Search PubMed⌕ Search

Biomedical subjects

L S Kaminsky

Publications and source records attributed to L S Kaminsky.

At least 19 recordsLinked to original sources
PubMed1998

Characterization of the cytochrome P450 CYP2J4: expression in rat small intestine and role in retinoic acid biotransformation from retinal.

The sites of expression in the small intestine and the function of CYP2J4, a recently identified rat cytochrome (P450) isoform found to be predominantly expressed in the small intestine, were characterized. Immunoblot analysis with a polyclonal antibody to heterologously expressed CYP2J4 revealed that expression of CYP2J4 was at the highest level in the distal duodenum and jejunum and decreased toward the ileum. Villous cells expressed higher levels of CYP2J4 than crypt cells. Isoform-specific RNA polymerase chain reaction indicated that a related P450 isoform, CYP2J3, was only a minor form in rat small intestine. Since the intestinal mucosa is exposed to high levels of dietary nutrients, we hypothesized that CYP2J4 may be active toward diet-derived factors. We determined that purified, heterologously expressed CYP2J4 is active toward all-trans- and 9-cis-retinal in reconstituted systems, producing the corresponding retinoic acids as the major products. Apparent K(m) values for the formation of retinoic acids were 54 and 49 microM, respectively, and apparent Vmax values were 20 and 21 nmol/min/nmol P450, respectively. These activities were readily inhibited by a polyclonal anti-CYP2J4 antibody. Rat enterocyte microsomes were also active with all-trans-retinal to produce all-trans-retinoic acid in the presence of NADPH, and the majority of retinoic acid synthesis activity was inhibited by the polyclonal anti-CYP2J4 antibody. These findings suggest that CYP2J4 plays a major role in intestinal microsomal metabolism of retinal to retinoic acid and may be involved in the maintenance of retinoid homeostasis in the small intestine in vivo.

Animals↗
PubMed1998

Caffeine based measures of CYP1A2 activity correlate with oral clearance of tacrine in patients with Alzheimer's disease.

AIMS: To study the potential utility of caffeine based probes of CYP1A2 enzyme activity in predicting the pharmokinetics of tacrine in patients with Alzheimer's disease. METHODS: The pharmokinetics of a single 40 mg oral dose of tacrine were measured in 19 patients with Alzheimer's disease. Each patient also received 2 mg kg(-1) [13C-3-methyl] caffeine orally and had breath and urine samples collected. RESULTS: Tacrine oral clearance (CL F(-1) kg(-1)), which varied 15-fold among the patients, correlated significantly with the 2 h total production of 13CO2 in breath (r=0.56, P=0.01), and with each of two commonly used urinary caffeine metabolite ratios: the 'paraxanthine/caffeine ratio' (1,7X + 1, 7U)/1,3,7X) (r=0.76, P=0.0002) and the 'caffeine metabolic ratio' (AFMU + 1X + 1U)/1, 7U)(r=0.76, P=0.0001). CONCLUSIONS: These observations support a central role for CYP1A2 in the in vivo disposition of tacrine and the potential for drug interactions when tacrine treated patients receive known inducers or inhibitors of this enzyme. The magnitude of the correlations we observed, however, are probably not sufficient to be clinically useful in individualizing tacrine therapy.

Administration, Oral↗
PubMed1998

16Alpha-hydroxylation of estrone by human cytochrome P4503A4/5.

