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

In this enzymatic method to analyze erythrocytes for D-sorbitol, the erythrocytes were separated from plasma by centrifugation, washed with isotonic saline (9 g/L NaCl), then diluted threefold with more saline. We lysed 1.5 mL of the diluted erythrocytes with chloroform and precipitated the protein with 58 g/L HClO4 solution. The resulting supernates were mixed with buffer, NAD+, and sorbitol dehydrogenase (EC 1.1.1.14). For standards, we added sorbitol to the erythrocytes before lysing. After 25 min of incubation at 37 degrees C, the fluorescence responses were recorded. Results for sorbitol were corrected for sample-blank and enzyme fluorescence, and were then normalized for hemoglobin content. Response was linear for a sorbitol concentration range of 1 to 30 mumol/L. Reproducibility was good, with an average CV of 2.5% at 10 mumol/L. Results for healthy individuals and diabetic patients are presented.