Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

Metabolism of 13-cis-retinoic acid: Identification of 13-cis-retinoyl-and 13-cis-4-oxoretinoyl-β-glucuronides in the bile of vitamin A-normal rats

The metabolism of 13-cis-[11-³H]retinoic acid has been examined in vitamin A-normal rats. Within 24 h after intravenous administration of the parent retinoid (15 μg/kg) to animals with biliary fistulas, 69 ± 9% of the dose was detected in the bile with 9 ± 6% being found in the urine. Analysis of the bile by reverse-phase high-pressure liquid chromatography demonstrated that the retinoic acid was being metabolized to several more polar compounds. A number of these compounds were sensitive to incubation with β-glucuronidase as evidenced by a change in their chromatographic behavior after treatment with the enzyme. Two of the metabolites have been identified as 13-cis-4-oxoretinoyl-β-glucuronide (8.1 ± 1.0% of the dose during the first 4 h after administration of the parent compound) and 13-cis-retinoyl-β-glucuronide (7.0 ± 4.4% of the dose). A comparison of the chromatographic profiles of bile from 13-cis- versus all-trans-retinoic acid-treated rats indicated a difference in their metabolism, with a greater proportion of the all-trans-retinoic acid being converted to compounds that eluted in the more polar regions of the column effluent.     

Proteolysis of chloroplast thylakoid membranes. II. Evidence for the involvement of the light-harvesting chlorophyll a/b-protein complex in thylakoid stacking and for effects of magnesium ions on photosystem II-light-harvesting complex aggregates in the absence of membrane stacking

Experimental evidence indicates that the major pathway of retinoic acid metabolism in hamster liver microsomes follows the sequence: retinoic acid → 4-hydroxy-retinoic acid → 4-keto-retinoic acid → more polar metabolites. Using all-trans-[10-³H]retinoic acid, it can be shown by reverse-phase high pressure liquid chromatographic analysis that the first and last steps of this sequence require NADPH, whereas the oxidation of 4-hydroxy to 4-keto-retinoic acid is NAD⁺ (or NADP⁺) dependent. Both NADPH-dependent steps, but not the NAD⁺-dependent dehydrogenase reaction, are strongly inhibited by carbon monoxide. The metabolism of retinoic acid but not of 4-hydroxy-retinoic acid is highly dependent on the vitamin A regimen of the animal. Retinoic acid is rapidly metabolized by liver microsomes either from vitamin A-normal hamsters or from vitamin A-deficient hamsters that have been pretreated with retinoic acid, but not by microsomes from vitamin A-deficient animals; in direct contrast, the rate of metabolism of 4-hydroxy-retinoic acid is equivalent in each of these microsomal preparations. Analysis of the kinetics of these reactions yields the following Michaelis constants with respect to the retinoid substrates: retinoic acid, 1 × 10^?6m; 4-hydroxy-retinoic acid, 2 × 10^?5m; and 4-keto-retinoic acid, 1 × 10^?7m. The 4-hydroxy to 4-keto-retinoic acid oxidation has been shown to be experimentally irreversible, to have a K_mNAD⁺of 2 × 10^?5m, to be strongly inhibited by NADH, and to be unaffected by the presence of retinoic acid or its 4-keto-derivative in an equimolar ratio to the 4-hydroxy-substrate.     

Preparation,characterization, biological activity and metabolism of all-trans retinoyl fluoride