The cytochrome P450 (P450) enzymes that catalyse metabolism of the estrogen, estrone (E1), to the putative carcinogen 16alpha-hydroxy E1 (16alpha-OHE1) in humans were determined. The potential of the most abundant circulating form of estrogen, estrone 3-sulfate (E1S), to be the substrate was also investigated. Human liver microsomal sulfatases convert E1S to E1, an essential prerequisite for formation of 16alpha-OHE1 from added E1S in this system. E1 metabolism to 16alpha-OHE1 in a panel of 15 human liver microsomal preparations correlated with total P450 concentrations (r2 = 0.63) and with activities associated with P450 forms CYP3A4 and 3A5 (r2 = 0.72). E1 16alpha-hydroxylase activity in human liver microsomes was inhibited by 75% by monoclonal anti human CYP3A4/5 antibodies at 4 mg antibody/nmol total P450, and by troleandomycin, a specific CYP3A4/5 inhibitor. Rates of E1 metabolism to 16alpha-OHE1 were 1.6-fold higher when E1 was generated in situ from E1S than when E1 was added. Microsomal preparations of cDNA expressed CYP3A4 or 3A5, with NADPH-P450-reductase co-expressed, both metabolized E1 to 16alpha-OHE1, and added cytochrome b5 increased the rates 5.1- and 7.5-fold, respectively. In these systems rates of E1 metabolism to 16alpha-OHE1 were 2.8-fold higher when E1 was generated in situ from E1S than when E1 was added. Kinetic values for E1 metabolism to 16alpha-OHE1 by human liver microsomes and for the expressed CYP3A4 system were Km 154 and 172 microM, respectively, and Vmax 238 pmol/min/nmol total P450 and 1050 pmol/min/nmol CYP3A4, respectively. Thus, formation of the putative carcinogen 16alpha-OHE1 is catalysed by CYP3A4 and 3A5 and stimulated by cytochrome b5. E1S is not a substrate but formation of E1 from E1S in situ stimulates formation of 16alpha-OHE1, possibly because E1S is more water soluble and in situ generation of E1 provides for facilitated exposure of E1 to the P450 substrate binding sites. Blocking of the pathway of E1 to 16alpha-OHE1 could provide a therapeutic approach for diminishing the risk of estrogen dependent breast cancer.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Warfarin analog inhibition of human CYP2C9-catalyzed S-warfarin 7-hydroxylation.

Human metabolism of the S-warfarin enantiomer is catalyzed primarily by cytochrome P4502C9 (CYP2C9), which, because of the enzyme's broad drug substrate specificity, leads to drug-S-warfarin interactions. Several warfarin analogs have been synthesized and used to determine whether they exhibit diminished interactions with CYP2C9. The kinetics of the warfarin analogs' inhibition of human liver microsomal CYP2C9 catalyzed metabolism of S-warfarin to S-7-hydroxywarfarin have been investigated. R- and S-7-fluorowarfarin were both predominantly competitive inhibitors, whereas racemic 6-fluorowarfarin and racemic 6,7,8-trifluorowarfarin were predominantly mixed inhibitors with some competitive inhibition. For the alcohols produced by reductive methylation of the side chain of R- and S-warfarin, the R-enantiomer did not inhibit S-warfarin metabolism, whereas the S-enantiomer was primarily a competitive inhibitor. The fluorine substituted warfarins and the S-warfarin alcohol apparently bind with high affinity to CYP2C9. Thus their use clinically (if efficacious) would not prevent CYP2C9 associated warfarin-drug interactions. The R-warfarin alcohol did not inhibit CYP2C9 catalyzed metabolism of S-warfarin and is less likely than warfarin to participate in CYP2C9 associated warfarin-drug interactions.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Alternative splicing of CYP2D mRNA in human breast tissue.

The human cytochrome P450 (CYP) 2D subfamily comprises the CYP2D6 gene and four pseudogenes, CYP2D7P1 and 2 and CYP2D8P1 and 2. The CYP2D6 gene product is a prominent drug-metabolizing enzyme, which is probably constitutive and has no known inducing agents. Alternative splicing of the pre-mRNAs of these genes has been detected in human liver and breast tissue. RNA-PCR, competitive RNA-PCR, Southern blotting, cDNA sequencing, and gene-specific PCR have been used to fully characterize the alternatively spliced forms of CYP2D mRNA in human breast tissue in the region of exon 5 to 8. Such alternative splicing could regulate the expression of CYP2D6 protein. A full-length mRNA (exons 5 to 8), and variants c (exon 6 deleted), b' (3' portion of exon 6 deleted), e (3' portion of exon 6 deleted, 3' 57-bp portion of intron 6 included), d (3' 57-bp portion of intron 6 included), and b (intron 6 included) were characterized and quantitated. Variant c was derived from CYP2D6, variants d, e, and b were from CYP2D7P, and variant b' and full-length mRNA were derived from both CYP2D6 and 2D7P. Full-length mRNA was a minor form in human breast tissue where variants b' and c predominated. Human breast tumor MCF-7 cells had CYP2D mRNA splice variant patterns similar to those of human breast tissue, while human liver tumor HepG2 cells had wild-type mRNA predominating. These results suggest that CYP2D6 could be regulated tissue specifically using tissue-specific alternative mRNA splicing.

