Determination of plasma glucose

Choice of blood specimen:

Because of the large number of determinations on individual diabetic patients, capillary blood has often been used.  Plasma glucose is higher than the concentration in whole blood. This may be corrected  by multiplying the whole blood glucose concentrations by 1.15 and adding 0.33 mmol/L to derive the plasma or serum glucose. 

    Plasma is preferred to serum as the blood can be added directly to the mixture of anticoagulant and preservative and then separated immediately. This gives a larger sample for analysis than serum from the same volume of blood and there is less glycolysis. If serum is used it must be separated as soon as possible, but not later than 30 to 40 min after blood collection.

Glucose Oxidase Methods

In these methods, the aldehyde group of β–D-glucose is oxidized by glucose oxidase to give gluconic acid and hydrogen peroxide.

Reagents:

1. Preparation of protein precipitant:

                   Dissolve 10 g sodium tungstate (Na₂WO₄,2H₂O)

               + 9 g sodium chloride

                       in about 800 ml water. 

Add about 125 ml 1.0 M hydrochloric acid to bring the pH to 3 using narrow range indicator paper. 

Add 1g phenol and make to 1 liter with water.

2. Preparation of color reagent:

    Mix 75 ml disodium hydrogen phosphate solution (40g Na₂HPO₄ /L)

    + 215 ml water

    + 5ml  peroxidase

    + 300 mg sodium azide

    + 100 mg 4-aminophenazone. 

3. Preparation of standard glucose solution:

    10 mM (1.8 g/l) in benzoic acid (10 g/L).

Technique:

• Add 100 µl plasma to 2.9 ml protein precipitant.
• Mix well, centrifuge and pipette 1.0 ml clear supernatant into a 1.6 cm test tube. 
• For the blank,  take 1.0 ml protein precipitant.
• For the standard, 1 ml of a mixture containing 100 µl glucose standard and 2.9 ml protein precipitant. 
• To each of sample, blank and standard add 3 ml color reagent.
• Incubate at 37 °C for 10 min, shaking briefly two or three times. 
• Read sample and standard against the blank at 515 nm.

Calculation: