The glucuronidation of mycophenolic acid by human liver, kidney and jejunum microsomes

AIMS: To estimate the relative contribution of liver, kidney and jejunum to MPA elimination via glucuronidation from in vitro kinetic data. METHODS: The kinetics of MPA glucuronidation by human liver, kidney and jejunum microsomes were characterized. Mycophenolic acid glucuronide (MPAG) concentrations in microsomal incubations were determined using a specific h.p.l.c. procedure. Non-specific microsomal binding of MPA was excluded using an equilibrium dialysis approach. RESULTS: Microsomes from all three tissues catalysed the conversion of MPA to MPAG. Mean microsomal intrinsic clearances for MPAG formation by liver, kidney and jejunum microsomes were 46.6, 73.5 and 24.5 microl (min mg)(-1), respectively. When extrapolated to the whole organ, however, hepatic intrinsic clearance was 21- and 38-fold higher than the respective intrinsic clearances for kidney and small intestine. CONCLUSIONS: The data suggest that the liver is the organ primarily responsible for the systemic clearance of MPA, with little contribution from the kidney, and that the small intestine would be expected to contribute to first-pass extraction to a minor extent only.     

Inhibition of mycophenolic acid glucuronidation by niflumic acid in human liver microsomes

OBJECTIVES: The first aim of this investigation was to study the variability of mycophenolic acid (MPA) glucuronidation rate in human liver. The second aim was to study the inhibition type of niflumic acid (NA) for MPA glucuronidation in human liver. The third aim was to study the variability of the IC(50) value of NA for MPA glucuronidation in human liver. METHODS: The rate of MPA glucuronidation was measured by employing an assay based on uridine 5'-diphosphate-[U-(14)C]-glucuronic acid (UDPGA), and MPA glucuronide was isolated by means of thin-layer chromatography. The necessary concentration for UDPGA and MPA was 1 mM. The rate of MPA glucuronidation was measured in 50 human liver samples. The inhibition type of NA for MPA glucuronidation was studied in 5 human liver samples. The NA IC(50) value was measured in 27 human liver samples using six concentrations of NA ranging from 1.05 microM to 34 microM. RESULTS: MPA glucuronidation rate was positively skewed, was not gender regulated and did not correlate with the liver donor's age. The rate of MPA glucuronidation varied 4.8-fold within the 5th and 95th percentiles, with a mean+/-SD and a median of 2.8+/-1.0 nmol/min/mg and 2.5 nmol/min/mg, respectively. The inhibition type of NA for MPA glucuronidation was mixed non-competitive. The Ki value of NA (mean+/-SD) was 15+/-10 microM and, in non-inhibited samples, the K(m) value for MPA was 0.41+/-0.06 mM. The distribution of NA IC(50) value varied 3.3-fold within the 5th and 95th percentiles with a mean+/-SD and a median of 5.6+/-2.1 microM and 5.2 microM, respectively. The distribution of NA IC(50) value did not deviate significantly from normality. CONCLUSION: The range of hepatic rate of MPA glucuronidation is narrow relative to those of ethinyloestradiol, testosterone and zidovudine glucuronidation obtained from literature. The Ki value of NA is one order of magnitude lower than the K(m) for MPA in non-inhibited samples. This indicates that the inhibitor affinity for glucuronosyl transferase is greater than that of the substrate. The range of variation of NA IC(50) values is narrow.     

