N-demethylation Activity Research Articles

Comparison of the properties of detergent-solubilized NADPH-cytochrome P-450 reductases from pig liver and kidney immunochemical, kinetic, and reconstitutive properties

NADPH-cytochrome P-450 reductases from pig liver and kidney and rabbit liver microsomes were purified to a specific activity of 50–62 μmol cytochrome c reduced/min/mg. All reductase preparations were separated into one major and one minor fraction on Sephadex G-200 columns. The molecular weights of the major fractions of the reductases were estimated to be 74,000, 75,000, and 75,500 for rabbit liver, pig kidney, and liver reductases, respectively, whereas the molecular weight of the minor fractions of these reductases, 67,000, was the same as that of the steapsin-solubilized pig liver reductase on SDS-polyacrylamide gel electrophoresis. K m values for NADPH and cytochrome c were: 20 and 29 μ m or 14 and 28 μ m for the pig kidney or liver reductase, respectively. Immunochemical studies, including Ouchterlony double diffusion experiments and inhibition of benzphetamine N-demethylation activity in microsomes by antibody against pig liver NADPH-cytochrome P-450 reductase, indicated the similarity of the purified liver and kidney reductases. There were no differences in the ability to reconstitute NADPH-mediated benzphetamine N-demethylation and laurate hydroxylation in reconstituted systems between the pig liver and kidney reductases, indicating that the reductase did not determine substrate specificity in these systems.

Purification of cytochrome P-450 from liver microsomes of phenobarbital-treated guinea pigs

Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b 5 and NADPH-cytochrome c reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome c reductase and phosphatidylcholine.

Effects of Polyamines on Ethylmorphine N-Demethylation in Rat Liver Microsomes

The time course linearity of ethylmorphine N-demethylation was improved by the addition of polyamines to the reaction mixture. The most remarkable effect on the time course linearity of ethvlmorphine N-demethylation was observed when spermine was used. The apparent stimulatory effect of spermine was decreased remarkably by the simultaneous addition of EDTA to inhibit lipid peroxidation. Similar results were observed when an additional lipid peroxidation inhibitor such as Co2+, Mn2+ or 2, 2’-bipyridine was used in place of EDTA. Hydrogen peroxide-dependent ethylmorphine N-demethylation activity in rat liver microsomes was not influenced by the addition of spermine. In addition, neither lipid peroxides formation nor the stimulatory effects of polyamines on ethylmorphine N-demethylation was observed in the reconstituted monooxygenase system. These results suggest that the inhibitory effects of polyamines on lipid peroxidation might be responsible for the stimulatory effects on drug oxidations.

Fatty Acid ω- and (ω-1)-Hydroxylation in Rabbit Intestinal Mucosa Microsomes

The microsomes from rabbit intestinal mucosa which had been washed quickly and thoroughly with phenylmethylsulfonyl fluoride were found to catalyze the hydroxylation of fatty acids in the presence of NADPH and molecular oxygen. Myristic and palmitic acids were converted to the corresponding omega-and (omega-1)-hydroxy fatty acids, whereas lauric acid was converted only to 12-hydroxylauric acid, and capric acid, to 9-and 10-hydroxycapric acids together with an unknown polar acid. Among these fatty acids, both myristic and lauric acids appeared to be the most efficient substrates. The inhibition of the hydroxylation by SKF 525-A and carbon monoxide suggested that the activity depended upon cytochrome P-450. The specific activity of the fatty acid hydroxylation was almost constant along the small intestine, while the aminopyrine N-demethylation activity and the cytochrome P-450 content were highest at the proximal end of the intestine and progressively declined toward the caudal end. The cytochrome P-450 was solubilized from the intestinal microsomes and purified by 6-amino-n-hexyl Sepharose 4B chromatography. The partially purified cytochrome P-450 was active in fatty acid hydroxylation in combination with intestinal NADPH-cytochrome c reductase and phosphatidylcholine.

