Cocaine decreases the expression of PSA-NCAM protein and attenuates long-term potentiation via glucocorticoid receptors in the rat dentate gyrus.

The present study investigated a potential role for glucocorticoid (GR) and mineralocorticoid (MR) receptors in the detrimental effects of single cocaine (COC) administration on both the number of polysialylated neural cell adhesion molecule (PSA-NCAM)-positive neurons and the induction of long-term potentiation (LTP) in the rat dentate gyrus (DG). The effects of COC (15 mg/kg i.p.) on the number of PSA-NCAM-positive neurons and the induction of LTP observed 2 days after COC administration were abolished either by depleting circulating corticosterone after administration of metyrapone (100 mg/kg s.c. given 3 h before COC) or by pharmacologically blocking GRs using mifepristone (RU 38486, 10 mg/kg s.c. given 1 h before COC). Administration of the MR blocker spironolactone (50 mg/kg s.c. given 1 h before COC) did not alter the effects of COC on the number of PSA-NCAM-positive neurons or LTP induction. Results have also shown that COC does not change the rate of cell proliferation, as measured by the presence of Ki-67 and the incorporation of bromodeoxyuridine (100 mg/kg i.p. given 2 h after COC) into the newly born cells in the DG 2 days after COC administration. Finally, we observed that GRs colocalized with some, but not all, PSA-NCAM-positive neurons, whereas MRs showed no colocalization with neurons positive for PSA-NCAM in the DG. These data indicate that a single dose of COC may arrest hippocampal susceptibility to plastic changes and lead to functional impairments through the alteration of hippocampal structure and the formation of memory traces.

Oral pharmacokinetics and in-vitro metabolism of metyrapone in male rats.

OBJECTIVES: The purpose of this study was to investigate the pharmacokinetics of a single oral administration of metyrapone (MP) and metabolites produced from it in male Wistar rats, and the major tissues and enzymes involved in the production of the MP metabolites. Furthermore, the MP metabolism in human liver subcellular fractions was compared with that in rats. METHODS: High-performance liquid chromatography with ultraviolet detection (HPLC-UV) was used to determine the concentrations of MP and its metabolites in plasma and urine after administration, and the production activity of MP metabolites in subcellular fractions of various tissues. KEY FINDINGS: Plasma concentration of MP was rapidly increased and decreased, and the primary metabolite, metyrapol (MPOL), was immediately produced. The production activity of MPOL was substantially inhibited by an 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitor in the rat and human liver microsomal and mitochondrial fractions. In the liver cytosolic fraction, the activity was inhibited by a carbonyl reductase inhibitor in the humans but not rats. CONCLUSIONS: In this study, we elucidated the plasma pharmacokinetics of MP and its metabolites in male rats after an oral administration. MPOL is most likely to be produced by 11ß-HSD1 in the male rats and humans.

Structure-activity relationship study of the inhibition of adrenal cortical 11 beta-hydroxylase by new metyrapone analogues.

Metyrapone, 2-methyl-1,2-di-3-pyridyl-1-propanone (1a), is a potent reversible inhibitor of the cytochrome P-450 11 beta-hydroxylase enzyme system (P-450(11) beta) of the adrenal cortex. The structural features of the metyrapone molecule have been systemically altered to determine the requirements necessary for inhibition of P-450(11) beta activity. Metyrapone and 14 analogues have been obtained or synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. The inhibition of P-450(11) beta activity with these derivatives demonstrated that (1) the A-ring phenyl derivatives 2a-d were better inhibitors than the respective dipyridyl analogues, (2) the ketone in the 1-position can be replaced by various functionalities without markedly reducing inhibition, and (3) at least one methyl group should be present in the 2-position to maintain inhibition. The observed inhibition of P450(11) beta activity with the metyrapone analogues suggest that A-ring phenyl metyrapone analogues 2a-d would be candidates for radioiodination and subsequently used as adrenal cortical imaging agents.

New inhibitors of steroid 11beta-hydroxylase. Structure--activity relationship studies of metyrapone-like compounds.