Lauric acid hydroxylation in human liver and kidney cortex microsomes

The ω- and (ω- 1 )-hydroxylation of the medium-chain fatty acid, dodecanoic or lauric acid, was studied in liver and kidney cortex microsomes from seven human cadavers. The rates of laurate hydroxylation in human liver microsomes were found to exceed the rates recorded in human kidney cortex microsomes by 4-to 30-fold. The mean specific activity of laurate hydroxylation from the seven human kidneys was six to fourteen times lower than the specific activities found in pig, rat or hamster kidney microsomes. The effects of several known inhibitors of the liver microsomal cytochrome P-450-dependent mono-oxygenase system were also studied. Metyrapone preferentially inhibited the (ω- 1)-hydroxylase activity of human liver microsomes, but did not affect the ω-hydroxylation reaction. In the presence of 7,8-benzoflavone, the human liver microsomal (ω- 1 )-hydroxylase activity was stimulated, but an inhibitory effect was observed on the ω-hydroxylation reaction. 2-Diethylaminoethyl-2,2-diphenylvaIerate (SKF 525A) inhibited both hydroxylase activities in human liver microsomes. Neither metyrapone nor SKF 525A inhibited the laurate hydroxylation reactions catalyzed by human kidney microsomes. These studies indicate that the cytochrome P-450-mediated hydroxylations of medium chain fatty acids in human kidney cortex microsomes are much less active than in kidneys of other species investigated. The effects of the inhibitors, metyrapone and SKF 525A, on ω- and (ω- 1)-hydroxylation of laurate in human liver and kidney microsomes were similar to the effects reported in other mammalian species.     

Glucuronide conjugation of 6-n-propyl-2-thiouracil and other antithyroid drugs by guinea pig liver microsomes in vitro.

Guinea pig liver microsome UDP glucuronyl transferase and UDPGA were incubated with the radioactive antithyroid drugs 6-n-propyl-2-thiouracil (PTU). 1-methy1-2-mercaptoimidazole (methimazole, MMI) and 2-thiouracil (TU). Radioactive metabolites were produced with PTU and thiouracil and, in each case, were identified as the corresponding β-glucuronide conjugate. No measurable glucuronidation of MMI was observed. Kinetic studies with the microsomal preparation demonstrated a K_m rmvalue of 7.2 × 10^?4 M for PTU and 6.7 × 10^?3 M for thiouracil. Glucuronide conjugation of PTU was linear for 1 hr. declining thereafter while conjugation of phenolphthalein was linear for 2 hr. Conjugation of phenolphthalein by microsomes stored in 0.154 M KCl at ?20° for 14 days was 41 per cent higher than in fresh microsomes, whereas conjugation of PTU was 67.4 per cent lower. PTU glucuronidation did not occur in the absence of UDPGA and was essentially linear with respect to enzyme concentrations. Under the same conditions, spontaneous N-glucuronidation of PTU by glucuronate was not measurable. The pH optimum for PTU glucuronidation was 8.0 and similar to the broad optimums of 7.3 to 7.9 for UDP glucuronyl transferases from a variety of sources rather than to non-enzymatic N-glucuronidation, which has a reported pH optimum of 3–4. The conjugating enzyme for PTU was located primarily in the guinea pig liver microsomes with this fraction exhibiting 75 per cent of the total activity of whole homogenates. PTU conjugation was inhibited by MMI but not by thiouracil, thiourea or 6-methy1-2-thiouracil. The results obtained demonstrate that β-glucuronide conjugation of the antithyroid drugs PTU and thiouracil, but not MMI. is readily catalyzed by a guinea pig liver microsomal UDP glucuronyl transferase in vitro.     

Glucuronidation of trans-resveratrol by human liver and intestinal microsomes and UGT isoforms