Alternative Splicing↗
PubMed1997

Comparisons of CYP2D messenger RNA splice variant profiles in human lung tumors and normal tissues.

Allelic variants of the CYP2D6 gene, a member of the cytochrome P450 gene superfamily, have been implicated in susceptibility to lung carcinogenesis. Human breast CYP2D6 and CYP2D7P (from a pseudogene) mRNAs were previously reported to be expressed as a series of splice variants. In this study, the expression of full-length and splice variants of these mRNAs in human lung tissue and tumors are reported for the first time and are compared in order to probe the potential for differential CYP2D6 regulation in lung normal tissue and tumors. The splice variant profiles differed within the same individual, but no consistent differences were detected.

Adult↗
PubMed1997

CDNA cloning, heterologous expression, and characterization of rat intestinal CYP2J4.

The small intestine is the major portal of entry of ingested xenobiotics. Previous studies from this and other laboratories indicated that at least 6 of the 33 xenobiotic metabolizing forms of P450 currently identified are expressed in rat small intestinal epithelial cells. In the present study, a previously unidentified rat P450, designated CYP2J4, was identified in rat small intestine using PCR. The full-length CYP2J4 cDNA contains an open reading frame for a protein of 501 residues and is 72.5 and 75.8% identical to rabbit CYP2J1 and human CYP2J2, respectively, in deduced amino acid sequences. The coding region of CYP2J4 cDNA has been cloned into a baculoviral expression vector (pVL1392) and expressed in cultured Spodoptera frugiperta (SF9) cells. The heterologously expressed CYP2J4 protein displayed a typical p450 CO-difference spectrum, with maximum absorbance at 449 nm. When purified to near electrophoretic homogeneity, it was active toward arachidonic acid in a reconstituted system with NADPH-P450 reductase and phospholipid, producing both hydroxyeicosatetraenoic and epoxyeicosatrienoic acids. RNA blot analysis with CYP2J4 cDNA as a probe detected two mRNA species, about 2.0 and 2.4 kb, respectively, in RNA preparations from liver, intestine, olfactory mucosa, kidney, heart, and lung. The 2.0-kb mRNA species was abundant in liver, small intestine, and olfactory mucosa, whereas the 2.4-kb mRNA species was predominant only in the olfactory mucosa. Immunoblot analysis of microsomal fractions from different rat tissues with a polyclonal anti-peptide antibody to CYP2J4 detected a protein with the same electrophoretic mobility as purified CYP2J4 most abundantly in small intestine and to a lesser extent in liver and other immunoreactive proteins with slightly higher electrophoretic mobility than purified CYP2J4 in a number of tissues, including small intestine, liver, kidney, lung, and olfactory mucosa. The predominant distribution of CYP2J4, which has activity toward arachidonic acid, is provocative, but its physiological function is as yet unknown.

Amino Acid Sequence↗
PubMed1997

Human P450 metabolism of warfarin.

The anticoagulant drug warfarin occurs as a pair of enantiomers that are differentially metabolized by human cytochromes P450 (CYP). R-warfarin is metabolized primarily by CYP1A2 to 6- and 8-hydroxywarfarin, by CYP3A4 to 10-hydroxywarfarin, and by carbonyl reductases to diastereoisomeric alcohols. S-warfarin is metabolized primarily by CYP2C9 to 7-hydroxywarfarin. Potential warfarin-drug interactions could occur with any of a very wide range of drugs that are metabolized by these P450s, and a number of such interactions have been reported. The efficacy of warfarin is affected primarily when metabolism of S-warfarin is altered.

