Human liver class I alcohol dehydrogenase γγ isozyme: the sole cytosolic 3β-hydroxysteroid dehydrogenase of iso bile acids

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Abstract
3β-Hydroxy (iso) bile acids are formed during enterohepatic circulation from 3α-hydroxy bile acids and constitute normal compounds in plasma but are virtually absent in bile. Isoursodeoxycholic acid (isoUDCA) is a major metabolite of UDCA. In a recent study it was found that after administration of isoUDCA, UDCA became the major acid in bile. Thus, epimerization of the 3β-hydroxy to a 3α-hydroxy group, catalyzed by 3β-hydroxysteroid dehydrogenases (HSD) and 3-oxo-reductases must occur. The present study aims to characterize the human liver bile acid 3β-HSD. Human liver cytosol and recombinant alcohol dehydrogenase (ADH) ββ and γγ isozymes were subjected to native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Activity staining with oxidized nicotinamide adenine dinucleotide (NAD+) or oxidized nicotinamide adenine dinucleotide phosphate (NADP+) as cofactors and various iso bile acids as substrates was used to screen for 3β-HSD activity. Reaction products were identified and quantified by gas chromotography/mass spectrometry (GC/MS). Computer-assisted substrate docking of isoUDCA to the active site of a 3-dimensional model of human class I γγ ADH was performed. ADH γγ isozyme was identified as the iso bile acid 3β-HSD present in human liver cytosol, with NAD+ as a cofactor. Values for kcat /Km were in the rank order isodeoxycholic acid (isoDCA), isochenodeoxycholic acid (isoCDCA), isoUDCA, and isolithocholic acid (isoLCA) (0.10, 0.09, 0.08, and 0.05 min-1 × μmol/L-1, respectively). IsoUDCA fits as substrate to the 3-dimensional model of the active-site of ADH γγ. ADH γγ isozyme was defined as the only bile acid 3β-HSD in human liver cytosol. Hydroxysteroid dehydrogenases are candidates for the binding and transport of 3α-hydroxy bile acids. We assume that ADH γγ isozyme is involved in cytosolic bile acid binding and transport processes as well.