Resveratrol (trans-resveratrol, trans-3,5,4'-trihydroxystilbene) is a naturally occurring stilbene analogue found in high concentrations in red wine. There is considerable research interest to determine the therapeutic potential of resveratrol, as it has been shown to have tumour inhibitory and antioxidant properties. This study was performed to investigate the glucuronidation of resveratrol and possible drug interactions via glucuronidation. Two glucuronide conjugates, resveratrol 3-O-glucuronide and resveratrol 4'-O-glucuronide, were formed by human liver and intestinal microsomes. UGT1A1 and UGT1A9 were predominantly responsible for the formation of the 3-O-glucuronide (Km = 149 microM) and 4'-O-glucuronide (Km = 365 microM), respectively. The glucuronide conjugates were formed at higher levels (up to 10-fold) by intestinal rather than liver microsomes. Resveratrol was co-incubated with substrates of UGT1A1 (bilirubin and 7-ethyl-10-hydroxycamptothecin (SN-38)) and UGT1A9 (7-hydroxytrifluoromethyl coumarin (7-HFC)). No major changes were noted in bilirubin glucuronidation in the presence of resveratrol. Resveratrol significantly inhibited the glucuronidation of SN-38 (Ki = 6.2 +/- 2.1 microM) and 7-HFC (Ki = 0.6 +/- 0.2 microM). Hence, resveratrol has the potential to inhibit the glucuronidation of concomitantly administered therapeutic drugs or dietary components that are substrates of UGT1A1 and UGT1A9.     

Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes.

Glucuronidation of thyroxine is a major metabolic pathway facilitating its excretion. In this study, we characterized the glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes, and identified human UDP-glucuronosyltransferase (UGT) isoforms involved in the activity. Human jejunum microsomes showed a lower K(m) value (24.2 microM) than human liver (85.9 microM) and kidney (53.3 microM) microsomes did. Human kidney microsomes showed a lower V(max) value (22.6 pmol/min/mg) than human liver (133.4 pmol/min/mg) and jejunum (184.6 pmol/min/mg) microsomes did. By scaling-up, the in vivo clearances in liver, intestine, and kidney were estimated to be 1440, 702, and 79 microl/min/kg body weight, respectively. Recombinant human UGT1A8 (108.7 pmol/min/unit), UGT1A3 (91.6 pmol/min/unit), and UGT1A10 (47.3 pmol/min/unit) showed high, and UGT1A1 (26.0 pmol/min/unit) showed moderate thyroxine glucuronosyltransferase activity. The thyroxine glucuronosyltransferase activity in microsomes from 12 human livers was significantly correlated with bilirubin O-glucuronosyltransferase (r = 0.855, p < 0.001) and estradiol 3-O-glucuronosyltransferase (r = 0.827, p < 0.0001) activities catalyzed by UGT1A1, indicating that the activity in human liver is mainly catalyzed by UGT1A1. Kinetic and inhibition analyses suggested that the thyroxine glucuronidation in human jejunum microsomes was mainly catalyzed by UGT1A8 and UGT1A10 and to a lesser extent by UGT1A1, and the activity in human kidney microsomes was mainly catalyzed by UGT1A7, UGT1A9, and UGT1A10. The changes of activities of these UGT1A isoforms via inhibition and induction by administered drugs as well as genetic polymorphisms may be a causal factor of interindividual differences in the plasma thyroxine concentration.     

Aldosterone glucuronidation by human liver and kidney microsomes and recombinant UDP-glucuronosyltransferases: Inhibition by NSAIDs

AIMS

To characterize: i) the kinetics of aldosterone (ALDO) 18β-glucuronidation using human liver and human kidney microsomes and identify the human UGT enzyme(s) responsible for ALDO 18β-glucuronidation and ii) the inhibition of ALDO 18β-glucuronidation by non-selective NSAIDs.

METHODS

Using HPLC and LC-MS methods, ALDO 18β-glucuronidation was characterized using human liver (n= 6), human kidney microsomes (n= 5) and recombinant human UGT 1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15, 2B17 and 2B28 as the enzyme sources. Inhibition of ALDO 18β-glucuronidation was investigated using alclofenac, cicloprofen, diclofenac, diflunisal, fenoprofen, R- and S-ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, S-naproxen, pirprofen and tiaprofenic acid. A rank order of inhibition (IC₅₀) was established and the mechanism of inhibition investigated using diclofenac, S-ibuprofen, indomethacin, mefenamic acid and S-naproxen.

