Determining Blood Sugar by Nelson and Somogyi’s Method

Principle
Blood proteins are precipitated by zinc hydroxide. The filtrate is heated with alkalinecopper reagent and the reduced Cu formed is treated with arsenomolybdate reagent, resulting in the formation of violet, which is read in the photometer.

Reagents

1. 5% ZnSO₄ solution
2. 0.34-N barium hydroxide These 2 solutions should be adjusted so that 5 mL ZnSO₄ require 4.7–4.8 mL Ba(OH)₂ for complete neutralization.
3. Alkaline copper reagent: Solution A: 25-gm anhydrous Na₂S₂O₃, 25-gm Rochelle salt, 20-gm NaHCO₃, and 200-gm anhydrous Na₂SO₄ were dissolved in about 800 mL of water and diluted to 1 liter. The solution was stored at room temperature and never dipped below 20°C. It was filtered before use if any sediment was formed. Solution B: 15% CuSO₄, 5H₂O containing 1 or 2 drops of concentrated H₂SO₄. On the day of use, 25 parts of solution A and part of solution B were mixed. That was the alkaline copper reagent.
4. Arsenomolybdate color reagent: Ammonium molybdate 25 gms was dissolved in 450 mL H₂O. 21 mL concentrated H₂SO₄ was added and mixed. Disodium orthoarsenate (Na₂H, ASO₄, 7H₂O) 3 gms was dissolved in 25 mL H₂O and added with stirring to the acidified molybdate solution. It was then placed in an incubator at 37°C for 24–48 hours and stored in a glass-stoppered brown bottle.
5. Standard glucose solution: (stock glucose solution) Exactly 0.1 gm of anhydrous pure glucose was dissolved in 10–15 mL 0.2% benzoic acid and diluted to 100 mL with benzoid acid solution.

Three working standards were prepared by diluting 0.5, 1.0, and 2.00 mL of the stock solution to 100 mL with benzoic acid. These solutions in benzoic acid kept indefinitely at room temperature.

Working standard: 5 mL made up to 100 mL yields 50 mg/M.

Procedure
Into a test tube containing 3.5 mL of water, 0.1 mL of blood was introduced through a clean dry micropipette and mixed well. To this tube, 0.2 mL of 0.3 N Ba(OH)₂ was added after the mixture turned brown. 0.2 mL ZnSO₄ was added and mixed. After 10–15 minutes, the mixture was filtered through a Whatman No. 1 filter paper.

Into 2 separate test tubes, 1 mL aliquot of the filtrate was transferred and 1 mL alkaline Cu reagent was then added. These tubes were covered with glass and placed in a boiling water bath for 20 minutes. The tubes were then cooled under running water, 1 mL arsenomolybdate reagent was added and the solution dilute to 25 mL with H₂O simultaneously standards in 0.2 to 1.0 mL range were prepared and a reagent blank were similarly prepared.

The intensity of color produced was red at 680 mm. The color was stable and reading may be taken at convenience.

Report:
The concentration of blood glucose in given sample is .............. mg/mL