Partial purification and properties of cytochrome P450 from homogenates of human fetal livers

Using ω-amino-n-octyl Sepharose 4B and hydroxylapatite columns, cytochrome P450 was purified to approx. 6.7 nmoles per mg of protein from the 105,000 g precipitate of homogenates of human fetal livers. The partially purified preparation of cytochrome P450 was free of detectable amounts of cytochrome b 5, NADPH-cytochrome P450 reductase and NADH-cytochrome b 5 reductase. The absolute spectrum of the preparation exhibited a peak at 417 nm in the Soret region, indicating that this cytochrome P450 is a low spin species. Binding of aniline and SKF 525-A to this partially purified preparation of cytochrome P450 produced a modified type II and a type I difference spectra, respectively. As judged by Ouchterlony double diffusion analysis, the cytochrome P450 preparation did not cross react with antibody against cytochrome P450 isolated from the livers of phenobarbital-pretreated rats. In reconstituted systems, the cytochrome exhibited considerable activity for aniline hydroxylation but only a low ethlymorphine N-demethylation activity compared to cytochrome P450 isolated from the livers of phenobarbital-pretreated rats.

Interaction between NADPH-cytochrome p-450 reductase and cytochrome p-450 in the membrane of phosphatidylcholine vesicles

Cytochrome P-450 and NADPH-cytochrome P-450 reductase, both purified from liver microsomes of phenobarbital-pretreated rabbits, have been incorporated into the membrane of phosphatidylcholine vesicles by the cholate dialysis method. The reduction of cytochrome P-450 by NADPH in this system is biphasic, consisting of two first-order reactions. The rate constant of the fast phase, in which 80–90% of the total cytochrome is reduced, increases as the molar ratio of the reductase to the cytochrome is increased at a fixed ratio of the cytochrome to phosphatidylcholine, suggesting that the rate-limiting step of the fast phase is the interaction between the reductase and the cytochrome. The rate constant of the fast phase also increases when the amount of phosphatidylcholine, relative to those of the two proteins, is decreased. This latter observation suggests that the interaction between the two proteins is effected by their random collision caused by their lateral mobilities on the plane of the membrane of phosphatidylcholine vesicles. The rate constant of the slow phase as well as the fraction of cytochrome P-450 reducible in the slow phase, on the other hand, remains essentially constant even upon alteration in the ratio of the reductase to the cytochrome or in that of the two proteins to phosphatidylcholine. No satisfactory explanation is as yet available for the cause of the slow-phase reduction of cytochrome P-450. The overall activity of benzphetamine N-demethylation catalyzed by the reconstituted vesicles responds to changes in the composition of the system in a similar way to the fast-phase reduction of cytochrome P-450, though the latter is not the rate-limiting step of the overall reaction.

Validation of 13CO 2 breath analysis as a measurement of demethylation of stable isotope labeled aminopyrine in man

Interval sampling of expired breath as a simple, non-invasive assessment of the effect of liver disease upon hepatic microsomal drug metabolism, has been demonstrated with [ 14C]dimethylaminoantipyrine (aminopyrine). In order to eliminate radiation risk we have validated the use of aminopyrine labeled with the stable, non-radioactive isotope 13C. Simultaneous oral administration of both [ 14C]- and [ 13C]aminopyrine to five adult subjects without liver disease as well as five patients with known liver disease, resulted in the excretion of label at nearly identical rates in both individual time collections ( r = 0.94) as well as cumulative excretion for three hours ( r = 0.97). An oral dose of 2-mg/kg of [ 13C]aminopyrine resulted in rates of production of 13CO 2 significantly greater than baseline variations in 13CO 2 production in the fasting, resting subject. Measurements of a single peak value at one half hour correlated closely with the determination of cumulative appearance over three hours ( r = 0.96). A consistent reproducible increase in the peak production of 13CO 2 was observed when five patients received phenobarbital. Stable isotope labeled aminopyrine may be used to detect the effects of disease and treatment upon hepatic N-demethylation activity in human subjects without incurring any risk from radiation. Furthermore, the availability of another isotopic carbon label should make possible the study of direct drug-drug interaction utilizing CO 2 analysis.