RESULTS

ALDO 18β-glucuronidation by hepatic and renal microsomes exhibited Michaelis-Menten kinetics. Mean (±SD) K_m, V_max and CL_int values for HLM and HKCM were 509 ± 137 and 367 ± 170 µm, 1075 ± 429 and 1110 ± 522 pmol min⁻¹ mg⁻¹, and 2.36 ± 1.12 and 3.91 ± 2.35 µl min⁻¹ mg⁻¹, respectively. Of the UGT proteins, only UGT1A10 and UGT2B7 converted ALDO to its 18β-glucuronide. All NSAIDs investigated inhibited ALDO 18β-G formation by HLM, HKCM and UGT2B7. The rank order of inhibition (IC₅₀) of renal and hepatic ALDO 18β-glucuronidation followed the general trend: fenamates > diclofenac > arylpropionates.

CONCLUSION

A NSAID-ALDO interaction in vivo may result in elevated intra-renal concentrations of ALDO that may contribute to the adverse renal effects of NSAIDs and their effects on antihypertensive drug response.     

Raloxifene glucuronidation in human intestine, kidney, and liver microsomes and in human liver microsomes genotyped for the UGT1A1*28 polymorphism

Raloxifene, a selective estrogen receptor modulator, exhibits quite large interindividual variability in pharmacokinetics and pharmacodynamics. In women, raloxifene is metabolized extensively by different isoforms of UDP-glucuronosyltransferase (UGT) to its glucuronides. To gain an insight into intestine, kidney, liver, and lung glucuronidation of raloxifene, human microsomes of all tested organs were used. Raloxifene-6-β-glucuronide (M1) formation followed the Michaelis-Menten kinetics in intestinal, kidney, and liver microsomes; meanwhile, raloxifene-4'-β-glucuronide (M2) formation followed the substrate inhibition kinetics. Human lung microsomes did not show any glucuronidation activity. The tissue intrinsic clearances for kidney, intestine, and liver were 3.4, 28.1, and 39.6 ml · min(-1) · kg(-1), respectively. The aim of our in vitro study was to explain the mechanism behind the observed influence of UGT1A1*28 polymorphism on raloxifene pharmacokinetics in a small-sized in vivo study (Br J Clin Pharmacol 67:437-444, 2009). Incubation of raloxifene with human liver microsomes genotyped for UGT1A1*28 showed a significantly reduced metabolic clearance toward M1 in microsomes from donors with *28 allele. On the contrary, no significant genotype influence was observed on the formation of M2 because of the high variability in estimated apparent kinetic parameters, although a clear trend toward lower glucuronidation activities was observed when UGT1A1*28 polymorphism was present. The liver intrinsic clearances of both homozygotes differed significantly, whereas the clearance of heterozygotes did not differ from the wild-type and the mutated homozygotes. In conclusion, our results show the high importance of the liver and intestine in raloxifene glucuronidation. Moreover, the significant influence of UGT1A1*28 polymorphism on metabolism of raloxifene was confirmed.     

Stereoselective oxidation and glucuronidation of carvedilol in human liver and intestinal microsomes

The aim of the present study was to investigate the mechanism for the stereoselective presystemic clearance of carvedilol. We examined the oxidation and glucuronidation of carvedilol in human liver microsomes (HLM) and human intestinal microsomes (HIM). The oxidation of carvedilol in HLM and HIM was evaluated in the presence of NADPH, whereas glucuronidation was evaluated in the presence of UDP-glucuronic acid. Oxidation of S-carvedilol in HLM and HIM was greater than that of R-carvedilol. In addition, the oxidation of R-carvedilol in HLM was inhibited by quinidine, whereas that of S-carvedilol was inhibited by both quinidine and furafylline. On the other hand, R- and S-carvedilol oxidation in HIM was inhibited by ketoconazole. Glucuronidation of S-carvedilol in HLM and HIM was also higher than that of R-carvedilol. These results suggested that cytochrome P450 (CYP) 2D6 and CYP1A2 are involved in the stereoselective oxidation of carvedilol in the liver, that CYP3A4 is involved in intestinal oxidation, and that glucuronidation in the liver and intestine is at least partly responsible for stereoselective presystemic clearance.     