All-trans retinoyl fluoride was prepared by treating all-trans retinoic acid with diethylaminosulfurtrifluoride. The crystalline product, which was characterized by melting point, infrared, ¹H-NMR, ¹⁹F-NMR and elementary analysis, showed λ_max at 382 nm in hexane (ε = 4.98·10⁴ M^?1·cm^?1) and at 392 nm in methanol (ε = 4.60·10⁴ M^?1·cm^?1). Its biological activity in the rat growth assay, relative to all-trans retinyl acetate, was 22% ± 10%. Upon oral administration for 5 days to vitamin A-depleted rats, retinoyl fluoride (1020 μg) was rapidly metabolized to a polar metabolite fraction and, in the intestine, to an unstable retinol-like metabolite, purpotedly 15-fluororetinol. Upon administering intraperitoneally smaller doses (47–94 μg) of [11-³H]retinoyl fluoride, which was synthesized from [11-³H] retinoic acid, radioactive retinoic acid was noted in the liver and plasma but not in the intestine. As expected, a radioactive polar fraction appeared in the intestine and liver, but radioactive retinol, retinyl ester and some common oxidation products were not detected. Of the administered radioactivity, 72% was excreted in the urine, and only 4% was found in the feces over a 7-day period. Hydrolysis of the urine gave a radioactive fraction with a polarity similar to that of retinoic acid. Retinoyl fluoride also reacts readily with glycine to yield N-retinoyl glycine. Thus, the biological activity of retinoyl fluoride can be attributed to the formation of retinoic acid, probably by way of N-retinoyl derivatives. A possible pathway for its metabolism is presented.     

Cellular retinoid-binding proteins transfer retinoids to human cytochrome P450 27C1 for desaturation

Cytochrome P450 27C1 (P450 27C1) is a retinoid desaturase expressed in the skin that catalyzes the formation of 3,4-dehydroretinoids from all-trans retinoids. Within the skin, retinoids are important regulators of proliferation and differentiation. In vivo, retinoids are bound to cellular retinol-binding proteins (CRBPs) and cellular retinoic acid–binding proteins (CRABPs). Interaction with these binding proteins is a defining characteristic of physiologically relevant enzymes in retinoid metabolism. Previous studies that characterized the catalytic activity of human P450 27C1 utilized a reconstituted in vitro system with free retinoids. However, it was unknown whether P450 27C1 could directly interact with holo-retinoid-binding proteins to receive all-trans retinoid substrates. To assess this, steady-state kinetic assays were conducted with free all-trans retinoids and holo-CRBP-1, holo-CRABP-1, and holo-CRABP-2. For holo-CRBP-1 and holo-CRABP-2, the k_cat/K_m values either decreased 5-fold or were equal to the respective free retinoid values. The k_cat/K_m value for holo-CRABP-1, however, decreased ∼65-fold in comparison with reactions with free all-trans retinoic acid. These results suggest that P450 27C1 directly accepts all-trans retinol and retinaldehyde from CRBP-1 and all-trans retinoic acid from CRABP-2, but not from CRABP-1. A difference in substrate channeling between CRABP-1 and CRABP-2 was also supported by isotope dilution experiments. Analysis of retinoid transfer from holo-CRABPs to P450 27C1 suggests that the decrease in k_cat observed in steady-state kinetic assays is due to retinoid transfer becoming rate-limiting in the P450 27C1 catalytic cycle. Overall, these results illustrate that, like the CYP26 enzymes involved in retinoic acid metabolism, P450 27C1 interacts with cellular retinoid-binding proteins.     

Simultaneous quantification of retinol, retinal, and retinoic acid isomers by high-performance liquid chromatography with a simple gradiation

A new method of high-performance liquid chromatography (HPLC) analysis to quantify isomers of retinol, retinal and retinoic acid simultaneously was established. The HPLC system consisted of a silica gel absorption column and a linear gradient with two kinds of solvents containing n-Hexane, 2-propanol, and glacial acetic acid in different ratios. It separated six retinoic acid isomers (13-cis, 9-cis, all-trans, all-trans-4-oxo, 9-cis-4-oxo, 13-cis-4-oxo), three retinal isomers (13-cis-, 9-cis-, and all-trans) and two retinol isomers (13-cis- and all-trans). Human serum samples were subjected to this HPLC analysis and at least, all-trans retinol, 13-cis retinol, and all-trans retinoic acid were detectable. This HPLC system is useful for evaluating retinoic acid formation from retinol via a two-step oxidation pathway. Moreover, it could be applied to monitoring the concentrations of various retinoids, including all-trans retinoic acid in human sera.     

