Fully enzymatic method for determining 1,5-anhydro-D-glucitol in serum

Abstract

We have developed a fully enzymatic method to measure 1,5 anhydro-D-glucitol (1,5-AG) in serum through use of pyranose oxidase (PROD: EC 1.1.3.10), glucokinase (EC 2.7.1.2), and an ATP-regenerating system. In a previous report (Clin Chem 1989;35:2039-43) the glucose interfering with the measurement of 1,5-AG was removed with a minicolumn. In the method used here, glucokinase and an ATP-regenerating system efficiently convert glucose to the unreactive compound, glucose 6-phosphate, making the method selective for 1,5-AG. The hydrogen peroxide produced in the oxidation of 1,5-AG by PROD is detected with a standard enzymatic color-developing system. The within-run and day-to-day precision (CV) of this method was 0.52-1.29% and 1.17-4.48%, respectively. The correlation (r) between the results obtained with our proposed method (y) and those obtained with the mini-column method (x) was 0.998 (y = 1.007x + 0.493 mg/L; n = 100; Sy/x = 0.641 mg/L). This newly developed method allows quicker and easier measurement of serum 1,5-AG than previously described methods.