In vitro metabolism of clindamycin in human liver and intestinal microsomes.

Incubations with human liver and gut microsomes revealed that the antibiotic, clindamycin, is primarily oxidized to form clindamycin sulfoxide. In this report, evidence is presented that the S-oxidation of clindamycin is primarily mediated by CYP3A. This conclusion is based upon several lines of in vitro evidence, including the following. 1) Incubations with clindamycin in hepatic microsomes from a panel of human donors showed that clindamycin sulfoxide formation correlated with CYP3A-catalyzed testosterone 6beta-hydroxylase activity; 2) coincubation with ketaconazole, a CYP3A4-specific inhibitor, markedly inhibited clindamycin S-oxidase activity; and 3) when clindamycin was incubated across a battery of recombinant heterologously expressed human cytochrome P450 (P450) enzymes, CYP3A4 possessed the highest clindamycin S-oxidase activity. A potential role for flavin-containing monooxygenases (FMOs) in clindamycin S-oxidation in human liver was also evaluated. Formation of clindamycin sulfoxide in human liver microsomes was unaffected either by heat pretreatment or by chemical inhibition (e.g., methimazole). Furthermore, incubations with recombinant FMO isoforms revealed no detectable activity toward the formation of clindamycin sulfoxide. Beyond identifying the drug-metabolizing enzyme responsible for clindamycin S-oxidation, the ability of clindamycin to inhibit six human P450 enzymes was also evaluated. Of the P450 enzymes examined, only the activity of CYP3A4 was inhibited (approximately 26%) by coincubation with clindamycin (100 microM). Thus, it is concluded that CYP3A4 appears to account for the largest proportion of the observed P450 catalytic clindamycin S-oxidase activity in vitro, and this activity may be extrapolated to the in vivo condition.     

Glucuronide and sulphate conjugation in isolated liver cells from control and phenobarbital- or PCB-treated rats

The conjugation of 4-methylumbelliferone, p-nitrophenol and o-aminophenol in isolated rat liver cells was studied. It was found that for maximum sulphate conjugation to take place a very high sulphate concentration (50 mM) was needed, indicating that the rate limiting step may be the formation of ‘active sulphate’. Under these conditions only a slight increase in the glucuronide/sulphate ratio with increasing substrate concentration was seen. Inducers of drug metabolism. PCB and phenobarbital, increased only glucuronidation. PCB was also found to cause qualitative changes in the metabolism of 4-methylumbelliferone.     

Mutual inhibition between carvedilol enantiomers during racemate glucuronidation mediated by human liver and intestinal microsomes

Carvedilol is administered orally as a racemic mixture of R(+)- and S(-)-enantiomers for treatment of angina pectoris, hypertension and chronic heart failure. We have reported that enzyme kinetic parameters for carvedilol glucuronidation by human liver microsomes (HLM) differed greatly depending on the substrate form, namely, racemic carvedilol and each enantiomer. These phenomena were thought to be caused by mutual inhibition between carvedilol enantiomers during racemate glucuronidation. The aim of this study was to clarify the mechanism of these phenomena in HLM and human intestinal microsomes (HIM) and its relevance to uridine 5'-diphosphate (UDP)-glucuronosyl transferase (UGT) 1A1, UGT2B4 and UGT2B7, which mainly metabolize carvedilol directly in phase II enzymes. HLM apparently preferred metabolizing (S)-carvedilol to (R)-carvedilol in the racemate, but true activities of HLM for both glucuronidation were approximately equal. By determination of the inhibitory effects of (S)-carvedilol on (R)-carvedilol glucuronidation and vice versa, it was shown that (R)-carvedilol glucuronidation was more easily inhibited than was (S)-carvedilol glucuronidation. UGT2B7 was responsible for (S)-carvedilol glucuronidation in HLM. Ratios of contribution to (R)-carvedilol glucuronidation were approximately equal among UGT1A1, UGT2B4 and UGT2B7. However, enzyme kinetic parameters were different between the two lots of HLM used in this study, depending on the contribution ratio of UGT2B4, in which (R)-glucuronidation was much more easily inhibited by (S)-carvedilol than was (S)-glucuronidation by (R)-carvedilol. Meanwhile, HIM preferred metabolizing (R)-carvedilol, and this tendency was not different between the kinds of substrate form.     