Metabolism and biological activity of all-trans 4,4-difluororetinyl acetate

All-trans [11-³H]4,4-difluororetinyl acetate was synthesized by treating methyl all-trans [11-³H]4-oxoretinoate with diethylaminosulfurtrifluoride, followed by reduction and acetylation of the product. After oral administration of the radioactive difluoro analog in oil to rats, difluororetinol, difluororetinyl palmitate and related esters, 4-oxoretinol, 4-oxoretinoic acid and polar conjugated derivatives were identified in the intestine, liver, kidney and / or blood. The major metabolic products were difluororetinyl palmitate and related esters, which were stored in the liver. The presence of the difluoro analog in liver oil from treated rats was confirmed by ¹⁹F-NMR spectroscopy. Neither retinol nor retinyl esters were detected as products of the metabolism of the difluoro analog. Nonetheless, all-trans difluororetinyl acetate showed 26 ± 12% of the biological activity of all-trans retinyl acetate in the rat growth assay. Presumably, the difluoro analog is active per se in growth rather than by conversion to retinol or to one of its known growth-promoting metabolites. In general, however, the difluoro analog was metabolized in a manner very similar to vitamin A. The vitamin A moiety of administered difluororetinyl acetate and retinyl acetate was poorly stored (1.8–3.3%) in the liver of vitamin A-depleted rats, confirming and extending past reports that the liver storage mechanism is severely impaired when initial liver stores are very low.     

The effect of ethanol on drug oxidations in vitro and the significance of ethanol-cytochrome P-450 interaction

The effect of ethanol on N-demethylation of aminopyrine in rat liver slices and in the microsomal fraction and on microsomal hydroxylation of pentobarbital and aniline was studied. With liver slices N-demethylation of aminopyrine was stimulated by 35–40% at low ethanol concentrations (2mm), whereas no stimulation occurred at high concentrations (100mm). With the liver microsomal fraction, an inhibitory effect was observed only at high ethanol concentrations (100mm). This was also observed with the other drugs studied. In agreement with these results, only at a high concentration did ethanol interfere with the binding of drug substrates to cytochrome P-450. Further, as previously reported, ethanol produced a reverse type I spectral change when added to the liver microsomal fraction. Evidence that this spectral change is due to removal of substrate, endogenously bound to cytochrome P-450, is reported. A dual effect of ethanol is assumed to explain the present findings; in liver slices, at a low ethanol concentration, the enhanced rate of drug oxidation is the result of an increased NADH concentration, whereas the inhibitory effect observed with the microsomal fraction at high ethanol concentration is due to the interference by ethanol with the binding of drug substrates to cytochrome P-450.     

Simultaneous determination of all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites and all-trans-retinol in human plasma by high-performance liquid chromatography

All-trans-retinoic acid (all-trans-RA) and 13-cis-retinoic acid (13-cis-RA), due to their effects on cell differentiation, proliferation and angiogenesis, improved treatment results in some malignancies. Pharmacokinetic studies of all-trans-RA and 13-cis-RA along with monitoring of retinoic acid metabolites may help to optimize retinoic acid therapy and to develop new effective strategies for the use of retinoic acids in cancer treatment. Therefore, we developed a HPLC method for the simultaneous determination in human plasma of the physiologically important retinoic acid isomers, all-trans-, 13-cis- and 9-cis-retinoic acid, their 4-oxo metabolites, 13-cis-4-oxoretinoic acid (13-cis-4-oxo-RA) and all-trans-4-oxoretinoic acid (all-trans-4-oxo-RA), and vitamin A (all-trans-retinol). Analysis performed on a silica gel column with UV detection at 350 nm using a binary multistep gradient composed on n-hexane, 2-propanolol and glacial acetic acid. For liquid-liquid extraction a mixture of n-hexane, dichloromethane and 2-propanolol was used. The limits of detection were 0.5 ng/ml for retinoic acids and 10 ng/ml for all-trans-retinol. The method showed good reproducibility for all components (within-day C.V.: 3.02–11.70%; day-to-day C.V.: 0.01–11.34%. Furthermore, 9-cis-4-oxoretinoic acid (9-cis-4-oxo-RA) is separated from all-trans-4-oxo-RA and 13-cis-4-oxo-RA. In case of clinical use of 9-cis-retinoic acid (9-cis-RA) the pharmacokinetics and metabolism of this retinoic acid isomer can also be examined.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats

The effects of retinoic acid (RA) (50 μg/100 g body wt. per day) on hepativ heme oxygenase activty, δ-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T₃ (10 μg/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P < 0.01) and microsomal heme content by 47% (P < 0.001). T₃ administration enhanced heme oxygenase activity by 72% (P < 0.001) and ALAS activity by 251% (P < 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P < 0.001) and heme levels by 75% (P < 0.001). When RA and T₃ were administered together, the retinoid markedly enhanced the T₃ stimulation of heme oxygenase activity; 173% above controls (P < 0.001), and 61% above T₃ alone (P < 0.001). However, RA failed to influence the effect of T₃ on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

An aryl hydrocarbon hydroxylating hepatic cytochrome P-450 from the marine fish Stenotomus chrysops

Hepatic microsomal cytochrome P-450 from the untreated coastal marine fish scup, Stenotomus chrysops, was solubilized and resolved into five fractions by ion-exchange chromatography. The major fraction, cytochrome P-450E (M_r = 54,300), was further purified to a specific content of 11.7 nmol heme/mg protein and contained a chromophore absorbing at 447 nm in the CO-ligated, reduced difference spectrum. NH₂-terminal sequence analysis of cytochrome P-450E by Edman degradation revealed no homology with any known cytochrome P-450 isozyme in the first nine residues. S. chrysops liver NADPH-cytochrome P-450 reductase, purified 225-fold (M_r = 82,600), had a specific activity of 45–60 U/mg with cytochrome c, contained both FAD and FMN, and was isolated as the one-electron reduced semiquinone.Purified cytochrome P-450E metabolized several substrates including 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene when reconstituted with lipid and hepatic NADPH-cytochrome P-450 reductase from either S. chrysops or rat. The purified, reconstituted monooxygenase system was sensitive to inhibition by 100 μM 7,8-benzoflavone, and analysis of products in reconstitutions with purified rat epoxide hydrolase indicated a preference for oxidation on the benzo-ring of benzo[a]pyrene consistent with the primary features of benzo[a]pyrene metabolism in microsomes. Cytochrome P-450E is identical to the major microsomal aromatic hydrocarbon-inducible cytochrome P-450 by the criteria of molecular weight, optical properties, and catalytic profile. It is suggested that substantial quantities of this aromatic hydrocarbon-inducible isozyme exist in the hepatic microsomes of some untreated S. chrysops. The characterization of this aryl hydrocarbon hydroxylase extends our understanding of the metabolism patterns observed in hepatic microsomes isolated from untreated fish.     

Metabolism of naphthalene by cell extracts of Cunninghamella elegans.

Microsomal preparations of Cunninghamella elegans oxidized naphthalene to trans-1,2-dihydroxy-1,2-dihydronaphthalene, 1-naphthol, and 2-naphthol. Enzymatic activity was dependent on the presence of reduced nicotinamide adenine dinucleotide phosphate and oxygen. Reduced microsomal preparations, when treated with carbon monoxide, showed absorption maxima at 450 and 420 nm. The inhibitor 1,2-epoxy-3,3,3-trichloropropane suppressed the formation of trans-1,2-dihydroxy-1,2-dihydronaphthalene and enhanced 1-naphthol formation. The results suggest that the metabolism of naphthalene by fungal microsomes may be analogous to the cytochrome P-450-dependent monooxygenase activity that is associated with mammalian liver microsomes.     

The direct oxidation of ethanol by a catalase- and alcohol dehydrogenase-free reconstituted system containing cytochrome P-4501.