A series of metyrapone analogues was synthesized for study as inhibitors of steroid 11beta-hydroxylase. Racemic mixtures of the new compounds were evaluated in vitro. Preliminary results revealed several analogues to be effective inhibitors of deoxycorticosterone hydroxylation. 2-(3-pyridyl)propiophenone (13) and alpha,beta-diphenyl-3-pyridineethanol (16) were the most active new compounds. Each was 65% as potent as metyrapone; 3-Pyridyl alpha-3-pyridylbenzyl ketone (3), 2-phenyl-2-(3-pyridyl)acetophenone (4), alpha-(diphenylmethyl)-3-pyridinemethanol (17), and 1,2-di-3-pyridyl-1-propanol (26) were 52, 32, 25, and 41% as inhibitory as metyrapone, respectively. Diphenylmethyl 3-pyridyl ketone (5), benzyl 3-pyridyl ketone (10), 2-(3-pyridyl)acetophenone (12), 2-phenyl-1-(3-pyridyl)-1-propanone (11), alpha,beta-di-3-pyridylphenethyl alcohol (15), and 1,2-di-3-pyridylethanol (27) had less than 25% the activity of metyrapone. All compounds displaying a metyrapone-like inhibition contained appropriately substituted alcoholic or ketonic functions. A phenyl or methyl group alpha to the carbon bearing the oxygen was necessary for appreciable activity. A 3-phridyl group alpha to the carbonyl carbon could be replaced by a phenyl group. For optimal activity, however, the other 3-pyridyl group of metyrapone could not be exchanged for a phenyl group.

Characterization of carbonyl reducing activity in continuous cell lines of human and rodent origin.

Carbonyl reduction was investigated in the continuous cell lines V79, NCI-H322 and C2REV7 by using the ketone compound metyrapone as a substrate. Metyrapone reducing enzymes were characterized by evaluating the cosubstrate requirement and by testing the sensitivity of this reaction to specific inhibitors. All cell lines were found to produce metyrapol at a linear rate over a time course of at least 48 hr, when tested in cultured monolayers. In general, cytosolic metyrapone reduction exceeds microsomal activity several-fold in all three cell lines. Quercitrin turned out to be the strongest inhibitor in all fractions, except in NCI-H322 microsomes where it had no effect. Consequently, carbonyl reductase is suspected to be responsible for metyrapone reduction in the cytosol and microsomes of V79 and C2REV7 cells as well as in the cytosol of NCI-H322 cells. Simultaneous sensitivity towards quercitrin, dicoumarol, indomethacin and 5 alpha-dihydrotestosterone in some cases points to the existence of different isozymes of carbonyl reductase. In NCI-H322 microsomes only dicoumarol and indomethacin decrease metyrapol formation, thus pointing to an isozyme of NAD(P)H:quinone-oxidoreductase. Concerning cosubstrate requirements metyrapone reducing enzymes show a strong preference for NADPH, thus confirming the involvement of carbonyl reductase in this reaction. In conclusion, carbonyl reduction of metyrapone in continuous cell lines is mediated by carbonyl reductases due to the common sensitivity towards the diagnostic inhibitor quercitrin and due to the strong preference for NADPH as cosubstrate. According to its maintenance in permanent cell lines carbonyl reductase seems to be an essential and constitutive enzyme, which probably fills an important role in normal cell physiology.

Carbonyl reduction of metyrapone in human liver.

Carbonyl reduction was investigated in cytosolic and microsomal fractions of human liver using the ketone metyrapone as a substrate. The cytosolic enzyme has a stronger preference for NADPH over NADH than the microsomal enzyme: the former shows only 14% of the NADPH-supported activity while the latter exhibits 36% activity with NADH. Barbitone and quercitrin, the classic inhibitors of carbonyl reductases, do not affect metyrapone reduction in either fraction. Dicumarol and indomethacin, the specific inhibitors of NAD(P)H: quinone-oxidoreductase and dihydrodiol dehydrogenase, respectively, only slightly decreased metyrapol formation. In contrast, 5 alpha-dihydrotestosterone, the active form of the androgen steroid testosterone, inhibited metyrapone reduction very strongly in the microsomal fractions and is postulated to be the physiological substrate of the enzyme. This resembles the situation in mouse liver [E. Maser and K. J. Netter, Biochem Pharmacol 38: 3049-3054, 1989] where microsomal metyrapone reductase was inhibited by steroids and the purified enzyme was demonstrated to mediate androsterone oxidation. Immunoblot analysis revealed antigenic cross-reaction of antibodies against the 34 kDa metyrapone reductase from mouse liver microsomes with the homologous protein in human liver microsomes pointing to structural homologies between the respective enzymes of the two species. These results--together with previous findings, which have shown that there exist functional as well as structural relationships between microsomal mouse liver metyrapone reductase and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni [E. Maser, U. Oppermann and K. J. Netter, Eur J Pharmacol 183:1366, 1990]--suggest that metyrapone reduction in human liver microsomes might be catalysed by a microsomal hydroxysteroid dehydrogenase.

