The Journal of Biochemistry, Vol. 59, No. 6, 1966

Possible Role of P-450 in the Oxidation of Drugs in Liver Microsomes*

By Ry RYUICHI KATO

(From the Department of Pharmacology, National Institute of Hygienic Scienecs, Tamagawayoga-machi, Setagaya-ku, Tokyo)

(Received for publication, November 17, 1965)

B r o die and coworkers (1) demonstrated where A represents the reducible component. that many drugs and other unphysiological Gillette assumed that A is a microsomal materials with high lipid solubility are flavoprotein, NADPH-cytochrome c reductase

oxidized by liver microsomes to more water- [EC 1.6.2.3], but gave no details on the soluble compounds. The microsomal systems nature of "active oxygen". On the other responsible for these reactions apparently hand, O m u r a and S a t o (7, 8) showed have a paradoxical requirement for NADPH that liver microsomes contain a hemoprotein, and molecular oxygen. The same require the reduced form of which combines with ments have also been demonstrated with the carbon monoxide to give a Soret peak at systems which hydroxylate phenylalanine and 45 mƒÊ. They have provisionally called this steroid hormones. It has been established hemoprotein as "P-450" and suggested its that atmospheric, but not aqueous, oxygen importance in electron transport in liver is in fact incorporated into the substrate microsomes. More recently, R e m m e r (9), molecules in the hydroxylation of phenyl Kato and Gillette (24)* and Orrenius alanine (2) and steroid hormones (3). and E r n s t e r (10) reported that admini Similarly, incorporation of atmospheric stration of phenobarbital to rats markedly oxygen into substrates has been demonstrated increase the content of P-450, the activity of in the oxidation of acetanilide (4) and NADPH-cytochrome c reductase and drug trimethylamine (5) by liver microsomes. metabolizing activity of liver microsomes. This suggests that in liver microsomes Furthermore, O r r e n i u s et al. (11) found NADPH reduces a component which in turn that carbon monoxide inhibits the oxidative reacts with molecular oxygen to form an N-demethylation of aminopyrine by liver "active oxygen" intermediate . The "active microsomes. These findings strongly suggest oxygen" seems to be involved in the oxida that P-450 may have some role in the oxida tion of a variety of substrates by a group of tion of drugs in liver microsomes. The non-specific enzymes in microsomes. present work was on the possible role of Gillette (6) formulated the following P-450 in the microsomal oxidation of drugs. coupled reactions for this :

MATERIALS AND METHODS 1) NADPH+H++A - -.AH2+NADP+ Female rats of the Sprague-Dawly strain were used 2) AH2+02 ->" active oxygen" unless otherwise specified. Sodium phenobarbital, 3) " active oxygen" +drugoxidized drug chlorcyclizine hydrochloride and chlorpromazine * Preliminary reports of part of this work have hydrochloride were dissolved in water, while glutethi snide and carisoprodol were suspended in 0.5% been given at the Federation Meeting of the American carboxymethylcellulose. These materials were injected Society for Experimental Biology and Medicine into animals intraperitoneally. (Chicago, April, 1964). The annual Meeting of the Biochemical Society of Japan (Nagoya, October, 1964) * Reported in the Federation Meeting of the and the Kanto Branch Meeting of the Biochemical American Society for Experimental Biology and Society of Japan (Tokyo, November, 1964). Medicine (Chicago, April, 1964) 574 P-450 and Microsomal Oxidation of Drugs 575

Preparation of Liver Microsomes-Rats were killed c reductase was determined by the method of by decapitation, and the liver was removed, chopped W i l l i a m s and K a m i n (21). All activities were into small pieces, washed well, and homogenized in a expressed as the amount of the compounds metabolized Teflon glass homogenizer with 4 volumes of ice cold per one g. wet weight of liver. 1.15% KCI solution. Estimation of P-450-The amount of P-450 was

The homogenate was centrifuged at 9,000•~g for determined by measuring the difference spectrum of

15 minutes and the supernatant fraction was then a microsomal preparation in a Beckman DK2 spectro

centrifuged at 105,000•~g for I hour to sediment the photometer with cuvettes of 1 cm. optical path. One microsomes. In the first experiment, the microsomal ml. of microsomal preparation, equivalent to 250mg.