Ethanol oxidation activity has been reconstituted in a system composed of NADPH-cytochrome c reductase, synthetic dilauroylglycerol-3-phosphorylcholine and cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats. This system is free of alcohol dehydrogenase and catalase activities. Furthermore, sodium azide (1 mm), a catalase inhibitor, is without effect on ethanol metabolism. There is a requirement for both NADPH-cytochrome c reductase and cytochrome P-450 and a partial requirement for phospholipid for ethanol oxidation by the reconstituted system. In addition, both NADPH and O₂ are required for catalysis. Under optimal reaction conditions, the rate of acetaldehyde formation if 25 to 50 nmol/min/nmol of cytochrome P-450. Cytochrome P-450 from other sources, including the homogeneous P-450LM₂ from phenobarbital-treated rabbits, have also been found to catalyze ethanol oxidation in reconstituted systems. Antibody prepared against cytochrome P-450 inhibits ethanol metabolism in the reconstituted system consistent with a cytochrome P-450-mediated reaction. Furthermore, cumene hydroperoxide can replace both NADPH and NADPH-cytochrome c reductase in ethanol oxidation and catalysis can be demonstrated in a system composed of only cytochrome P-450, lipid, ethanol, and cumene hydroperoxide. These data implicate cytochrome P-450 in the direct oxidation of ethanol by this system.     

Effects of lipid peroxidation on adrenal microsomal monooxygenases

Incubation of guinea pig adrenal microsomes with 10^?6 M ferrous (Fe²⁺) ion and adrenal cytosol initiated high levels of lipid peroxidation as measured by the production of malonaldehyde. Cytosol or Fe²⁺ alone had little effect on microsomal malonaldehyde formation. When microsomes were incubated in the presence of Fe²⁺ and cytosol, malonaldehyde levels continued to increase for at least 60 min. Accompanying the lipid peroxidation was a decline in adrenal microsomal monooxygenase activities. The rates of metabolism of xenobiotics (benzphetamine demethylase, benzo[α]pyrene hydroxylase) as well as steroids (21-hydroxylation) decreased as malonaldehyde levels increased. In addition, cytochrome P-450 levels, NADPH- and NADH-cytochrome c reductase activities, and substrate interactions with cytochrome(s) P-450 decreased as lipid peroxidation progressed. Inhibition of lipid peroxidation by increasing microsomal protein concentrations during the incubation period prevented the changes in microsomal metabolism. Malonaldehyde had no direct effects on adrenal microsomal enzyme activities. The results indicate that lipid peroxidation may have significant effects on adrenocortical function, diminishing the capacity for both xenobiotic and steroid metabolism.     

Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid)

Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity.     

Measurement of the metabolism of cytochrome P-450 in cultured hepatocytes by a quantitative and specific immunochemical method

We have defined conditions that permit quantitative and specific measurement of the metabolism of the major phenobarbital-inducible form of cytochrome P-450 protein in primary non-proliferating monolayer cultures of adult rat hepatocytes. Isolated antibodies specifically directed against phenobarbital cytochrome P-450 are used to immunoprecipitate the cytochrome from lysates of cultured hepatocytes pulse-labelled with [³H]leucine. Phenobarbital cytochrome P-450 protein is then isolated from the immunoprecipitate by electrophoresis on polyacrylamide gradient slab gels. Specificity of the assay for phenobarbital cytochrome P-450 was established by competition experiments involving other forms of purified cytochrome P-450 as well as by testing antibodies directed against these other forms of the cytochrome. Using purified phenobarbital cytochrome P-450, radiolabelled in both its haem and apoprotein portions, as an internal standard, we demonstrated that, with this immunoassay, recovery of cytochrome P-450 from microsomal samples is nearly complete. Basal rates of synthesis of phenobarbital cytochrome P-450 representing as little as 0.02–0.05% of total cellular protein synthesis were reliably and reproducibly detected in hepatocyte culture maintained in serum-free medium for 72h. Moreover, inclusion of phenobarbital in the culture medium for 96h stimulated not only synthesis de novo of phenobarbital cytochrome P-450 protein, but also accumulation of spectrally and catalytically active cytochrome P-450. Advantages of this immunoassay are that metabolism (synthesis or degradation) of the haem or protein of this important form of the cytochrome can be measured conveniently in the small samples available from cultured cells without the necessity of preparing subcellular fractions.