Reduction of 3-keto-5β-cholanoic acid to lithocholic and isolithocholic acids by human liver cytosol in vitro

Abstract

The formation of lithocholic and isolithocholic acids from 3-keto-5β-cholanoic acid by human liver cytosol was examined in vitro. Liver cytosol was incubated at various pH levels with 3-keto-5β-cholanoic acid in a phosphate buffer containing NADPH or NADH; the products formed were analyzed by gas chromatography. Results showed that human liver cytosol reduced 3-keto-5β-cholanoic acid to lithocholic acid at a pH level of 7.0 or above and to isolithocholic acid at a pH level of 6.0 or below when NADPH was used as a coenzyme, and it was reduced to isolithocholic acid only when NADH was used. Furthermore, two peaks for the reducing enzymes could be clearly found by column chromatography of Affi-Gel ® Blue. These results indicate that human liver cytosol contains two enzymes acting on reduction of 3-keto-5β-cholanoic acid to lithocholic and isolithocholic acids, which are dependent on the pH level and the use of NADPH or NADH in vitro. Since the 3β-dehydrogenation was inhibited by the addition of pyrazole, an alcohol dehydrogenase inhibitor or ethanol, and the major peak of 3β-hydroxysteroid dehydrogenase coincided with the peak of alcohol dehydrogenase on Affi-Gel ® Blue chromatography, at least some of the cytosolic 3β-hydroxysteroid dehydrogenase seemed to be identical to or to have characteristics similar to alcohol dehydrogenase.