pellet was resuspended in ice cold 1.15% KCI and of liver and 1.8 ml. of 0.1 M phosphate buffer (pH 7.4) recentrifuged to eliminate adsorbed hemoglobin. were placed in Thunberg-type cuvettes and chilled in

However, this was omitted in later experiments, because an ice cold water bath. Then 0.2 ml. of NADPH

recentrifugation decreased enzyme activities and did (2.0ƒÊmoles) was added and the gas-phase was not eliminate appreciable hemoglobin as judged by immediately replaced with oxygen-free nitrogen

measuring its absorption in the form of CO-hemoglobin (reference cell) or oxygen-free carbon monoxide (sample at 419mƒÊ. It was also found that a perfusion of liver cell). The difference spectrum was measured at the

in situ with isotonic KCI or NaCI significantly (about time of maximal absorbance. The P-450 content was 20%) decreased the recovery of microsomes. expressed as the difference between the optical densities The microsomal pellet was suspended in ice cold at 450 my and 490 my per 1 g. wet weight of liver. 1.15% KCI at a concentration such that 1.0ml. of suspension was equivalent to 400 mg. of liver. RESULTS Enzyme Assays-Microsomes (2.5ml.) equivalent to 1. Effect of Phenobarbital and Methyl 1.0g. of liver were mixed with 2.05 ml. of a solution cholanthreneon Drug Metabolism and the P-450 containing NADP (1.5lƒÊmoles), glucose-6-phosphate Contentof Microsomes-Administration of pheno (50 ƒÊmoles), glucose-6-phosphate dehydrogenase [EC 1. 1. 1.49] (0.5 ƒÊunit), magnesium chloride (25 ƒÊmoles), barbital, methylcholanthrene, benzopyrene, or nicotinamide (100 ƒÊmoles), potassium phosphate buffer various other drugs have been shown to

(280 ƒÊmoles, pH 7.4) and various substrates (5 ƒÊmoles stimulate the oxidative metabolism of many of aminopyrine, methylaniline, aniline ; 4 ƒÊmoles of drugs in liver microsomes (22, 23). hexobarbital ; 3 ƒÊmoles of zoxazolamine ; or 2 ƒÊmoles As shown in Table I, it was in fact of pentobarbital) and 0.45m1. of 1.15% KCI. found that phenobarbital administration The mixtures were incubated in a Dubnoff significantly increased the P-450 content in metabolic shaker for 30 minutes at 37°C under air. liver microsomes even by 6 hours after its Demethylation of aminopyrine was determined by injection and the maximal increase in the mearsuring the formation of 4-aminoantipyrine, as P-450 content was attained after 24 to 36 described by Brodie and Axelrod (12). hours. Subsequently, the P-450 gradually Demethylation of N-methylaniline was estimated decreased, finally returning to the normal by measuring the amount of formaldehyde formed level after 120 hours. The microsomal hy according to the method of N a s h (13) as modified by droxylation of pentobarbital and the C o c h i n and A x e l r o d (14 ). Aromatic hydroxylation N-demethylation of aminopyrine followed of aniline was determined by measuring T-aminophenol similar patterns of increase and decrease in formation by the phenolindophenol method of B rod i e activity. However, 6 hours after administra and Axe 1 r o d (15) modified as reported previously tion when the P-450 content had increased (16). Aromatic hydroxylation of zoxazolamine was significantly these enzyme activates had not determined by measuring the disappearance of substrate yet increased. by the method of C o n n e y et al. (17). Aliphatic A similar discrepancy between the induc hydroxylations of hexobarbital and pentobarbital were tion of P-450 synthesis and drug-oxidizing determined by measuring the disappearance of the substrates according to the methods of Cooper and activity was demonstrated more clearly on Brodie (18) and Brodie et al. (19), respectively. administration of methylcholanthrene. As Microsomal NADPH dehydrogenase [EC 1.6. 99. 1] was shown in Table II, injection of this com- assayed spectrophotometrically as described by pound markedly increased the demethylation Gillette et al. (20). Microsomal NADPH-cytochrome of N-methylaniline and hydroxylation of 576 R. KATO

TABLE I Effect of Phenobarbital Treatment on Aminopyrine N-Demethylation and Pentobarbital Hydroxylation Activities and Amount of P-450

Male rats weighing about 70g. were treated with phenobarbital (70 mg. /kg.) and killed 6, 12, 24, 48, 72 and 96 hours later. The livers from 2 rats were combined for each microsomal preparation. The

determination of the enzyme activity and amount of P-450 were carried out as described in the methods. The amount of P-450 is expressed by the difference between in optical density at 450mƒÊ and 490mƒÊ.

