1982 Volume 30 Issue 10 Pages 3796-3799
A new colorimetric method for the determination of total 3α-OH bile acids in serum by means of an enzymatic cycling reaction is described. Bile acids were oxidized by 3α-hydroxysteroid dehydrogenase from Pseudomonas testosteroni, and after the destruction of the remaining nicotinamide-adenine dinucleotide (NAD)+, the formed NADH was determined. The cycling system consisted of two enzymes, alcohol dehydrogenase from yeast and diaphorase from Clostridium kluyverii. The formed NADH was oxidized by diaphorase in the presence of 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and then was reduced again by alcohol dehydrogenase and ethanol. Subsequently, formazan produced as a final product was determined by rate assay. As the proposed method dose not require as extraction step, it is simpler and more accurate than the conventional methods.
