Warfarin metabolites: Stereochemical aspects of protein binding and displacement by phenylbutazone

The in vitro human serum albumin binding characteristics of the enantiomers of the major metabolites of warfarin [6-hydroxywarfarin (6-HW), 7-hydroxywarfarin (7-HW), (S)-warfarin alcohols [(S,S)- and (S,R)-WA], and (R,S)-warfarin alcohol [(R,S)-WA]] have been studied, using a stereospecific HPLC assay. Warfarin metabolites are less bound both within plasma and a 40 g/liter solution of human serum albumin than the enantiomers of warfarin. The reduced warfarin metabolites have a lower fraction unbound [1.33% for (S,R)-WA, 2.09% for (S,S)-WA, and 1.04% for (R,S)-WA] than hydroxylated metabolites [3.24% for (R)-6-HW, 4.26% (S)-6-HW, 4.49% for (R)-7-HW and 4.27% for (S)-7-HW] to HSA. Phenylbutazone produced a concentration-dependent increase in the unbound fraction of all metabolites. It was possible to predict the unbound fraction of warfarin metabolites based on the unbound fraction of warfarin enantiomers. © 1993 Wiley-Liss, Inc.     

Analysis of warfarin enantiomers: Chromatographic separation of six stereoisomeric esters prepared from (−)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid

Reaction of rac-warfarin, (?)-(1S,2R,4R)-endo-1,4,5,6,7,7-hexachlorobicyclo[2.2.1]hept-5-ene-2- carboxylic acid [(?)-HCA] and carbodiimide reagents gave two noncyclic ketonic diastereoisomeric derivatives whereas rac-warfarin and (?)-HCA acid chloride with 4-(dimethylamino)pyridine gave four cyclic hemiketal diastereoisomeric ester derivatives. The structure and stereochemistry of diastereoisomeric esters prepared from warfarin and p-chlorowarfarin were determined from ¹H- and ¹³C-NMR spectra, mass spectra, and hydrolysis to warfarin and p-chlorowarfarin enantiomers. The structure and stereochemistry of one of the cyclic hemiketal diastereoisomeric derivatives of warfarin are supported by an X-ray crystallographic determination. Mechanisms for the formation of all products are proposed. © 1994 Wiley-Liss, Inc.     

Metabolic enantiomeric interactions: the inhibition of human (S)-warfarin-7-hydroxylase by (R)-warfarin

Inhibition of the metabolism of (S)-warfarin, the more pharmacologically active enantiomer of the racemic drug, by (R)-warfarin was investigated in microsomes obtained from three human livers. In each case the production of both (S)-6- and (S)-7-hydroxywarfarin was found to be competitively inhibited by (R)-warfarin. The KiS for inhibition of (S)-6- and (S)-7-hydroxylation by (R)-warfarin ranged from 7.0 to 8.4 microM and from 6.0 to 6.9 microM, respectively, while the KmS for the 6- and 7-hydroxylation of (S)-warfarin ranged from 3.6 to 3.8 microM and from 3.3 to 3.9 microM, respectively. In contrast, except for the 4'-hydroxylation pathway (S)-warfarin was found to be a weak inhibitor of the metabolism of (R)-warfarin. Possible implications of these findings include the following: (1) the kinetic parameters defining the interactions of two enantiomers of a racemic drug with the cytochrome P-450s or other macromolecular systems in the living organism can only be properly defined from experiments with the pure enantiomers, (2) an enantiomer of a racemic drug may contribute significantly to biological effect not by its inherent activity but by altering the pharmacokinetics of the eutomer, and (3) enantiomeric interactions are not easily detected unless directly sought and may be relatively common.     

Measurement of the (R)- and (S)-isomers of warfarin in patients undergoing anticoagulant therapy.

An achiral/chiral high-performance liquid chromatographic system for the analysis of total warfarin together with the (R)- and (S)-enantiomers in clinical samples has been developed. The achiral analysis is achieved using a C8 column, which is coupled to a chiral stationary phase, alpha 1-acid glycoprotein (AGP), thereby allowing for analysis of warfarin isomers without interfering serum peaks. A 0.015 M phosphate buffer mobile phase with 15% v/v propan-2-ol (pH 7.0) was used on the C8/AGP system. UV analysis at 308 nm was used for quantitation of total warfarin on the C8 column and fluorescence (excitation 300 nm, emission 390 nm) detection was employed for isomer quantitation on the AGP. Retention time of total warfarin on the C8 column was 5.95 min, while that of the (S)- and (R)-warfarin on the AGP column was 10.38 and 12.69 min, respectively. Peak resolution of the warfarin isomers was 1.64. All serum samples were subjected to solid-phase extraction. Data from two patients in a single dose study indicate that a two-compartmental model could represent the warfarin concentration-time data with enterohepatic circulation. In some patients studied during steady state therapy, concentrations of (S)-warfarin were greater than (R)-warfarin indicating that the clearance of the former is slower in these patients.     