The results are expressed as averages±S.D. (standard deviation) of the values of 5 preparations. The figures in brackets indicate percentage differences from the control.

TABLE II Effect of Methylcholanthrene Treatment on N-Methylaniline N-Demethylation and Zoxazolamine Hydroxylation Activities and Amount of P-450

Male rats weighing about 70g. were treated with methylcholanthrene (40mg./kg.) and killed 6, 12, 24, 36, 48, 72 and 96 hours later. P-450 and Microsomal Oxidation of Drugs 577

TABLE I I I Effect of Phenobarbital Treatment on Aminopyrine N-Demethylation, Pentobarbital Hydroxylation and NADPH Dehydrogenase Activities and Amount of P-450 of Rats Fed on Chow or Sucrose or Fasted

Female rats weighing about 160g. were divided into three groups 72 hours before sacrifice. The first group was fed on standard chow diet, the second group was fasted and the third group was fed on sucrose only. Half the rats in each group were treated with phenobarbital 72 hours and 48 hours before sacrifice. The livers from 2 rats were combined for each microsomal preparation. The results are expressed as averages±S.D. of the results of 5 preparations. The figures in brackets indicate percentage differences from the values of rats on the chow diet.

zoxazolamine in microsomes, whereas the of aminopyrine by liver microsomes in an microsomal content of P-450 showed only a atmosphere consisting of 5% oxygen, 40% slight increase. carbon monoxide and 55% nitrogen. Ex It may be seen from Table III that the periments were therefore made on the induction caused by phenobarbital administ correlation between the inhibition of micro ration was affected by starvation and the somal drug-metabolizing enzymes and the diet of the animals. binding of P-450 to carbon monoxide. The Starvation increased the activities of aniline hydroxylation and aminopyrine N aminopyrine N-demethylation, pentobarbital demethylation activities decreased progres hydroxylation, and NADPH oxidation as sively with increase in the amount of carbon well as the content of P-450 even when monoxide. However, the decrease in the phenobarbital was not administered. On content of free P-450 (not bound to CO) the other hand, a sucrose diet depressed was generally greater than the decrease in these activities as well as the P-450 content. the enzymatic activities. For example, in Injection of phenobarbital into animals on a an atmosphere of 50% carbon monoxide the variety of diets invariably caused increases P-450 was completely bound to carbon in all the above activities and also in the monoxide, while the activities of the micro P-450 content. Fasted rats showed a bigger somal drug-metabolizing enzymes were not response to the injection than rats fed on a completely inhibited. chow diet or on sucrose. The data in Table These results indicate that microsomal III also show that there is a good correla P-450 is responsible for the drug-metaboliz tion between the content of P-450 and the ing activities of microsomes and that CO- enzymatic activites, thus supporting the idea inhibition is not strictly related to binding that P-450 is related to microsomal drug- of P-450 with CO. This suggests the possib metabolism. ility that at least part of the drug oxidation 2. Effect of Carbon Monoxide on the may occur via a different metabolic pathway Activities of Drug-metabolizing Enzymes in which does not involve P-450. Microsomes-O r r e n i u s et al. (11) observed 3. Distribution of Microsomal P-450, complete inhibition of the N-demethylation NADPH Dehydrogenase and Drug-metabolizing 578 R. KATo

TABLE IV Effect of Carbon Monoxide Concentration on Hydroxylation of Aniline and N-Demethylation of Aminopyrine by Liver Microsomes

Male rats weighing about 70 g. were used. The livers obtained from 8 rats were pooled for each microsomal preparation. Incubations were carried out for 15 min., instead of 30 min. in atmospheres containing various concentrations of carbon monoxide, keeping the concentrantion of oxygen constant at 4%. The amount of free P-450 is calculated from the difference between the difference spectrum in an atmosphere of 100% carbon monoxide and the given carbon monoxide concentration. The results are expressed as averages ± S. D. of the values of 3 experiments. The 'figures in brackets indicate the percentage differences from the values obtained in an atmosphere free from 'carbon monoxide or of 100% carbon monoxide.