Metabolism of coumarin by rat,gerbil and human liver microsomes

1. o-Hydroxyphenylacetaldehyde was the major metabolite of coumarin (1 mM) in rat, gerbil and human liver microsomes.

2. Treatment of rats with phenobarbitone (PB) or β-naphthoflavone increased the o-hydroxyphenylacetaldehyde formed. 3-Hydroxycoumarin was the other main metabolite produced by rat liver microsomes.

3. Liver microsomal metabolism of coumarin in gerbil was extensive with 3-, 5-, 6-, 7-and 8-hydroxycoumarins, and 3,7- and 6,7-dihydroxycoumarins produced, in addition to o-hydroxyphenylacetaldehyde. The profile of the hydroxy metabolites was altered by in vivo treatment of gerbils with cytochrome P-450 inducers, but there was no increase of coumarin metabolism.

4. Coumarin was metabolized by human liver microsomes to o-hydroxyphenylacetaldehyde, 7-hydroxycoumarin, 3-hydroxycoumarin, and trace amounts of 5-, 6-and 8-hydroxycoumarins.

5. At low substrate concentrations (0-10 μM) hepatic microsomal metabolism of coumarin in gerbil resembled that in man, with 7-hydroxycoumarin being a major metabolite. However, the production of o-hydroxyphenylacetaldehyde was greater in gerbil than human liver microsomes.

6. At higher substrate concentrations (1 mM) metabolism of coumarin by liver microsomes from PB-treated gerbils most closely resembled that by human liver microsomes.

7. The gerbil would appear to be a more appropriate animal model than rat for studies to assess the toxicological hazard of coumarin for man.     

Enantioselective hydroxylation of nortriptyline in human liver microsomes, intestinal homogenate, and patients treated with nortriptyline

The enantioselectivity of hydroxylation of nortriptyline (NT) to E-10-hydroxynortriptyline (E-10-OH-NT) was studied in human liver microsomes, intestinal homogenate, and patients treated with NT. The rate of formation of (-)-E-10-OH-NT was higher than that of (+)-E-10-OH-NT both in the liver microsomes and in the intestinal homogenate. Quinidine, a prototype competitive inhibitor of the cytochrome P450IID6 ("debrisoquin hydroxylase"), inhibited the formation of (-)-E-10-OH-NT in a concentration-dependent manner in liver microsomes, while the formation of (+)-E-10-OH-NT was hardly affected. This indicates that P450IID6 catalyzes the hydroxylation of NT in a highly enantioselective manner to (-)-E-10-OH-NT in the liver. Another P450 isozyme besides IID6 seems to be responsible for the formation of the (+)-enantiomer in the liver. In intestinal homogenate, the formation of both enantiomers of E-10-OH-NT was inhibited to about the same extent by quinidine, the maximum inhibition being much less than in the liver. In the urine of six patients treated with NT, the (-)-enantiomer accounted for 91 +/- 2% of the unconjugated E-10-OH-NT, and for 78 +/- 6% of the glucuronide conjugates. The study shows that NT is hydroxylated in a highly enantioselective way, probably catalyzed by the polymorphic P450IID6, to (-)-E-10-OH-NT both in vitro in human liver as well as in vivo in patients treated with the drug.     