Ginkgo biloba extract attenuates warfarin-mediated anticoagulation through induction of hepatic cytochrome P450 enzymes by bilobalide in mice

Ginkgo biloba extract (GBE) is a popular herbal ingredient used worldwide, but it is reported to induce bleeding as a serious adverse event. In this study we examined whether GBE induced spontaneous bleeding or accelerated warfarin anticoagulation via herb-drug interaction. Mice were given GBE or various active components of GBE orally for 5 days and blood coagulation parameters and hepatic cytochrome P450 enzymes (CYPs) were measured. Mice also received warfarin (racemate, (S)- or (R)-enantiomer) for the last 3 days of the 5-day regimen to examine GBE-warfarin interactions. Neither GBE (up to 1000 mg/kg) nor ginkgolide B (up to 140 mg/kg), a platelet-activating factor antagonist, influenced blood coagulation parameters. In contrast, GBE attenuated the anticoagulant action of warfarin. Bilobalide, a component of GBE that markedly induced hepatic CYPs including (S)-warfarin hydroxylase, showed similar effects. For (S)-warfarin, the anticoagulation action and the interaction with GBE was clear, while the influence on metabolism was greater for (R)-warfarin than for (S)-warfarin, which corresponded to the CYP types induced by GBE. These results suggest that GBE and ginkgolide B have no influence on blood coagulation in vivo, and that GBE attenuates the anticoagulation action of warfarin via induction of hepatic CYPs by bilobalide.     

Highly sensitive and specific high-performance liquid chromatographic analysis of 7-hydroxywarfarin, a marker for human cytochrome P-4502C9 activity

The formation of 7-hydroxywarfarin in incubations of (S)-warfarin with human liver microsomes reflects their cytochrome P-4502C9 activity. This paper describes a rapid high-performance liquid chromatographic method for the determination of 7-hydroxywarfarin with high sensitivity, selectivity, and a simple sample clean-up procedure. Separation was achieved with a C₁₈ reversed-phase column and quantification by fluorometric detection. The method employs an internal standard resulting in good accuracy and precision. The limit of detection is 150 fmol for 7-hydroxywarfarin.     

Structural forms of phenprocoumon and warfarin that are metabolized at the active site of CYP2C9

Possible reasons for the observed differences in metabolic behavior and drug interaction liability between the structurally similar oral anticoagulants warfarin and phenprocoumon were explored. Incubating (S)-phenprocoumon with human liver microsomes and cDNA-expressed CYP2C9 and determining its metabolism both in the absence and presence of the CYP2C9 inhibitor, sulfaphenazole, confirmed that phenprocoumon is a substrate for CYP2C9. Comparing the metabolic behavior of (S)- and (R)-warfarin, (S)- and (R)-phenprocoumon, and fixed structural mimics of the various tautomeric forms [(S)- and (R)-4-methoxyphenprocoumon, (S)- and (R)-2-methoxyphenprocoumon, (S)- and (R)-4-methoxywarfarin, (S)- and (R)-2-methoxywarfarin, and 9(S)- and 9(R)-cyclocoumarol] available to these two drugs with expressed CYP2C9 provides compelling evidence indicating that the ring closed form of (S)-warfarin and the ring opened anionic form of (S)-phenprocoumon are the major and specific structural forms of the two drugs that interact with the active site of CYP2C9. The conclusion that (S)-warfarin and (S)-phenprocoumon interact with CYP2C9 in very different structural states provides a clear basis for the significant differences observed in their metabolic profiles. Moreover, in accord with a previously established CoMFA model these results are consistent with the hypothesis that the active site of CYP2C9 possesses at least two major substrate binding sites, a pi-stacking site for aromatic rings and an ionic binding site for organic anions. An additional electrostatic binding site also appears to contribute to the orientation of coumarin analogs in the CYP2C9 active site by interacting with the C2-carbonyl group of the coumarin nucleus.     