Enzymes in Various Tissues-It is well known 4. Amount of P-450, NADPH Dehydro that the activity for oxidation of drugs is genase and Drug-metabolizing Enzymes in Rats mainly localized in liver microsomes. The of Different Ages-In the previous paper (25) concentrations of P-450 in various tissues of we reported that the oxidative activities of rat resembled that of microsomal NADPH drugs and NADPH of liver microsomes of dehydrogenase and drug-metabolizing en the fetus and newborn rats were extremely zymes. For example, the liver had the highest low but that they increased progressively up content and also the greatest enzyme activities, to the age of 30 days and later gradually followed by the kidney and lung with much decreased. Therefore, the correlations be lower P-450 contents and enzymatic activities. tween the aminopyrine N-demethylation, Heart, muscle and brain had very little pentobarbital hydroxylation, NADPH dehydro P-450 or enzyme activites. genase activities and the amount of P-450 in

TABLE V Distribution of Aniline Hydroxylation, Hexobarbital Hydroxylation, Aminopyrine N-Demethylation and NADPH Dehydrogenase Activities and Amount of P-450 in Various Tissues

The liver, kidney, lung, heart, muscle and brain obtained from 2 rats were pooled for each microsomal preparation. The figures in the table show the values relative to those of female rats, which were taken as 100% for each of the enzymes and for the P-450 content. The results were given as averages±S.D. of 4 experiments. P-450 and Microsomal Oxidation of Drugs 579

TABLE VI Aminopyrine N-Demethylation, Pentobarbital Hydroxylation and NADPH Dehydrogenase Activities and Amount of P-450 in Rats of Different Ages

Female rats of different ages were used. In this experiments livers obtained from 2-6 female rats or 12 fetuses (18 days old) were pooled for preparation of microsomes. The figures in the table show the values relative to those of 30 days old rats, which were taken as 100% for each enzyme and for the P-450 content. The results are given as averages±S.D. of 4 experiments. rats of different ages were investigated. As glutathimide, chiorcyclizine, chlorpromazine shown in Table VI there were good correla or carisoprodol increased the metabolism of tions between the activities of drug-metaboliz drugs, the activity of NADPH-dehydrogenase ing enzymes, and of NADPH oxidase and the (26-28), and the activity of NADPH-cyto amount of P-450. chrome c reductase (24). It was therefore of 5. Amount of P-450, and Aminopyrine interest to investigate whether the abilities of N - demethylation, Hexobarbital Hydroxylation, various compounds to increase the above NADPH Dehydrogenaseand NADPH-cytochromec activities was related with the P-450 content. ReductaseActivities after Administrationof Various Table VII shows that phenobarbital had the Drugs-In the previous papers we reported most effect in increasing both the amount of that the administration of phenobarbital, P-450 and aminopyrine N-demethylation,

TABLE VII Aminopyrine N-Demethylation, Hexobarbital Hydroxylation and NADPH Dehydrogenase Activities and Amount of P-450 after Administration of Various Drugs

Male rats weighing about 70g. were treated with phenobarbital (70 mg. /kg.), glutethimide (60 mg. /kg.), chlorcyclizine (25 mg. /kg.), chlorpromazine (15 mg. /kg.) or carisoprodol (180mg. /kg.) 60 and 36 hours before sacrifice. Livers of 2 rats were combined for each microsome preparation. The figures in the table show the values relative to those of female rats, which were taken as 100% for each enzyme and for the P-450 content. The results are given as averages±S.D. of 4 experiments. 580 R. KATO

TABLE V111 Arninopyrine N-Demethylation, Aniline Hydroxylation, NADPH Dehydrogenase and NADPH-cytochrome c Reductase Activities and Amount of P-450 in Different Species of Animals

