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

We developed an improved enzymatic assay of D-sorbitol in human erythrocyte s by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp . (EC 1.1.1.14) and replacing perchloric acid (HClO4) and potassium carbona te (K2CO3), generally used for deproteinization, with sodium hydroxide (NaO H) and zinc sulphate (ZnSO4). In this assay, erythrocytes were separated fr om plasma by centrifugation and washed once with physiological saline. Subs equently, the erythrocytes were lysed with distilled water and proteins pre cipitated with NaOH and ZnSO4. After centrifugation, the resulting colourle ss supernatant was mixed with a glycine buffer (pH 9.0) containing NAD(+) a nd D-sorbitol dehydrogenase. After incubation for 30 min at 37 degreesC, th e NADH produced was measured fluorimetrically. The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normal ized for haemoglobin content. The method had an analytical range of 1-180 mu mol/L. The intra- and inter- assay precisions were < 3.3% and < 5.8%, respectively. The detection limit was 0.65 mu mol/ L. In terms of the linearity, precision and sensitivity, t he improved method using NaOH and ZnSO4 was superior to the conventional me thod using HClO4 and K2CO3.