The effects of UDP-sugars, UDP and Mg2+on uridine diphosphate glucuronosyltransferase activity in human liver microsomes.

1. The UDP-glucuronosyltransferase (UGT) enzymes are important in the metabolism, elimination and detoxification of many xenobiotics and endogenous compounds. As extrapolation of in vitro kinetics of drug metabolizing enzymes to predict in vivo clearance rates becomes more sophisticated, it is important to ensure proper optimization of enzyme assays. The luminal location of the enzyme active site (i.e. latency), and the complexity of UGT kinetics, results in consistent under-prediction of clearance of drugs metabolized by glucuronidation. 2. We examined inhibition of UGT activity in alamethicin-disrupted human liver microsomes (HLM) by uridine diphosphate (UDP), a UGT reaction product, and its reversal by Mg2+ ions. We also determined whether UDP-sugars other than the co-substrate UDP-glucuronic acid (UDP-GlcA) affected glucuronidation. 3. We show that other UDP-sugars do not significantly influence glucuronidation. We also demonstrate that UDP inhibits HLM UGT activity and that this is reversed by including Mg2+ in the assay. The Mg2+ effect can be offset using EDTA, and is dependent on the concentration of UDP-GlcA in the assay. 4. We propose that formation of a Mg2+-UDP complex prevents UDP from affecting the enzyme. Our results suggest that 5 mM UDP-GlcA and 10 mM Mg2+ be used for UGT assays in fully disrupted HLM.

The ontogeny and population variability of human hepatic dihydronicotinamide riboside:quinone oxidoreductase (NQO2).

Dihydronicotinamide riboside:quinone oxidoreductase (NQO2) is an enzyme that performs reduction reactions involved in antioxidant defense. We hypothesized that NQO2 hepatic drug clearance would develop in children over time, similar to NQO1. Using human liver cytosol (n = 117), the effects of age, sex, ethnicity, and weight on NQO2 expression and activity were probed. No significant correlations were observed. Biochemical activity of NQO2 was as high at birth as in adults (0.23 ± 0.04 nmol/min/mg protein, mean ± SEM, range 0-1.83). In contrast, modeled hepatic clearance through the NQO2 pathway was up to 10% of adult levels at birth, reaching predicted adult levels (0.3 ± 0.03 L/h) at 14 years of age. Comparisons between NQO1 and NQO2 in the same livers showed that neither protein (P = 0.32) nor activity (P = 0.23) correlated, confirming both orthologs are independently regulated. Because hepatic clearance through NQO2 does not mature until teenage years, compounds detoxified by this enzyme may be more deleterious in children.

ATP-binding cassette proteins BCRP, MRP1 and P-gp expression and localization in the human umbilical cord.

1. The umbilical cord is a direct conduit to the fetus hence transporters could have roles in partitioning substances between the maternal-placental-fetal units. Here we determined the expression and localization of the ATP-Binding Cassette (ABC) transporters BCRP (ABCG2), P-gp (ABCB1) and MRP1 (ABCC1) in human umbilical cords. 2. The mRNA for BCRP and MRP1 was detected in 25/25 samples, but P-gp was detected in only 5/25. ABC transporter mRNA expression relative to 18S was 25.6 ± 0.3, 26.5 ± 0.6 and 22.2 ± 0.2 cycles for BCRP, MRP1 and P-gp respectively. 3. Using a subset of 10 umbilical cords, BCRP protein was present in all samples (immunoblot) with positive correlation between mRNA and proteins (p = 0.07, r = 0.62) and between immunoblotting and immunohistochemistry (IHC) (p = 0.03, r = 0.67). P-gp protein was observed in 4/10 samples by both immunoblot and IHC, with no correlation between mRNA and protein (p = 0.45, r = 0.55) or immunoblotting and IHC (p = 0.2, r = 0.72), likely due to small sample size. MRP1 protein was not observed. 4. Localization of BCRP and P-gp proteins was to Wharton's jelly with no specific staining in arterial or venous endothelia. 5. Understanding ABC transporter expression in the umbilical cord may be useful for determining fetal exposures to xenobiotics if functional properties can be defined.