Clinical Chemistry, Vol 14, 463-476, Copyright © 1968 by the American Association for Clinical Chemistry

The Coulometric Determination of Glucose in Human Serum

¹ Department of Chemistry, University of Maryland, College Park, Md. 20740.

The coulometric titration method is combined with the use of an enzymatic analytic reagent for the determination of glucose in human serum. The glucose in 25 µl. of serum is determined in a protein-free filtrate (PFF) with an accuracy of ± 3% and a coefficient of variation of approximately 2%. The procedure routinely covers a concentration range of 25-250 mg/100 ml. Calibrations are linear to at least 450 mg./100 ml. with zero intercept. Glucose oxidase specifically catalyzes the aerobic oxidation of glucose to hydrogen peroxide. The peroxide reacts with iodide, in the presence of molybdenum (VI) catalyst, to form iodine. A known excess of thiosulfate reduces the iodine as it is produced. The reagents and the sample are incubated at 25.0° and pH 5.1. After 15 min., the pH is adjusted to 8.0 with phosphate reagent to stop the enzymatic reaction. The residual thiosulfate is titrated coulometrically with iodine at pH 8.0 to a dead-stop end point at a generating current of 0.4825 ma. The difference between the sample and thiosulfate reagent titers is proportional to the glucose concentration. The method is empirical. Peroxide-reducing impurities in the glucose oxidase preparation and mutarotation equilibrium prevent the complete recovery of glucose under the conditions of the experiment. Calibrations are reproducible from day to day and week to week. Reagents and the PFF constitute a negligible titration blank. Only 1 calculation is necessary. A simplified apparatus and procedure for the preparation of PFF’s permits 15 manual determinations per hour. Coulometric assays of commercial serum controls are accurate to within 3-4%. Data indicate that the precision of the coulometric method exceeds that of the Auto-Analyzer, Folin-Wu, Glucostat, and Nelson-Somogyi procedures. The proposed method is free from interferences at normal serum levels.