Abstract
This chapter describes an assay method for the synthesis of aldose reductase from human tissues. Aldose reductase and polyol dehydrogenase constitute the sorbitol pathway converting glucose to fructose in extrahepatic tissues. Aldose reductase catalyzes the reduction of other sugar aldehydes and of several aliphatic and aromatic aldehydes. Aldose reductase is assayed spectrophotometrically by recording the decrease in nicotinamide adenine dinucleotide phosphate dehydrogenase (NADPH) absorbance at 340 nm. With crude enzyme solutions, blank reactions occur with NADPH in the absence of any aldehyde substrate; consequently the rate of the reaction is recorded before the addition of exogenous aldehyde. Depending on the substrate and on the tissue analyzed, other dehydrogenases, notably aldehyde reductase, may interfere. Interference with aldehyde reductase is diminished by the addition of diphenylhydantoin or phenobarbitone to the assay medium. The ratio of activities measured with D-xylose, DL-glyceraldehyde, and D-glucuronate is used to distinguish between the two enzymes. The chapter discusses the procedure used to isolate and purify carbonyl reducing enzymes from human tissues, along with the preparation of brain aldose reductase. Brains, either whole or without cortex and cerebellum, are obtained from legal medical autopsies.
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