HPLC法测定人血浆中麦考酚酸的浓度

目的建立人血浆中麦考酚酸酯活性代谢物麦考酚酸的HPLC测定法。方法色谱柱为HYPERSIL C18(150 mm×5.0mm,5μm),预柱为岛津ODS(5.0 mm×3.9 mm,10μm)。流动相为甲醇-磷酸二氢钠缓冲液(pH=3.25)-乙腈(40∶40∶20,V/V),流速0.8 mL.min-1,检测波长254 nm,柱温25℃。结果麦考酚酸的血浆样品为0.23~46.13 mg.L-1时线性良好(r=0.999 7)。方法回收率96%~106%,日内RSD<6%,日间RSD<7%。结论本方法简单、灵敏、重现性好,适用于临床血药浓度监测,以及人体药动学的研究。     

The hydrolysis of acetylsalicylic acid by liver microsomes

Pretreatment of rats and guinea-pigs with phenobarbitone or phenylbutazone leads to a decrease in the rate of hydrolysis of acetylsalicylic acid in vitro by liver microsomes, treatment with phenacetin does not.     

Lindane metabolism by human and rat liver microsomes

1. Human liver microsomes convert lindane (gamma isomer of 1,2,3,4,5,6-hexachlorocyclohexane) to four major primary metabolites; gamma-1,2,3,4,5,6-hexachlorocyclohex-1-ene (3,6/4,5-HCCH), gamma-1,3,4,5,6-pentachlorocyclohex-1-ene (3,6/4,5-PCCH), beta-1,3,4,5,6-pentachlorocyclohex-1-ene (3,4,6/5-PCCH), and 2,4,6-trichlorophenol (2,4,6-TCP); and two major secondary metabolites; 2,3,4,6-tetrachlorophenol (2,3,4,6-TTCP) and pentachlorobenzene (PCB). 2. Under the same conditions, rat liver microsomes produce 3,6/4,5-HCCH, 2,4,6-TCP and 2,3,4,6-TCCP at rates similar to human liver microsomes. 3,4,6/5-PCCH is produced at much lower rates and 3,6/4,5-PCCH and PCB are not detected when lindane is incubated with rat liver microsomes for up to 30 min. 3. The identity of 3,4,6/5-PCCH, previously not identified as a mammalian metabolite of lindane, is confirmed by column chromatography and g.l.c.-mass spectrometry by comparison with authentic material. 4. It is concluded that there is potentially substantial hepatic metabolism by humans of lindane, a topically used scabicide and pediculicide.     

Phenobarbital N-glucosylation by human liver microsomes.

Glucosylation of xenobiotics in mammals has been observed for a limited number of drugs. Generally, these glucoside conjugates are detected as urinary excretion products with limited information on their formation. An in vitro assay is described for measuring the formation of the phenobarbital N-glucoside diasteriomers ((5R)-PBG, (5S)-PBG) using human liver microsomes. Human livers (n = 18) were screened for their ability to N-glucosylate PB. Cell viability, period of liver storage, prior drug exposure, serum bilirubin levels, age, sex and ethnicity did not appear to influence the specific activities associated with the formation of the PB N-glucosides. The average rate of formation for both PB N-glucoside was 1.42 +/- 1.04 (range 0.11-4.64) picomole/min/mg-protein with an (5S)-PBG/(5R)-PBG ratio of 6.75 +/- 1.34. The apparent kinetic constants, Km and Vmax, for PB N-glucosylation for eight of the livers ranged from 0.61-20.8 mM and 2.41-6.29 picomole/min/mg-protein, respectively. The apparent Vmax/Km ratio for PB exhibited a greater than 20 fold variation in the ability of the microsomes to form the PB N-glucosides. It would appear that the formation of these barbiturate N-glucoside conjugates in vitro are consistent with the amount of barbiturate N-glucosides formed and excreted in the urine in prior drug disposition studies.