Reductive metabolism of metyrapone by a quercitrin-sensitive ketone reductase in mouse liver cytosol.

Mouse liver cytosol catalyses the reduction of metyrapone to the corresponding alcohol metabolite metyrapol. The enzyme involved was characterized as a NADPH-dependent carbonyl reductase which is strongly inhibited by the plant flavonoid quercitrin but which shows no sensitivity to phenobarbital. Thus, by inhibitor subdivision of carbonyl reductases the metyrapone reductase in mouse liver cytosol has to be classified as a ketone reductase rather than an aldehyde reductase, as it was shown previously for the analogous enzyme in mouse liver microsomes based on the same pattern of inhibitor classification. Moreover, immunological comparison of the metyrapone reductases from the two subcellular fractions reveal no common antigenic determinants indicating the structural difference between these enzymes. In conclusion, metyrapone undergoes reductive biotransformation mediated by two clearly distinct carbonyl reductases located in different subcellular compartments of mouse liver cells. Considering the convenient and sensitive HPLC-method for direct metyrapol determination, metyrapone may serve as a useful tool in the investigation of these enzymes, although their physiological roles remain to be determined.

Antibiotic resistance and enhanced insecticide catabolism as consequences of steroid induction in the gram-negative bacterium Comamonas testosteroni.

The effects of steroid induction on antibiotic resistance against the fungal steroid fusidic acid (ramycin; 16-(acetyloxy)-3 alpha,11 alpha-dihydroxy-29-dammara-17(20), 24-dien-21-oic-acid) as well as on carbonyl reduction and degradation of the novel anti-insect agent NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) were studied in the Gram-negative soil bacterium Comamonas testosteroni strain ATCC 11996. Cells grown with testosterone as inducing agent showed a 5-6-fold elevation of antibiotic resistance against the fungal steroid fusidic acid. Furthermore, testosterone induction caused a faster uptake and different metabolism of the anti-insect agent NKI 42255 compared to control cultures, revealing carbonyl reduction of the substrate keto group as an initial degradation step in induced cells. It is concluded that the formerly described steroid inducible hydroxysteroid dehydrogenases/carbonyl reductases present in Comamonas testosteroni contribute to these altered phenotypes, thus establishing steroid-inducible catabolic pathways as important defense processes against natural and synthetic toxicants in certain bacteria, which are present in the intestinal microflora of mammalian species as well as in soil samples.

Alteration of the inhibitory effect of metyrapone by reduction to metyrapol during the metabolism of methacetin in vivo in mice.

Metyrapone is known as an inhibitor of the oxidative drug metabolism in vitro. We have used the exhalation analysis as a tool to study the influence of this inhibitor on the demethylation of 14C-methacetin in vivo. In parallel we investigated the reductive metabolism of metyrapone in mice by measuring the concentrations of metyrapone and its reduced metabolite metyrapol with a HPLC-method. 50 mg of metyrapone/kg b. wt. resulted in a 90% inhibition of 14CO2 exhalation when given 2 min before the exhalation analysis was started. The prolongation of the intervals between i.p. metyrapone and substrate administration leads to a diminution of the in vivo inhibition. We found that the hepatic metyrapone concentration falls rapidly and passes the detection limit at 120 min. Transiently metyrapol reaches a maximal concentration 15 min after the application of metyrapone. The rapid reduction of metyrapone was confirmed in vitro with fresh mouse liver homogenates. The administration of metyrapol itself in vivo causes a decrease in 14CO2 exhalation, too. The 14CO2 exhalation curves after metyrapol correspond to the curves after metyrapone, when sufficient time was allowed for its reduction to metyrapol. It can be concluded that not only metyrapone itself but also its reductive metabolite metyrapol is an effective, however weaker inhibitor.

Comparison of non-isotopic exchange methods for n.c.a. labelling of 2-iodophenyl-metyrapone.

