ESTIMATION OF GLUCOSE IN BLOOD
FOLIN-WU METHOD:

PRINCIPLE: the protein-free blood filtrate is heated with an alkaline copper solution, using a special tube to prevent reoxidation. The cuprous oxide formed is treated with a phosphomolybdic acid solution, and the blue color is obtained which is compared with that of the standard.
(spectrophotometric method)

REAGENTS REQUIRED:

1. ALKALINE COPPER SOLUTION:

dissolve 40g of anhydrous sodium carbonate in about 400ml of water and transfer to a liter flask. Add 7g of tartaric acid, and when the latter has dissolved add 4.5g of crystallized copper sulfate. Mix and make up to 1 liter with the remaining volume of water.

2. PHOSPHO MOLYBDIC ACID SOLUTION:

to 35g of molybdic acid and 5g of sodium tungstate, add 200 ml of 10% sodium hydroxide solution and 200 ml of water. boil vigorously for 20 min-40 min. cool and dilute to about 350 ml and add 125 ml of phosphoric acid(85%). Dilute 500 ml with the remaining volume of water.

3. STANDARD SUGAR SOLUTIONS:

3 standard sugar solutions should be on hand:
(a). A STOCK SOLUTION:
1% glucose made up in saturated benzoic acid solution
(b). a solution containing 2mg of sugar in 1ml
solutions containing 0.2 and 0.4 mg of sugar in 2 ml.

PROCEDURE:

transfer 2ml of tungstic acid blood filtrate (or 1ml filtrate+1ml water ) to a folin wu sugar tube graduated at 25ml and to other similar tubes add 2ml of standard sugar solutions containing 0.2 and 0.4 mg respectively of glucose. To each tube add 2ml of alkaline copper solution. The surface of the mixture now has reached the constricted part of the tube. Transfer the tubes to a rapidly boiling water bath and heat for 8min. Cool in running water without shaking. To each tube add 2ml of phosphomolybdic acid reagent. After about 1min dilute to the mark (25ml) with water and mix. Transfer the solutions to suitable containers and determine the absorbances at 680nm using a visible spectrophotometer or photoelectric colorimeter with a blank.
        The concentration of glucose in the unknown sample=En/Es x concentration of glucose in standard

   where Eu= absorbance for sample
              Es= absorbance for standard sugar solutions

CLINICAL SIGNIFICANCE:

elevated levels of blood glucose are most frequently found in diabetes 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

NORMAL VALUES:

serum/plasma glucose:100mg/100ml.