Covalent binding in vitro of polychlorinated biphenyls to microsomal macromolecules: Involvement of metabolic activation by a cytochrome P-450-linked mono-oxygenase system

Incubation of 14C-labeled polychlorinated biphenyls (PCBs) with rat, mouse or rabbit liver microsomes in the presence of an NADPH-generating system and molecular oxygen caused covalent binding of radioactive metabolites of PCBs to microsomal macromolecules. The binding was more pronounced with liver microsomes from animals pretreated with inducers of the microsomal mono-oxygenase system. The order of induction effect of the inducers used was KC-500 (a PCB preparation containing 55% chlorine) ≥ phenobarbital (PB) ⪢ 3-methylcholanthrene (3-MC) in rats, PB > KC-500 > 3-MC in mice, and PB > KC-500 in rabbits. [ 14C]KC-300 (a PCB preparation containing 42% chlorine) was more effective than [ 14C]KC-500 as substrate for all the microsomal preparations. The binding reaction was dependent on both NADPH and oxygen, sensitive to carbon monoxide, glutathione, cysteine, hexobarbital, and aniline, and enhanced by EDTA, which inhibits lipid peroxidation. The addition of NADH, which was by itself a very poor electron donor, caused a synergistic increase of the NADPH-dependent binding of PCBs. It is concluded that the conversion of PCBs to active metabolites by the cytochrome P-450-linked mono-oxygenase system is prerequisite to the binding reaction. A survey of the effects of various inducers suggested that a cytochrome P-450 having a high aminopyrine N-demethylation activity is mainly responsible for the metabolic activation of PCBs in liver microsomes. Kidney and lung microsomes from untreated rats were virtually devoid of the PCB-binding capacity, but in kidney microsomes this capacity could be induced by pretreatment with 3-MC or KC-500, though not with PB.

Different developmental profiles for the hepatic N-demethylation of N-methylamphetamine and N, N-dimethylamphetamine in the rat

1. No parallelism in the rate of development was found between the oxidative N-demethylation of N-methyl and N, N-dimethylamphetamine in rat liver. A gradual increase in apparent V max values was observed for the conversion of N, N-dimethylamphetamine by hepatic 9000 g supernatants in the period from 2 until about 60 days after birth, whereas the N-demethylating activity of the same liver preparation using N-methylamphetamine as a substrate remained low during the first 20 days after birth and suddenly increased in the 20–30-day period. 2. The mono-oxygenase system of the developing rat liver appeared to be stereoselective with respect to the enantiomers of N-methylamphetamine. The S(+)-isomer was the preferred substrate. No stereoselectivity, however, was observed in the metabolism of the enantiomers of N, N-dimethylamphetamine by the male rats, unlike the female rats. 3. Another difference refers to a decrease in the apparent K m values of both isomers of N-methylamphetamine, whereas the K m values of the N, N-dimethylamphetamines remained unchanged during the maturation. 4. These findings strongly support the concept that multiple forms of N-demethylating enzymes exist, which develop and maturate independently from each other.

Stimulative Effects of Chelating Agents, 2, 2’-Bipyridine and 1, 10-Phenanthroline, on Lipid Peroxidation in Rat Liver Microsomes

Well known lipid peroxidation inhibitors, 1, 10-phenanthroline and 2, 2’-bipyridine, stimulated microsomal NADPH- and ascorbic acid-dependent lipid peroxidation when low concentrations of these chelating agents were added to incubation mixture. The stimulatory effects of the chelating agents on lipid peroxidation were enhanced when ferrous ion was added together with the chelating agents to the mixture at a molar ratio of 1 : 1. Ethylenediaminetetraacetic acid (EDTA) had no stimulatory effect on lipid peroxidation. Ferrous ion-EDTA complex increased lipid peroxidation by only 20–30%, which was lower than that obtained by addition of the same concentration of ferrous ion alone. On the other hand, manganese and calcium ions, which are also inhibitors of lipid peroxidation, had no ability to stimulate lipid peroxidation even in the presence of extra ferrous ions. Changes in the lipid peroxidation by chelating agents affected the apparent activity of ethylmorphine N-demethylation.

