’s disease, hypoglycemia, and hypopituitarism.
_______ mg of glucose is present in 100 ml of the given blood sample.
Mg of glucose / 100ml of blood = mg of glucose in standard X (OD of test / OD of Standard) X 100/ 0.2
- Elevated levels of blood glucose are most frequently found in diabetis mellitus.
- Hyperglycemia may also occur as a result of hypothyroidism, hyperpituitarism, liver disease Cushing’s syndrome, traumatic injury, and infection.
- Decreased blood glucose levels may result from hyperinsulinism, liver disease and deficiencies of adrenal, thyroid or pituitary hormones.