Stereochemistry of the microsomal glutathione S-transferase catalyzed addition of glutathione to chlorotrifluoroethene

The stereochemistry of S-(2-chloro-1,1,2-trifluoroethyl)glutathione formation was studied in rat liver cytosol, microsomes, N-ethylmaleimide-treated microsomes, 9000g supernatant fractions, purified rat liver microsomal glutathione S-transferase, and isolated rat hepatocytes. The absolute configuration of the chiral center generated by the addition of glutathione to chlorotrifluoroethene was determined by degradation of S-(2-chloro-1,1,2-trifluoroethyl)glutathione to chlorofluoroacetic acid, followed by derivatization to form the diastereomeric amides N-(S)-alpha-methylbenzyl-(S)-chlorofluoacetamide and N-(S)-alpha-methylbenzyl-(R)-chlorofluoroacetamide, which were separated by gas chromatography. Native and N-ethylmaleimide-treated rat liver microsomes, purified rat liver microsomal glutathione S-transferase, rat liver 9000g supernatant, and isolated rat hepatocytes catalyzed the formation of 75-81% (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione; rat liver cytosol catalyzed the formation of equal amounts of (2R)- and (2S)-S-(2-chloro-1,1,2-trifluoroethyl)glutathione. In rat hepatocytes, microsomal glutathione S-transferase catalyzed the formation of 83% of the total S-(2-chloro-1,1,2-trifluoroethyl)glutathione formed. These observations show that the microsomal glutathione S-transferase catalyzes the first step in the intracellular, glutathione-dependent bioactivation of the nephrotoxin chlorotrifluoroethene.     

Site I on human albumin: differences in the binding of (R)- and (S)-warfarin.

The binding of drugs known to interact with area I on human serum albumin (HSA) was investigated using a chiral stationary phase obtained by anchoring HSA to a silica matrix. In particular, this high-pressure affinity chromatography selector was employed to study the binding properties of the individual enantiomers of warfarin. The pH and composition of the mobile phase modulate the enantioselective binding of warfarin. Displacement chromatography experiments evidenced significant differences in the binding of the warfarin enantiomers to site I. The (S)-enantiomer was shown to be a direct competitor for (R)-warfarin, while (R)-warfarin was an indirect competitor for the (S)-enantiomer. Salicylate directly competed with (R)-warfarin and indirectly with (S)-warfarin. This behavior was confirmed by difference CD experiments, carried out with the same [HSA]/[drug] system in solution.     

Determination of warfarin enantiomers and hydroxylated metabolites in human blood plasma by liquid chromatography with achiral and chiral separation

An assay comprising two simple, selective and isocratic HPLC methods with UV detection was developed and validated for measuring warfarin enantiomers and all five warfarin monohydroxylated metabolites in patient blood plasma. Following liquid/liquid extraction from 1 ml of blood plasma a baseline separation of analytes was achieved on chiral (alpha(1) acid glycoprotein - AGP) and achiral (C(18)) column. Both methods were consistent (R.S.D.<6.9% for warfarin enantiomers and<8.9% for monohydroxylated metabolites) and linear (r>0.998). The limits of detection were 25 ng/ml for warfarin enantiomers, 25 ng/ml for 4'-, 10-, 6- and 7-hydroxywarfarin, 35 ng/ml for 8-hydroxywarfarin and 50 ng/ml for racemic warfarin. In a clinical study in 204 patients, it was confirmed that the assay is appropriate for evaluation of influences of genetic polymorphisms, demographic factors and concomitant drug treatment on warfarin metabolism.     

The proximate carcinogen trans-3,4-dihydroxy-3,4-dihydro-dibenz[c,h]acridine is oxidized stereoselectively and regioselectively by cytochrome 1A1, epoxide hydrolase and hepatic microsomes from 3-methylcholanthrene-treated rats.

Metabolism of the proximate carcinogen trans-3,4-dihydroxy-3,4-dihydrodibenz[c,h]acridine has been examined with rat liver enzymes. The dihydrodiol is metabolized at a rate of 2.4 nmol/nmol of cytochrome P450 1A1/min with microsomes from 3-methylcholanthrene-treated rats, a rate more than 10-fold higher than that observed with microsomes from control or phenobarbital-treated rats. Major metabolises consisted of a diastereomeric pair of bis-dihydrodiols (68-83%), where the new dihydrodiol group has been introduced at the 8,9-position, tetraols derived from bay region 3,4-diol-1,2-epoxides (15-23%), and a small amount of a phenolic dihydrodiol(s) where the new hydroxy group is at the 8,9-position of the substrate. A highly purified monooxygenase system reconstituted with cytochrome P450 1A1 and epoxide hydrolase (17 nmol of metabolites/nmol of cytochrome P450 1A1/min) gave a metabolite profile very similar to that observed with liver microsomes from 3-methylcholanthrene-treated rats. Study of the stereoselectivity of these microsomes established that the (+)-(3S,4S)-dihydrodiol gave mainly the diol epoxide-1 diastereomer, in which the benzylic 4-hydroxyl group and epoxide oxygen are cis. The (-)-(3R,4R)-dihydrodiol gave mainly diol epoxide-2 where these same groups are trans. The major enantiomers of the diastereomeric bis-dihydrodiols are shown to have the same absolute configuration at the 8,9-position. Correlations of circular dichroism spectra suggest this configuration to be (8R,9R). The (8R,9S)-oxide may be their common precursor.     