Catalysis↗
PubMed1997

The molecular epidemiology of lung cancer.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation; tissue source, race, statistical power, and ethical implications are addressed.

Biomarkers, Tumor↗
PubMed1996

Characterization of purified human recombinant cytochrome P4501A1-Ile462 and -Val462: assessment of a role for the rare allele in carcinogenesis.

Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val. The rare allele has been associated with enhanced susceptibility to lung cancer. To resolve its role in cancer we have constructed CYP1A1-Val462 cDNA by site-directed mutagenesis from CYP1A1-Ile462, as confirmed by sequencing and allele-specific PCR. Both alleles were expressed in Escherichia coli, and CYP1A1-Ile462 and -Val462 were purified to electrophoretic homogeneity. The secondary structures of both forms were virtually identical, with high alpha helix content, as assessed by circular dichroism. The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)- warfarin, in reconstituted systems, with very similar profiles. Both P450s produced (R)-6- and 8-hydroxy-warfarin with Km values of 0.40 +/- 0.06 and 0.43 +/- 0.05 mM, respectively, and Vmax values of 84.0 +/- 6.8 and 137.7 +/- 8.9 pmol/min/nmol CYP1A1-Val462, respectively, 1.0 +/- 0.1 and 1.0 +/- 0.1 mM, respectively, and 46.7 +/- 2.5 and 80.0 +/- 4.4 pmol/min/nmol CYP1A1-Ile462, respectively. Reconstituted CYP1A1-Val462 catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile462; Vmax values were 4.4 +/- 0.6 and 3.1 +/- 0.3 nmol/min/nmol CYP1A1, respectively. However, with the carcinogen benzo(a) pyrene as substrate, reconstituted CYP1A1-Ile462 together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val462. Thus, the apparently greater susceptibility of the CYP1A1-Val462 genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation.

Alleles↗
PubMed1996

The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism.

Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway. The primary P450 isozyme responsible for this metabolism is thought to be CYP2C9. Population studies have indicated the existence of slow metabolizers of tolbutamide (approximately 1 in 500) suggesting a rare polymorphism associated with 2C9. Several allelic variants of 2C9 have been identified; however, the effect of these allelic variations on metabolism in vivo is not established. In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers. One individual was homozygous for Leu359/Leu359 and the other individual was heterozygous for Arg144/Cys144 and for Ile359/Leu359. No other genetic variations in 2C9 were detected in these individuals. PCR-RFLP tests showed that Arg144 Tyr358 Ile359 Gly417 is the principle CYP2C9 allele. Frequencies of the rarer Leu359 and Cys144 alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and 0.005 and 0.01 respectively in African-Americans. The frequency of the Leu359 allele was 0.026 in Chinese-Taiwanese, but the Cys144 allele was not detected in this population. Studies in a recombinant yeast expression system showed that the Leu359 variant had the highest Km and the lowest Vmac for hydroxylation of tolbutamide of all the CYP2C9 allelic variants. This allelic variant also had the highest Km for the 7-hydroxylation of S-warfarin. The present data suggest that the incidence of the Leu359 allelic variant of CYP2C9 may account for the occurrence of poor metabolizers of tolbutamide.

Alleles↗
PubMed1996

Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.

In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.

Base Sequence↗
PubMed1996

Expression of cytochromes P450 in human breast tissue and tumors.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Breast↗
PubMed1995

Characterization of human cytochromes P450 involved in theophylline 8-hydroxylation.

