Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure

Abstract

We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO 4) and potassium carbonate (K2CO3), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO4). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO4. After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9·0) containing NAD+ and D-sorbitol dehydrogenase. After incubation for 30 min at 37°C, the NADH produced was measured fluorimetrically. The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content. The method had an analytical range of 1-180 µmol/L. The intra- and inter-assay precisions were < 3·3% and < 5·8%, respectively. The detection limit was 0·65 µmol/L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO4 was superior to the conventional method using HClO4 and K2CO23.