Hepatic Aminopyrine N-Demethylase System: Further Studies of Assay Procedure

Rat hepatic aminopyrine N-demethylase activity was measured by detecting the amount of formaldehyde produced from aminopyrine. Some optimal conditions for the N-demethylation were determined using both isolated microsomes and whole homogenates, and the standard assay method is described. Formaldehyde production from the substrate by microsomal enzyme system was linear to the amount of microsomes added during 3 min reaction time, whereas long-time incubation caused a decrease in the apparent activity of aminopyrine N-demethylation. The N-demethylase activity observed in normal rat liver homogenate was quite similar to that in microsomes when the activity was expressed on the basis of cytochrome P-450 as molecular activity. Pretreatment of animals with typical inducers, phenobarbital and 3-methylcholanthrene, resulted in alteration of the aminopyrine N-demethylase system, which was detectable in both microsomes and whole homogenates.

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography.

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.

The use of 8-aminooctyl sepharose for the separation of some components of the hepatic microsomal electron transfer system.

Hepatic microsomes from phenobarbital-pretreated rats were treated with sodium cholate in the presence of glycerol and the solubilized proteins were absorbed on a column of 8-aminooctyl derivative of Sepharose 4B. Washing the column stepwise with buffers containing suitable detergents in addition to cholate resulted in the elution of cytochrome P-450, NADH-cytochrome b5 reductase [EC 1.6.2.2], NADPH-cytochrome c reductase [EC 1.6.2.4], and cytochrome b5; the former two enzymes were eluted with Emulgen 913 (a polyoxyethylene nonylphenyl ether), and then the latter two were obtained separately by increasing the concentration of added deoxycholate. Thus, cytochrome P-450, NADPH-cytochrome c reductase, and cytochrome b5 were purified to specific contents (or activities) of 9--10 nmoles per mg of protein, 15-16 units per mg of protein, and nearly 35 nmoles per mg of protein, respectively (about 40--50% pure), in 40--50% yields. Practically no mutual contamination of the three enzymes was detected. Benzphetamine N-demethylation activity could be reconstituted on mixing the cytochrome P-450 and NADPH-cytochrome c reductase preparations in the presence of cholate. NADH-cytochrome C reductase activity could be observed at high efficiency under appropriate conditions in a system containing the cytochrome b5 and NADH-cytochrome b5 reductase preparations. Various derivatives of Sepharose 4B were compared for effectiveness in the separation of the microsomal election transfer enzymes.

Inhibition of hepatic drug metabolizing enzyme by cadmium in mice.

The effect of cadmium on hepatic drug metabolizing enzymes and the involvement of metallothionein in the protection of the impaired activity of these enzymes was studied. Administration of cadmium (cadmium chloride) to mice significantly inhibited the activity of aminopyrine N-demethylation in 24 and 48 hr, but the activity returned to nearly control values in 72 hr. The intensity of the inhibition of drug metabolizing enzymes was dose-dependent, suggesting that the inhibition was a result of a direct action of cadmium upon the drug metabolizing enzymes. The inhibition of hepatic drug metabolizing enzymes and the decrease in cytochrome P-450 content by the challenging dose were diminished when mice were pre-dosed with cadmium chloride. The degree of protection against the challenging dose was correlated with the pre-dose levels. The effect of the challenging dose on hepatic drug metabolizing enzymes and cytochrome P-450 content was completely abolished when mice were pre-dosed with 3 mg/kg dose level of cadmium chloride. It is considered that metallothionein may be involved in the biological protection of animals against cadmium poisoning. (ND)

Studies on lung tumours III. oxidative metabolism of dimethylnitrosamine by rodent and human lung tissue