Studies were undertaken to determine which human P450 enzymes catalyze the metabolism of theophylline to 1,3-dimethyluric acid (1,3-DU), to facilitate predictions of theophylline drug-drug interactions, and to develop a noninvasive test for human P4501A2. Microsomes from a human cell line transfected individually with human P450 cDNAs for P4501A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1, or 3A4 were used to demonstrate that only P4501A2 exhibited catalytic activity for theophylline metabolism to 1,3-DU with high affinity and low capacity (Km = 0.6 mM, Vmax = 37.8, pmol/min/mg), while P4502D6, 2E1, and 3A4 (Km = 14.4, 19.9, and 25.1 mM, respectively, and Vmax = 219.8, 646.4, and 20.8 pmol/min/mg, respectively) exhibited activities with low affinity and variable capacities. Correlations of rates of theophylline 8-hydroxylation to 1,3-DU with other P450 form-specific activities, in a series of ten human liver microsomal preparations, at 5 and 40 mM theophylline concentrations, revealed that at low concentrations the metabolism was catalyzed primarily by P4501A2, while at high substrate concentrations P4502E1 was primarily responsible for catalysis. The results with individually expressed P450s and hepatic microsomal preparations were consistent, indicating that the former system provides a qualitatively accurate reflection of the function of the heterogeneously expressed liver P450s. At pharmacologic theophylline concentrations achieved in vivo, its metabolism must thus be catalyzed primarily by P4501A2.

Cell Line↗
PubMed1995

Determination of theophylline and its metabolites in rat liver microsomes and human urine by capillary electrophoresis.

A capillary electrophoretic (CE) method has been developed for the determination of theophylline and all of its identified and potential metabolites. The method is rapid, resolves all metabolites to baseline, and requires extraction of only some biological fluids. It has been applied to the analysis of theophylline metabolism by hepatic microsomes from rats treated with a variety of inducing agents for different forms of P450 enzymes which metabolize theophylline, and to human urine spiked with theophylline and its metabolites, and concentrated by solid-phase extraction.

Animals↗
PubMed1995

Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence.

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Base Sequence↗
PubMed1995

Human cytochromes P4501A1 and P4501A2: R-warfarin metabolism as a probe.

Two forms of the cytochrome P450 enzyme superfamily, P4501A1 and P4501A2, that are heterogeneously distributed in populations and are induced in response to environmental factors are important because of their capacity to bioactivate procarcinogens. Phenotyping P4501A1 and P4501A2 in individuals will thus provide assessments of those individuals' susceptibility to procarcinogens. The anticoagulant drug warfarin is metabolized by human P4501A1 and P4501A2, and we have characterized this metabolism for the R-warfarin enantiomer as a potential in vivo probe. cDNA-expressed human P4501A1 and P4501A2 are regioselective for R-warfarin 6- and 8-hydroxylation with very similar KM values: 1.4 mM (6-hydroxylation), 1.2 mM (8-hydroxylation), 1.6 mM (6-hydroxylation), and 1.4 mM (8-hydroxylation), respectively, indicating possible binding competition for R-warfarin between the two forms. However, when comparing 6- and 8-hydroxylation, P4501A1 showed weak regioselectivity for 8-hydroxylation, whereas P4501A2 exhibited strong regioselectivity for 6-hydroxylation, with 6-hydroxylation/8-hydroxylation ratios of 0.6 and 5.0, respectively. These findings were confirmed by using microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated HepG2 and MCF-7 cells expressing only P4501A1 (ratios of 0.7), and from human hepatic microsomal preparations containing only P4501A2 (average ratios of 4.0). P4501A2 levels in the liver preparations, as assessed by densitometry of immunoblots, correlated with R-warfarin 6-hydroxylation rates (r2 = 0.83) and caffeine 3-demethylation rates (r2 = 0.67), but not with R-warfarin 8-hydroxylation rates. P450s 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4 did not yield either 6- or 8-hydroxy-warfarin from R-warfarin. We conclude that R-warfarin 6-hydroxylation rates are markers for human hepatic P4501A2, whereas ratios of 6-hydroxylation/8-hydroxylation could be used in vitro as a marker for P4501A1.

Base Sequence↗