The glucuronidation of Δ4-3-keto C19- and C21-hydroxysteroids by human liver microsomal and recombinant UDP-glucuronosyltransferases (UGTs):: 6α- and 21-hydroxyprogesterone are selective substrates for UGT2B7

The stereo- and regioselective glucuronidation of 10 Delta(4)-3-keto monohydroxylated androgens and pregnanes was investigated to identify UDP-glucuronosyltransferase (UGT) enzyme-selective substrates. Kinetic studies were performed using human liver microsomes (HLMs) and a panel of 12 recombinant human UGTs as the enzyme sources. Five of the steroids, which were hydroxylated in the 6 beta-, 7 alpha-, 11 beta- or 17 alpha-positions, were not glucuronidated by HLMs. Of the remaining compounds, comparative kinetic and inhibition studies indicated that 6 alpha- and 21-hydroxyprogesterone (OHP) were glucuronidated selectively by human liver microsomal UGT2B7. 6 alpha- OHP glucuronidation by HLMs and UGT2B7 followed Michaelis-Menten kinetics, whereas 21-OHP glucuronidation by these enzyme sources exhibited positive cooperativity. UGT2B7 was also identified as the enzyme responsible for the high-affinity component of human liver microsomal 11 alpha-OHP glucuronidation. In contrast, UGT2B15 and UGT2B17 were the major forms involved in human liver microsomal testosterone 17 beta- glucuronidation and the high-affinity component of 16 alpha-OHP glucuronidation. Activity of UGT1A subfamily enzymes toward the hepatically glucuronidated substrates was generally low, although UGT1A4 and UGT1A9 contribute to the low-affinity components of microsomal 16 alpha- and 11 alpha-OHP glucuronidation, respectively. Interestingly, UGT1A10, which is expressed only in the gastrointestinal tract, exhibited activity toward most of the glucuronidated substrates. The results indicate that 6 alpha- and 21-OHP may be used as selective probes for human liver microsomal UGT2B7 activity and, taken together, provide insights into the regio- and stereoselectivity of hydroxysteroid glucuronidation by human UGTs.