The Cu(I)-assisted aromatic halogen exchange proved useful for preparation of macroscopic amounts of 2-iodophenyl-metyrapone as well as for the n.c.a. radioiodinated analogue. Semi-preparative HPLC-isolation provided the compound with high purity for use as chromatographic standard and for the determination of the inhibition constant of the 2-iodo-analogue. The non-isotopic exchange led to a high specific activity (> 5000 GBq/mumol) of 2-[123I]iodophenyl-metyrapone. A reaction in acetic acid at elevated temperatures proved superior to an exchange in aqueous solution with in-situ reduction of Cu2+. Even without Cu+ high radiochemical yields of > 80% were obtained, while addition of Cu+ provided the radiotracer with 95% radiochemical yield within 5 minutes in acetic acid.

Synthesis of 2-[131I]iodophenyl-metyrapone using Cu(I)-assisted nucleophilic exchange labelling: study of the reaction conditions.

2-Bromophenyl-metyrapone has been synthesized as a precursor for Cu(I)-assisted labelling with radioiodine. A labelling yield of > 95% was obtained and the specific activity of the purified product was 120 GBq/mumol. The iodo for bromo exchange requires an excess of reducing agents to maintain the Cu(I) redox potential. The effects of the amount of reactants, temperature and time were studied. The labelling yield showed a direct dependence on the amount of precursor and Cu(+)-catalyst used for the reaction, and an increase with reaction time (optimal at 60 min) and temperature (optimal at 100 degrees C). Studies of the stability, lipophilicity and binding of 2-[131I]iodophenyl-metyrapone to serum protein indicated high in vitro stability, high lipophilicity (log P = 2.19) and a loose association with serum proteins.

11beta-Hydroxysteroid dehydrogenase type 1: tissue-specific expression and reductive metabolism of some anti-insect agent azole analogues of metyrapone.

The azole analogues of metyrapone are novel candidates for selective anti-insect agents that inhibit the synthesis of 20-hydroxyecdysone (20E), the moulting hormone of insects. Metyrapone, which is a model substrate for studying the reductive properties of oxidoreductases, is itself effectively reduced to the corresponding alcohol by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). For this reason, the ability of 11beta-HSD 1 to metabolize the metyrapone analogues as well was studied. In addition, the expression (by Western blots) and activity (reduction/oxidation of dehydrocorticosterone/corticosterone) of 11beta-HSD 1 in different male and female mouse tissues were investigated. Xenobiotic carbonyl reductase activities in these tissues were assessed with metyrapone as a model substrate. The kinetic parameters of 11beta-HSD 1 with metyrapone analogues as substrates were calculated after high-pressure liquid chromatography (HPLC) determination of the product alcohols. Our results indicate that the novel insecticides are extensively metabolized by mouse 11beta-HSD 1. Moreover, the resulting alcohols are not only less toxic than the parent ketones but also have the potential, owing to the newly formed hydroxyl group, to be eliminated from the body by consecutive phase II reactions. Thus, the new metyrapone analogues may be potential anti-insect agents, safer for humans due to their reductive detoxification, mainly by the hepatic 11beta-HSD 1, and selectively affecting insect development by inhibiting ecdysone 20-monooxygenase (E-20-M).

Carbonyl reduction of an anti-insect agent imidazole analogue of metyrapone in soil bacteria, invertebrate and vertebrate species.

Carbonyl reduction to the respective alcohol metabolites of the anti-insect agent imidazole analogue of metyrapone, NKI 42255 (2-(1-imidazolyl)-1-(4-methoxyphenyl)-2-methyl-1-propanone) and its parent compound metyrapone was characterized in subcellular fractions previously described bacterial and mammalian hydroxysteroid dehydrogenases/carbonyl from soil bacteria, as well as insect, invertebrate and teleost species. The enzymes involved in this metabolic step were characterized with respect to their cosubstrate specificities, inhibitor susceptibilities, and immunological crossreactivities with antibodies directed against reductases (HSD/CR). All fractions investigated rapidly reduced metyrapone, with highest specific activities found in insect, invertebrate and vertebrate fractions. Except for the insect fractions, all species examined reduced the NKI compound. Cosubstrate dependence and inhibitor specificities suggest that the enzymes described belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) or aldo-keto reductases (AKR). Immunological crossreactions to the previously established subgroup of HSD/CRs were found in trout liver microsomes and insect homogenates, but not in all bacterial extracts or earthworm microsomes. These findings suggest that the high CR activities found in these fractions belong to different subgroups of SDR or AKR.

Spectral and chromatographic properties of 2-methoxyphenylmetyrapone and its potential metabolites.