Stereochemical studies of chiral resolving agents, M9PP and H9PP acids

The stereoselective Grignard reaction of (1R,2S,5R)-(-)-2-isopropyl-5-methylcyclohexyl pyruvate (menthyl pyruvate) with 9-phenanthrylmagnesium bromide yielded diastereomeric hydroxy-esters, where intramolecular OH em leader O=C hydrogen bond was observed in IR and (1)H NMR spectra. The alkaline hydrolysis of the major product gave (+)-2-hydroxy-2-(9-phenanthryl)propionic acid (H9PP acid (3)), whose absolute configuration was assigned as S based on the chemical correlation with (1R,2S,5R)-2-isopropyl-5-methylcyclohexyl ester of (S)-2-methoxy-2-(9-phenanthryl)propionic acid (M9PP acid (2)); the absolute configuration of 2 had been previously established by X-ray crystallography. The enantioresolution of (+/-)-6-methyl-5-hepten-2-ol, sulcatol, an insect pheromone, was carried out using (S)-(+)-M9PP acid 2.     

Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin

The basis for our previous observations [Kaminsky, L.S., Guengerich, F.P., Dannan, G.A. & Aust, S.D. (1983) Arch. Biochem. Biophys. 225, 398-404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F greater than P-450PB-D greater than or equal to P-450UT-A greater than or equal to P-450BNF/ISF-G greater than P-450PB/PCN-E greater than P-450PB-B greater than or equal to P-450PB-C greater than or equal to P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations.     

Eggs of the sea urchin, Strongylocentrotus purpuratus, contain a prominent (11R) and (12R) lipoxygenase activity

Recent work has shown that oocytes of the starfish synthesize (8R)-hydroxyeicosatetraenoic acid and that this eicosanoid has a potent and highly specific action in induction of oocyte maturation. These striking results prompted us to examine the lipoxygenase activity of eggs of the sea urchin Strongylocentrotus purpuratus. Four hydroxyeicosanoids were formed in homogenates of sea urchin eggs; their structures and stereochemistry were characterized by high pressure liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as (11R)-hydroxy-5,8,12,14-ZZEZ-eicosatetraenoic acid and (12R)-hydroxy-5,8,10,14-ZZEZ-eicosatetraenoic acid (from arachidonic acid) and the corresponding (11R)- and (12R)-hydroxy analogs of eicosapentaenoic acid. The formation of these egg products was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), and their precise structures are consistent with their formation by a lipoxygenase reaction. Eicosapentaenoic acids with a prochiral tritium label in the 10-D or 10-L position were used to investigate the mechanism of biosynthesis. The formation of (12R)-hydroxyeicosapentaenoic acid proceeded with the stereoselective abstraction of the 10-D hydrogen from the substrate. This reaction was shown to be opposite to the (12S) oxygenation catalyzed by porcine leukocyte 12-lipoxygenase. These results with S. purpuratus eggs constitute the first demonstration of (11R)- or (12R)-lipoxygenase activity in any cell type or tissue.     

Enzymatic determinants of the substrate specificity of CYP2C9: role of B'-C loop residues in providing the pi-stacking anchor site for warfarin binding

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.     

Stereoselective kinetics of warfarin binding to human serum albumin: effect of an allosteric interaction

Kinetic and equilibrium binding studies were performed on the interaction of warfarin enantiomers with human serum albumin (HSA) in the absence and presence of lorazepam acetate (LoAc) enantiomers. Binding kinetics were followed by recording changes in the fluorescence of warfarin upon binding to HSA using the stopped-flow technique. The binding of (R)-warfarin displayed an exponentially increasing fluorescence, satisfying the two-step mechanism reported previously for the racemate, i.e., a diffusion controlled pre-equilibrium is followed by a slower rearrangement of the complex. In the case of (S)-warfarin, the signal was biphasic: a fast fluorescence enhancement was followed by a slow decline. The different kinetic features indicate that the equilibrium conformations of the [(S)-warfarin-HSA] and [(R)-warfarin-HSA] complexes are achieved via different mechanisms. The phenomenon was seen in buffers of different pH and compositions. Equilibrium binding measurements indicated significantly lower molar intrinsic fluorescence for the (S)-warfarin complex, suggesting differences in the microenvironments of the bound enantiomers. In the presence of (S)-LoAc, the allosterically enhanced binding of (S)-warfarin manifested itself in accelerated relaxation kinetics. In accordance with the low molar intrinsic fluorescence determined for the stable ternary complex, the amplitude of the decline in fluorescence became larger.     