In the experiments livers of 6 mice, 2 rats, 2 rabbits or 2 cats were pooled for preparation of microsomes. The figures in the table show the values relative to those of female rats, which were taken as 100% for each enzyme and for the P-450 content. The results are given as averages±S.D. of 4 experiments. hexobarbital hydroxylation and NADPH-cyto and molecular oxygen. The enzyme systems chrome c reductase activities and that the effects responsible for the oxidation of drugs are of the various drugs in increasing the enzyme called microsomal drug-metabolizing enzymes. activities were in parallel with these effects in One of the characteristics of these enzymes elevating the P-450 content (Table VII). is their relatively low specificity. The mech 6. Amount of P-450, and Microsomal Drug anism of oxidation of drugs by liver micro metabolizing Enzymes, NADPH Dehydrogenase somes is not yet clear, but it is generally and NADPH-cytochromec Reductase Activities in accepted that NADPH acts as a reductant Livers of Different Species of Animals-The for an intermediate of the electron transport aminopyrine N-demethylation, aniline hy system and then the reduced intermediate droxylation, NADPH dehydrogenase and activates molecular oxygen, thus oxidizing NADPH-cytochrome c reductase activities and the drug. Gillette et al. (20) reported the amount of P-450 in the livers from male that liver microsomes oxidized NADPH to and female mice, male and female rats, male NADP in the absence of any exogenous rabbits and male cats are shown in Table electron acceptor, such as cytochrome c or a VIII. Microsomes from mice (male and drug. Moreover, they (20) showed that female) had the highest aminopyrine N-deme hydrogen peroxide was formed during the thylation, aniline hydroxylation, NADPH reaction. Gillette et al. (29) also found dehydrogenase and NADPH-cytochrome c that 4, 4'-diaminodiphenyl sulfide decreased reductase activities and the most P-450. The the formation of hydrogen peroxide by an enzyme activities were qualitatively though amount equivalent to the 4, 4'-diaminodi not quantitatively parallel to the P-450 con- phenyl sulfoxide formed. However, various tents in the various species of animals, as other lines of evidence indicate that hydrogen shown in Table VIII. peroxide itself is not "active oxygen" (20). Thus, the nature of the "active oxygen" is DISCUSSION still obscure. Recently, O m u r a and Sat o Highly lipid-soluble compounds are oxi presented evidence that the microsomal CO- dized by microsomes of mammalian liver to binding pigment, "P-450" was a hemoprotein more water-soluble compounds. These oxida and showed that it was reduced by the addi tions include hydroxylation of the aromatic tion of NADPH or NADH (7, 8). On the ring, alkyl-side chain, N-dealkylation, and other hand, E s t a b r o o k et al. (30) reported O-dealkylation, and require reduced NADP evidence suggesting that P-450 in microsomes P-450 and Microsomal Oxidation of Drugs 581 of the adrenal cortex is responsible for the N - demethylation, aniline hydroxylation, activation of oxygen in the steroid C-21 NADPH dehydrogenase and NADPH-cyto hydroxylase [EC 1.14.1.8] reaction. These chrome c reductase activities in the livers of observations suggest that liver microsomal different species of animals are similar to P-450 may have the function of activating those of their P-450 contents (Table VIII). oxygen in the oxidation of drugs. The However, in several cases the amount of results of the present studies strongly support P-450 is not always exactly correlated with this view, and suggest that the "active the enzymatic activities of the microsomes. oxygen" involved in the oxidation of drugs For example, the increse in the amount may be oxidized P-450 the latter suggestion of P-450 preceded the increse in the amino is based on the following evidence. pyrine N-demethylation and pentobarbital 1. Administration of phenobarbital in- hydroxylation activities following pheno creases the aminopyrine N-demethylation barbital injection (Table I). Further the and pentobarbital hydroxylation activities N-methylaniline N-demethylation and zoxazo and the amount of P-450 in similar ways lamine hydroxylation activities increased by (Table I). 141 and 340%, respectively, 24 hours after 2. The variations in the aminopyrine treatment with methylcholanthrene, while N-demethylation, pentobarbital hydroxyla the amount of P-450 increased by only about tion and NADPH dehydrogenase activities 50%. Moreover, the percentage inhibition and P-450 content in various kind of diet of aniline hydroxylation and aminopyrine and following phenobarbital injection are N-demethylation by carbon monoxide is similar (Table III). W a d a el al. (31) also markedly less than that of binding of P-450 reported an increse in the P-450 content of with carbon monoxide (Table VII). The liver microsomes in fasting rats. incomplete inhibition of aniline hydroxyla 3. The hydroxylation of aniline and tion and aminopyrine N-demethylation when N-demethylation of aminopyrine are in P-450 is compltely bound to carbon monoxide hibited by increase in the percentage of is not accord with the result of O r r e n i u s carbon monoxide in the atmosphere (Table et al. (11). These results strongly suggest IV). This agrees with the report by that oxidized P-450 likely plays a role as Cooper et al. (32) that carbon monoxide "active oxygen" in the • oxidation of drugs competitively inhibits the oxidation of drugs in liver microsomes and that the rate of drug in liver microsomes. oxidation is influenced by the amount of 4. The distributions of aniline hydroxyla P-450. The rate of drug oxidation may tion, hexobarbital hydroxylation, aminopyrine also be influenced by the amount of each of N-demethylation and NADPH dehydrogenase the respective enzymes with rather low activities in different tissues is similar to that specificity for the drugs and of NADPH-P- of P-450 (Table V). 450 reductase, as well as by the structural 5. The changes in the aminopyrine and steric characteristics of the substrates N-demethylation, pentobarbital hydroxyla and the drug oxidizing enzymes, P-450 and tion and NADPH dehydrogenase activities NADPH-P-450 reductase. Thus in some cases with age in female rats are similar to that there may well be no strict relationship of P-450 (Table VI). between the amount of P-450 and the rate 6. The percentage increases in amino of drug oxidation while in other cases, where pyrine N-demethylation, hexobarbital hy the amount of P-450 is a rate limiting step, droxylation, NADPH dehydrogenase and the rate of drug oxidation is strictly correlated NADPH-cytochrome c reductase activities on with the amount of P-450. In accordance administration of various drugs are similar with our view, S u g i m u r a et al. (33) to the increase in the amount of P-450 observed that the aniline hydroxylation (Table VII). activity and the amount of P-450 in 7. The differences in the aminopyrine hepatoma of minimum deviation decreased 582 R. KATO

