Reaction-rate assay of serum sorbitol dehydrogenase activity of 37 degrees C

The sorbitol dehydrogenase (EC 1.1.1.14) activity of human serum and liver was investigated at 37 degrees C. Various buffers were examined but tris(hydroxymethyl)aminomethane HCI in a final concentration of 90 mmol/liter (pH 6.6, at 37 degrees C) was the one with which activity was the greatest. The enzyme is inhibited by its substrate, D-fructose, and activity was greatest with a substrate concentration (in the final assay mixture) of 500 mmol/liter and an NADH concentration of 247 mumol/liter. The specimen volume was 100 mul. The enzyme from liver and serum was shown to have similar Km values and activation energies, and we conclude that the liver enzyme was unchanged on passing into the extrahepatic space. Normal values for this assay are 0--2.6 U/liter (2.5 and 97.5 percentiles).