In the search for new metyrapone derivatives as radioligands for the functional diagnosis of adrenal pathology, 2-methoxyphenylmetyrapone [2-MPMP, 1-(2-methoxyphenyl)-2-methyl-2-(3-pyridyl)-1-propanone] (1), and related 2-substituted phenylmetyrapone derivatives, have been separated as potent inhibitors of adrenal 11 beta-hydroxylase, with high affinity for adrenal mitochondrial binding sites. Surprisingly, 2-[11C]MPMP showed a rapid loss of the radioactive label, which prompted investigation of its metabolism. Synthetic 2-MPMP (1) and its seven potential metabolites (2-8) have been identified spectroscopically (1H- and 13C-NMR and mass spectrometry) and further characterised by chromatography (TLC and gradient reversed-phase HPLC). Chromatographic and mass analysis of urinary extracts from rats dosed with 2-MPMP have confirmed the major metabolites as 2-hydroxyphenylmetyrapone (2-OHPMP), 2) and its N-oxide (2-OHPMP-NO, 6), which are present predominantly as conjugates.

Synthesis of N-oxide derivatives of metyrapone and their detection as in vitro metabolites.

A variety of possible N-oxidation products of 2-methyl-1, 2-bis(3-pyridyl)propan-1-one (metyrapone) have been synthesized by peracid oxidation, and characterized using various spectroscopic techniques. Specific and sensitive chromatographic techniques have been developed for the separation and identification of its in vitro metabolites. Incubation of metyrapone with rat or mouse hepatic microsomes, in the presence of a NADPH-regenerating system, leads to the formation of metyrapol (keto-reduction), and two mono-N-oxides.

Effects of anti-ecdysteroid quaternary derivatives of azole analogues of metyrapone on the post-embryonic development of the red cotton bug (Dysdercus cingulatus F).

In order to improve the larvicidal activity of the azole analogues of metyrapone, previously found to have a strong inhibitory activity on ecdysone 20-monooxygenase (E-20-M) from the fleshfly Neobellieria bullata Parker, soft-alkylated compounds (3-(1,1-dimethyl-2-oxo-2-phenylethyl)-1-dodecanoyloxymethyl-1H-imidazolium chloride, sPIM) and (1-(1,1-dimethyl-2-oxo-2-phenylethyl)-4-dodecanoyloxymethyl-1H-1,2,4-triazolium chloride, sPTM), derivatives of phenyl-imidazolyl-metyrapone (PIM) and phenyl-1,2,4-triazolyl-metyrapone (PTM), respectively, were synthesized. Both sPIM and sPTM, designed as propesticides, inhibited E-20-M in vitro at 10(-4) M concentration, which was unexpected since they had been expected to be inactive in vitro and to gain activity only within the organism. sPTM significantly delayed the pupariation of N. bullata larvae and this effect could be reversed by the simultaneous application of 20-hydroxyecdysone (20E), supporting the hypothesis that sPTM can act by interfering with the moulting hormone system. Due to this in vitro activity, sPTM and sPIM cannot be considered to be simple drug precursors, and their structure should contain structural elements (pharmacophores) responsible for the observed biological effects. In order to examine this hypothesis, derivatives of sPTM and sPIM were synthesised in which the hydrolytically labile N(+)-CH2O(CO)- moiety was changed to the more stable N(+)-CH2CH2(CO)-group. In three new stable derivatives, a dodecylamino or a phenyl group, respectively, is attached to the carbonyl group to obtain PTM and PIM derivatives quaternised with a 2-dodecylcarbamoylethyl or a 3-oxo-3-phenylpropyl group. In one derivative, the 2-oxo-2-phenylethyl quaternising group has one fewer carbon atom. In addition to their moderate activity (LC50 = 10(-6)-10(-5) M) against the red cotton bug Dysdercus cingulatus F, they delayed development and caused developmental abnormalities, including mortality in the pharate phase, mortality during moulting and wing deformations. These symptoms and the delay in development are characteristic of known compounds inhibiting the synthesis of 20E or interfering in the moulting processes. The facts that the frequent appearance of insects with developmental abnormalities and the delay in development could be reversed by co-application of 20E indicate that the moulting system might be the site of action. We presume that the quaternary azole derivatives of PIM and PTM can themselves also interact with the moulting system.

Pharmacokinetics of 2-methoxyphenylmetyrapone and 2-bromophenylmetyrapone in rats.

