Human liver class I alcohol dehydrogenase gamma gamma isozyme: The sole cytosolic 3 beta-hydroxysteroid dehydrogenase of iso bile acids

(3)beta-Hydroxy (iso) bile acids are formed during enterohepatic circulatio n from 3 alpha-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a m ajor metabolite of UDCA. In a recent study it was found chat after administ ration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3 beta-hydroxy to a 3 alpha-hydroxy group, catalyzed by 3 beta-hydro xysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur, The present study aims to characterize the human liver bile acid 3 beta-HSD. Human liv er cytosol and recombinant alcohol dehydrogenase (ADN) beta beta and gamma gamma isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinami de adenine dinucleotide (NAD(+)) or oxidized nicotinamide adenine dinucleot ide phosphate (NADP(+)) as cofactors and various iso bile acids as substrat es was used to screen for 3 beta-HSD activity. Reaction products were ident ified and quantified by gas chromotography/mass spectrometry (GC/MS). Compu ter-assisted substrate docking of isoUDCA to the active site of a 3-dimensi onal model of human class I gamma gamma ADN was performed. ADH gamma gamma isozyme was identified as the iso bile acid 3 beta-HSD present in human liv er cytosol, with NAD(+) as a cofactor. Values for k(cat)/K-m were in the ra nk order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0.05 min(- 1) x mu mol/L-1, respectively). IsoUDCA fits as substrate to the 3-dimensio nal model of the active-sire of ADH gamma gamma. ADH gamma gamma isozyme wa s defined as the only bile acid 3 beta-HSD in human liver cytosol. Hydroxys teroid dehydrogenases are candidates for the binding and transport of 3 alp ha-hydroxy bile acids. We assume that ADH gamma gamma isozyme is involved i n cytosolic bile acid binding and transport processes as well.