in parallel according to degrees of deviation, on the oxidation of NADPH, the reduction while S a t o showed a good correlation of P-450, the oxidation of drugs and the between the decrease in the rate of reduc formation of hydrogen peroxide , and also tion of P-450 by NADPH and the decrease studies on electron spin resonance of micro in hydroxylation of aniline on treatment of somal Fe.. microsomes with steapsin (34). The perferryl ion formed in the P-450 SUMMARY molecule may be considered as the "active It was postulated from evidence obtained oxygen". According to I n g r a h a m (35) in the present studies that liver microsomal ferrous ion reacts with molecular oxygen cytochrome binding carbon monoxide forming the perferryl ion (FeO22+}). The , pro- visionally called P-450 , may act as an perferryl ion stabilized by the various ionic oxygen-activating component in the oxida resonance structures may be represented as tion of drugs in microsomes . follows. I. The administration of phenobarbit al to rats increased the amount of P-450 in liver microsomes and this found t The bond between the oxygen molecule o be i and the perferryl ion is weak, so that the ncrease was almost parallel to that of d rug ferryl ion (FeO2+) may liberate an oxygen oxidation activity. atom. It was suggested that in the hy 2. Starvation caused a slight increase in droxylation of salicylic acid by the peroxidase the amount of P-450 , while a sucrose diet [EC 1. 11. 1.71 in the presence of molecular caused a decrease . The effect of pheno oxygen and 2, 3-dihydroxyfumaric acid the barbital in increasing the amount of P -450 perferryl ion formed from molecular oxygen was greatly increased in fasting rats . These changes in P-450 were similar to those in and the peroxidase is reduced by 2, 3-di- hydroxyfumaric acid and the ferryl ion oxidation activity. hyroxylates the salicylic acid (36). Thus as 3. There was little or no P-450 in the a mechanism of oxidation of drugs in liver liver of the fetus and new-born rats but the microsomes the following two possible amout increased progressively up to 30 days schemes are possible. of age and decreased again late . These changes are also similar to the changes in drug oxidation activity. 4. The amount of P-450 was highest in the liver and very low in the kidney and lung. It was negligibly small in the brain, muscle and heart. Drug oxidation activity also varied in the some way. 5. Administration of phenobarbital and various other compounds increased the amount of P-450 and the degree of this increase was parallel to that of drug oxida tion activity. 6. In a survey of the distributions of P-450 and drug oxidation activity in different animals the amount of P-450 was found to be highest in the liver of the mouse and lowest in the liver of the cats and drug Futher investigations are needed to oxidation activity varied similarly. elucidate the mechanism of drug oxidation 6. Drug oxidation activity was inhibited in more detail including stoichiometric studies by increase in the concentration of carbon P-450 and Microsomal Oxidation of Drugs 583

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