The pharmacokinetics of two 2-substituted phenylmetyrapone analogues, 2-methoxyphenylmetyrapone (2-MPMP) and 2-bromophenylmetyrapone (2-BrPMP), developed as potential adrenal imaging agents, were investigated in conscious male rats following an intravenous dose of 25 mg/kg. Arterial blood samples (0.25 ml) were collected at various intervals for up to 7 h after dose and subjected to reversed-phase HPLC analysis. Blood concentrations versus time profile for each compound was determined and the pharmacokinetic parameters calculated using the model-independent approach. Blood concentrations of 2-MPMP declined biexponentially with mean initial (t1/2alpha) and terminal (t1/2beta) half-lives of 3.6 and 23.1 min, respectively. The corresponding area under the curve (AUC(0-infinity)) was 159.3 microg x min/ml, the total blood clearance (CI) was 158.3 ml/min and the volume of distribution (Vd) was 5.2 l. Two metabolites of 2-MPMP, namely 2-hydroxyphenylmetyrapone (2-OHPMP) and 2-methoxyphenylmetyrapone N-oxide (2-MPMP-NO), were detected in the blood and their elimination from blood was almost parallel to that of the parent compound. The maximum blood concentrations (Cmax) of 2-OHPMP and 2-MPMP-NO were approximately 0.9 and 1.7 microg/ml, respectively. Blood concentrations of 2-BrPMP declined monoexponentially with a mean t1/2beta of 12.0 min. The pharmacokinetic parameters for 2-BrPMP were: AUC(0-infinity), 193.7 microg x min/ml; Cl, 131.7 ml/min and Vd, 2.3 l. 2-Bromophenylmetyrapone N-oxide was the only one metabolite detected in the blood, its Cmax and AUC0-infinity were 10.1 microg/ml and 1690.0 microg x min/ml, respectively.

Determination of the plasma levels of metyrapone and its enantiomeric metyrapol metabolites by direct plasma injection and multidimensional achiral-chiral chromatography.

The development and validation of a direct injection high-performance liquid chromatographic (HPLC) method, with column switching, for the determination of metyrapol enantiomers and metyrapone in human plasma is described. The system used in this work was composed of a restricted access media (RAM) bovine serum albumin (BSA) octyl column coupled to an amylose tris(3,5-dimethoxyphenylcarbamate) chiral column. Water was used as eluent for the first 5 min at a flow rate of 1.0 ml/min for the elution of the plasma proteins and then acetonitrile-water (30:70 v/v) for the transfer and analysis of metyrapol enantiomers and metyrapone, which were detected by UV at lambda = 260 nm. The total analysis time was about 32 min. The calibration curves for each enantiomer and for the metyrapone were linear in the ranges 0.075-0.75 microg/ml and 0.150-1.50 microg/ml, respectively. Recoveries, intra- and interday precision and accuracy were determined using three quality controls, one low (0.18 microg/ml), one medium (0.75 microg/ml), and one high (1.35 microg/ml) plasma concentration. Quantitative recoveries and good precision and accuracy were obtained. The limit of quantitation were 0.045 microg/ml for both enantiomers and for the metyrapone.

Determination of metyrapone and the enantiomers of its chiral metabolite metyrapol in human plasma and urine using coupled achiral-chiral liquid chromatography.

A coupled achiral-chiral liquid chromatographic assay has been developed to determine the concentrations of metyrapone and the enantiomers of its chiral metabolite metyrapol in plasma and urine. The chromatographic system consisted of a silica precolumn (75 x 4.6 mm I.D.) coupled in-line to a 250 x 4.6 mm I.D. column containing cellulose tris(4-methylbenzoate) coated on silica gel (Chiralcel OJ-CSP). When plasma samples were analyzed, the mobile phase was hexane-ethanol (92:8, v/v) modified with 0.1% diethylamine and when urine samples were analyzed the mobile phase was hexane-ethanol (94:6, v/v) modified with 0.2% diethylamine. Under these chromatographic conditions the chromatographic retentions [expressed as capacity factors (k')] for metyrapone were k' = 2.35 (plasma) and 2.52 (urine); for (-)-metyrapol k' = 4.22 (plasma) and 4.62 (urine); for (+)-metyrapone k' = 5.16 (plasma) and 5.86 (urine); enantioselectivities (alpha) were 1.09 (plasma) and 1.13 (urine). The assay has been validated for use in metabolic studies. The analyses of plasma and urine samples from one subject following oral administration of 750 mg of metyrapone indicated that the enzymatic reduction of myterapone by aldo-keto reductase was enantiospecific.