The oxidative metabolism of the carcinogen dimethylnitrosamine (DMN) was studied in mouse, rat, hamster and human respiratory tissue. [ 14C]DMN was purified by Dowex-1-bisulfite column chromatography to remove a contaminant (probably [ 14C]formaldehyde) interfering with the enzyme assay. Since formaldehyde and methyl carbonium ions — yielding methanol with water — are considered to be the primary products of DMN metabolism, tissue slices were assayed for the production of [ 14C]CO 2 from 14C-labelled methanol, formaldehyde, formate, and DMN. Oxidation of formaldehyde to formate was not, but oxidation of formate to CO 2 was very much rate-limiting. This rate-limiting step was circumvented by introducing quantitative chemical oxidation of formate to CO 2 by mercury(II)chloride following the enzymic reaction. Since oxidation of methanol to CO 2 proved to be insignificant, production of CO 2 from DMN by lung tissue enzymes and HgCl 2 may serve as a parameter for N-demethylating activity and the production of the suspected carcinogenically active methyl carbonium ions. The DMN- N-demethylating activities of lung tissue slices of two mouse strains with widely different susceptibilities to formation of lung adenomas by DMN differed significantly, but the difference seemed too small to explain the divergence in tumourigenic response. The enzymatic activities decreased in hamster bronchus, hamster trachea, hamster lung, GRS/A mouse lung, C3Hf/A mouse lung, human lung, Sprague-Dawley rat lung, in that order. The reported resistance of the hamster

Binding of homogeneous cytochrome b5 to rat liver microsomes effect on N-demethylation reactions

Incubation of rat homogeneous detergent-solubilized cytochrome b 5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b 5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b 5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b 5 preparations reconstituted from heme and apocytochrome b 5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b 5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b 5 contains contaminating amounts of tightly bound detergent which is responsibe for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.

Cobalt stimulation of heme degradation in the liver. Dissociation of microsomal oxidation of heme from cytochrome P-450

The administration of cobalt to rats caused a marked increase in the oxidative degradation of heme (hematin, iron protoporphyrin-IX) BY HEPATIC MICROSOMAL ENZYMES. The onset of this enzyme stimulation was very rapid, beginning within 2 hours after injection of the metal and reaching its maximum in 16 to 24 hours. During the rapid phase of stimulation, i.e. the first 2 to 4 hours, when heme oxidation was 450% above control values, there was a significant decrease in microsomal oxidative N-demethylation activity and in microsomal oxidative Ndemethylation activity and in microsomal content of heme with an insignificant decrease in cytochrome P-450 content. Within 24 hours the oxidative activity of the microsomal electron transport chain for drugs was decreased to about 30% of the control. However, during the same period the oxidation of heme approached levels 800% above control. During this period there was a further decrease in the microsomal content of heme with a significant decrease in cytochrome P-450 content and an increase in the activity of delta-aminolevulinate synthetase. The activity of delta-aminolevulinate synthetase reached its maximum within 8 hours after cobalt treatment. Repeated injections (at 24-hour intervals) of cobalt were necessary to maintain these changes in microsomal enzyme activities since, after single injections of the metal, these parameters returned to normal within 72 hours. The inducing effect of cobalt on the oxidation of heme could be inhibited by the administration of actinomycin D and puromycin. Furthermore, this stimulatory effect could not be elicited by in vitro treatment of microsomes with cobalt nor could the effect be attributed to any soluble components of the cytoplasm. Cobalt protoporphyrin-IX was less effective than cobalt chloride in stimulating heme oxidation. 3-Amino-1, 2, 4-triazole did not enhance hepatic heme oxidation activity, while allylisopropylacetamide decreased this activity. The oxidative degradation of heme was found not to be cytochrome P-450 dependent since the highly increased levels of heme oxidation in microsomes from cobalt-treated animals could be retained despite the fact that the cytochrome P-450 content of such microsomes was decreased to spectrally undetectable amounts and drug oxidation was eliminated by treatment of the microsomes with 4 M urea. These findings exclude an obligatory role for cytochrome P-450 in the oxidation of heme compounds, although the possibility that this process is a heme-dependent oxidation is not ruled out.

