Abstract
Human UDP-glucuronosyltransferases (UGTs) are important enzymes in metabolic elimination of endo- and xenobiotics. It was recently shown that addition of fatty acid free bovine serum albumin (BSA) significantly enhances in vitro activities of UGTs, a limiting factor in in vitro–in vivo extrapolation. Nevertheless, since only few human UGT enzymes were tested for this phenomenon, we have now performed detailed enzyme kinetic analysis on the BSA effects in six previously untested UGTs, using 2–4 suitable substrates for each enzyme. We also examined some of the previously tested UGTs, but using additional substrates and a lower BSA concentration, only 0.1%. The latter concentration allows the use of important but more lipophilic substrates, such as estradiol and 17-epiestradiol. In five newly tested UGTs, 1A7, 1A8, 1A10, 2A1, and 2B15, the addition of BSA enhanced, to a different degree, the in vitro activity by either decreasing reaction’s K m, increasing its V max, or both. In contrast, the activities of UGT2B17, another previously untested enzyme, were almost unaffected. The results of the assays with the previously tested UGTs, 1A1, 1A6, 2B4, and 2B7, were similar to the published BSA only as far as the BSA effects on the reactions’ K m are concerned. In the cases of V max values, however, our results differ significantly from the previously published ones, at least with some of the substrates. Hence, the magnitude of the BSA effects appears to be substrate dependent, especially with respect to V max increases. Additionally, the BSA effects may be UGT subfamily dependent since K m decreases were observed in members of subfamilies 1A, 2A and 2B, whereas large V max increases were only found in several UGT1A members. The results shed new light on the complexity of the BSA effects on the activity and enzyme kinetics of the human UGTs.
Highlights
The human UDP-glucuronosyltransferase enzymes (UGTs) are located in the membrane of endoplasmic reticulum and play important roles in the metabolic elimination of lipophilic organic molecules of either external or internal origin [1,2]
The desire to include the two estrogens in this study, due to their suitability for studies with many human UGTs [24] was one of the major reasons to use a lower bovine serum albumin (BSA) concentration than previously used in most studies, since results from our laboratory demonstrated that most of the BSA effects are detectable in the presence of 0.1% and there is no real need to use 2% BSA in such assays [8]
Out of the ten UGT enzymes, 1A7, 1A8, 1A10, 2A1, 2B15, and 2B17 have not been tested for BSA effects before, while the remaining four, UGTs 1A1, 1A6, 2B4, and 2B7, were tested previously, mostly with other substrates [4,5,6,10,12] and partly serve as positive controls in this study
Summary
The human UDP-glucuronosyltransferase enzymes (UGTs) are located in the membrane of endoplasmic reticulum and play important roles in the metabolic elimination of lipophilic organic molecules of either external (xenobiotic) or internal origin [1,2]. These enzymes transfer the glucuronic acid moiety from the cosubstrate, UDP-a-D-glucuronic acid (UDPGA), to nucleophilic groups on aglycone substrates that can vary considerably in chemical structure and physicochemical properties. The main goal of in vitro glucuronidation assays is to accurately predict in vivo drug glucuronidation, both qualitatively and quantitatively [2]. The inclusion of fatty acid free bovine serum albumin (abbreviated here as BSA even if not all BSA types are fatty acid free) was previously reported to significantly increase the in vitro activities of UGT1A9 and 2B7, regardless of whether recombinant enzymes or human liver microsomes (HLM) were used as an enzyme source [4,5,6,7,8,9]
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