Absolute configuration of a chiral CHD group via neutron diffraction: confirmation of the absolute stereochemistry of the enzymatic formation of malic acid

Neutron diffraction has been used to monitor the absolute stereochemistry of an enzymatic reaction. (-)(2S)malic-3-d acid was prepared by the action of fumarase on fumaric acid in D2O. After a large number of cations were screened, it was found that (+)(R) alpha-phenylethylamine forms the large crystals necessary for a neutron diffraction analysis. The subsequent structure determination showed that (+)(R) alpha-phenylethylammonium (-)(2S)malate-3-d has an absolute configuration of R at the CHD site (i.e., the C3 carbon of malate). This result confirms the absolute stereochemistry of fumarate-to-malate transformation as catalyzed by the enzyme fumarase.     

ω-Hydroxymodin, a major hepatic metabolite of emodin in various animals and its mutagenic activity

The hepatic microsomes derived from various animal species transformed emodin (1,3,8-trihydroxy-6-methylanthraquinone), and anthraquinoid pigment present in fungal metabolites and a constituent of plant medicines, into an unidentified anthraquinone h, along with 2-hydroxy-, 4-hydroxy- and 7-hydroxyemodins. TLC, UV, MS and NMR clarified this unidentified major metabolite as ω-hydroxy-emodin (1,3,8-trihydroxy-6-hydroxymethylanthraquinone). Among 7 animal species, the highest activity to produce this ω-hydroxyemodin was observed in the hepatic microsomes of guinea pig and rat, followed by mouse and rabbit. The microsomal activity to convert emodin into ω-hydroxyemodin was accelerated by the pretreatment of animals with phenobarbital, and inhibited by SKF 525A. The microsomal hydroxylation reactions of the methyl residue and the anthraquinoid nucleus of emodin were presumed to be catalyzed regiospecifically by multiple forms of cytochrome P-450.

ω-Hydroxyemodin was not mutagenic to Salmonella typhimurium in the absence of S9, but exhibited mutagenicity in the presence of an activating system. This genotoxic potential was comparable to 2-hydroxyemodin, a direct-acting mutagen.     

Stereochemistry of the epoxidation of fatty acids catalyzed by soybean peroxygenase

The stereochemistry of C18 unsaturated fatty acids epoxidation catalyzed by detergent-solubilized and partially purified soybean peroxygenase was determined by chiral phase HPLC. Linoleic acid was oxidized into 9, 10- and 12,13-cis-epoxyoctadecenoic acids with a high enantiofacial selectivity. A 5.2:1 and 2.3:1 ratio respectively in favor of the 9(R), 10(S)- and 12(R), 13(S)-epoxy enantiomers was observed. These epoxy-derivatives of linoleic acid have the chirality of metabolites known to be involved in plant defense against fungi. This finding is of importance in establishing a physiological role for the peroxygenase.     

omega-Hydroxylation of epoxy- and hydroxy-fatty acids by CYP94A1: possible involvement in plant defence

The C(18) fatty acid derivatives 9,10-epoxystearic acid and 9,10-dihydroxystearic acid were hydroxylated on the terminal methyl by microsomes of yeast expressing CYP94A1 cloned from Vicia sativa. The reactions did not occur in incubations of microsomes from yeast transformed with a void plasmid or in the absence of NADPH. After incubation of a synthetic racemic mixture of 9,10-epoxystearic acid, the chirality of the residual epoxide was shifted to 66:34 in favour of the 9S,10R enantiomer. Both the 9S,10R and 9R,10S enantiomers were incubated separately. We determined respective K(m) and V(max) values of 1.2+/-0.1 microM and 19.2+/-0.3 nmol/min per nmol of cytochrome P450 for the 9R,10S enantiomer and of 5.9+/-0.1 microM and 20.2+/-1.0 nmol/min per nmol of cytochrome P450 for the 9S,10R enantiomer. This demonstrated that CYP94A1 is enantioselective for the 9R,10S, which is preferentially formed in V. sativa microsomes. Cutin analysis of V. sativa seedlings revealed that it is mainly constituted of derivatives of palmitic acid, a C(16) fatty acid. Our results suggest that CYP94A1 might play a minor role in cutin synthesis and could be involved in plant defence. Indeed, 18-hydroxy-9,10-epoxystearic acid and 9,10,18-trihydroxystearic acid have been described as potential messengers in plant-pathogen interactions.