Rate-limiting steps in drug metabolism by microsomes from CCl 4-cirrhotic rat liver

Comparative studies have been conducted of the activity of microsomal mixed-function oxidases from livers of normal, precirrhotic and cirrhotic rats linked with the metabolism of type-I (aminopyrine, hexobarbital), type-II (aniline, metyrapone) and "modified type-II" (corticosterone) substrates. The following factors were investigated: the possible role of cytochrome P-450 content, the state of the "substrate-binding protein" of this enzyme, the degree of affinity of this hemoprotein for both type-I and type-II substrates and finally, the activity of the enzymes of the microsomal electron-transport chain (both in the absence and in the presence of type-I substrate) -- as rate-limiting reactions, "tight spots" in the biotransformation of drugs in experimental microsomes. It was found that the hydroxylation activity for type-II and "modified type-II" substrates during the entire period of liver cirrhosis development is determined by the cytochrome P-450 content and the amplitude of maximal spectral changes observed in the presence of excess substrate. Type-I substrate metabolism, however, is limited in the precirrhotic phase by the state of the "substrate-binding protein" contained in P-450 as well as by the NADPH-cytochrome P-450 reductase activity. On the other hand, the N-demethylating activity in CCl-4-cirrhotic liver microsomes does not depend on either the concentration of P-450, on the amplitude of the maximal spectral changes or on the Ks value. The rate-limiting step in this case is the rate of reduction of the P-450-substrate complex by NADPH.

Polychlorinated biphenyl-induced alteration of biologic parameters in the rat

Aroclor 1242, a commercial polychlorinated biphenyl (PCB) mixture, was administered ip to rats for 10 weeks. Principal findings included: loss of body weight; hepatic and renal damage, with some animals exhibiting renal papillary epithelial hyperplasia; slight reduction in erythrocyte count, diameter, and hemoglobin content, with an elevation in serum iron; diminished plasma corticosteroid and glucose concentrations; increased urinary excretion of protein, sugars, and coproporphyrin. The effect of PCBs on several hepatic microsomal enzymatic parameters was also evaluated. Maximal hydroxylation and N-demethylation activities were observed 3–10 days following a single ip injection (100 mg/kg). Each remained significantly higher than control values after 20 days, with hydroxylation activity still 150% of controls after 40 days. The minimal effective single dose for induction of hydroxylation activity was approximately 5 mg/kg. Induction of hydroxylation activity was found to be dose-dependent; however, a smaller degree of correlation was noted between N-demethylation activity and dose. Values for cytochromes P 450 and b 5 and NADPH-cytochrome c reductase activity were observed to roughly parallel hydroxylation and N-demethylation activities, the highest degree of correlation was manifest between enzymatic activity and cytochrome P 450 values.

Riboflavin and drug metabolism in adult male and female rats

Oxidative metabolism of drugs in vitro in liver 9000 g supernatant fraction from riboflavin-deficient adult male and female rats was investigated. A significant decrease in overall oxidation of aminopyrine, ethylmorphine, N-methylaniline, aniline and acetanilide was observed. Studies at two deficiency levels indicate that a marked decrease in specific activities of N-demethylation of aminopyrine. ethylmorphine. N-methylaniline and hydroxylation of aniline and acetanilide occurs in both male and female rats. The levels of drugmetabolizing enzymes were further lowered with increase in deficiency. The levels of flavin, NADPH cytochrome c reductase, cytochrome P-450 and cytochrome b 5 were reduced in riboflavin-deficient (7 weeks) male rats as compared to normal rats. The activity of drug enzymes from riboflavin-deficient rats was stimulated by pretreatment with phenobarbital. A significant reversal of drug enzyme activities was noted when riboflavin was administered to deficient animals.