Effect of substituents on microsomal reduction of benzo(c)fluorene N-oxides

The potential benzo(c)fluorene antineoplastic agent benfluron (B) displays high activity against a broad spectrum of experimental tumours in vitro and in vivo. In order to suppress some of its undesirable properties, its structure has been modified. Benfluron N-oxide (B N-oxide) is one of benfluron derivatives tested. The main metabolic pathway of B N-oxide is its reduction to tertiary amine B. A key role of cytochrome P4502B and P4502E1 in B N-oxide reduction has been proposed in the rat. Surprisingly, B N-oxide is reduced also in the presence of oxygen although all other N-oxides undergo reduction only under anaerobic conditions. With the aim to determine the influence of the N-oxide chemical structure and its redox potential on reductase affinity, activity and oxygen sensitivity five relative benzo(c)fluorene N-oxides were prepared. A correlation between the redox potential measured and the non-enzymatic reduction ability of the substrate was found, but no effect of the redox potential on reductase activity was observed. Microsomal reductases display a high affinity to B N-oxide (apparent K(m) congruent with0. 2 mM). A modification of the side-chain or nitrogen substituents has led to only a little change in apparent K(m) values, but a methoxy group substitution on the benzo(c)fluorene moiety induced a significant K(m) increase (ten-fold). Based on kinetic study results, the scheme of mechanism of cytochrome P450 mediated benzo(c)fluorene N-oxides reduction have been proposed. All benzo(c)fluorene N-oxides under study were able to be reduced in the presence of oxygen. Changes in the B N-oxide structure caused an extent of anaerobic conditions preference. The relationship between the benzo(c)fluorene N-oxide structure and the profile of metabolites in microsomal incubation was studied and important differences in the formation of individual N-oxide metabolites were found.     

Biochemical strategy of sequestration of pyrrolizidine alkaloids by adults and larvae of chrysomelid leaf beetles

Tracer feeding experiments with (14)C-labeled senecionine and senecionine N-oxide were carried out to identify the biochemical mechanisms of pyrrolizidine alkaloid sequestration in the alkaloid-adapted leaf beetle Oreina cacaliae (Chrysomelidae). The taxonomically closely related mint beetle (Chrysolina coerulans) which in its life history never faces pyrrolizidine alkaloids was chosen as a 'biochemically naive' control. In C. coerulans ingestion of the two tracers resulted in a transient occurrence of low levels of radioactivity in the hemolymph (1-5% of radioactivity fed). With both tracers, up to 90% of the radioactivity recovered from the hemolymph was senecionine. This indicates reduction of the alkaloid N-oxide in the gut. Adults and larvae of O. cacaliae sequester ingested senecionine N-oxide almost unchanged in their bodies (up to 95% of sequestered total radioactivity), whereas the tertiary alkaloid is converted into a polar metabolite (up to 90% of total sequestered radioactivity). This polar metabolite, which accumulates in the hemolymph and body, was identified by LC/MS analysis as an alkaloid glycoside, most likely senecionine O-glucoside. The following mechanism of alkaloid sequestration in O. cacaliae is suggested to have developed during the evolutionary adaptation of O. cacaliae to its alkaloid containing host plant: (i) suppression of the gut specific reduction of the alkaloid N-oxides, (ii) efficient uptake of the alkaloid N-oxides, and (iii) detoxification of the tertiary alkaloids by O-glucosylation. The biochemical mechanisms of sequestration of pyrrolizidine alkaloid N-oxides in Chysomelidae leaf beetles and Lepidoptera are compared with respect to toxicity, safe storage and defensive role of the alkaloids.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Generation of superoxide anion and hydrogen peroxide during redox cycling of 5-(4-nitrophenyl)-penta-2,4-dienal by mammalian microsomes and enzymes

5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.     

Stimulatory effect of FMN and methyl viologen on cytochrome P-450 dependent reduction of tertiary amine N-oxide.

FMN or methyl viologen stimulated anaerobic reduction of tertiary amine N-oxides by liver microsomes and this stimulatory effect was completely inhibited by carbon monoxide. Spectral study indicated that FMN or methyl viologen is reduced by NADPH-cytochrome c reductase and reduced FMN or methyl viologen is reoxidized by cytochrome P-450 in the presence of tertiary amine N-oxides. In the presence of FMN, xanthine oxidase-hypoxanthine system rapidly reduced tiaramide N-oxides through the reduction of cytochrome P-450: the maximum reduction rate of tiaramide N-oxide was about 100 nmoles/mg protein/min.     

Properties of soluble cyclic AMP-dependent protein kinase activity of renal inner medulla

Studies of the chromatographic distribution of soluble protein kinase in rat kidney demonstrated that the type I isoenzyme predominates in cortex, whereas activity in outer and inner medulla is almost exclusively the type II form. The type II isoenzyme also predominates (95% or greater) in human, canine, bovine, porcine and rabbit inner medulla. Compared to soluble type I activities from rat renal cortex or medulla, type II activity of inner medulla demonstrates a marked resistance to activation by NaCl and/or urea in subcellular preparations. However, with respect to solute activation, the resistance of the type II enzyme of inner medulla does not differ from that of type II activities from other tissues. In contrast to the effects on basal activity, NaCl and urea potentiated inner medullary type II activation by cyclic AMP and also delayed the rate of subunit reassociation after chromatographic removal of cyclic AMP. Incubation of inner medullary slices in high osmolality buffer (NaCl and urea) did not alone activate soluble protein kinase, an observation which implied that the enzyme was also resistant to solute activation in the intact cell system. Moreover, at 1650 mosM, vasopressin activation of soluble protein kinase was enhanced compared to responses at 750 mosM despite comparabel levels of cyclic AMP accumulation at the two osmolalities. However, a cyclic AMP-independent action of high osmolality to reduce the rate of inactivation of arginine vasopressin-stimulated protein kinase was not demonstrable in inner medullary slices.The present data suggest the possibility that the resistance of inner medullary protein kinase to solute activation could be related to the isomeric form of enzyme (type II) present in this tissue. The high concentrations of NaCl and urea routinely found in inner medulla during hydropenia also influenced protein kinase responses to arginine vasopressin, and may do so in part by directly potentiating the action of cyclic AMP on subunit dissociation.     

Inhibitory effects of nitrogen oxides on a mixed methanogenic culture

The effect of nitrate, nitrite, nitric oxide (NO), and nitrous oxide on a mixed, mesophilic (35 degrees C) methanogenic culture was investigated. Short-term inhibition assays were conducted at a concentration range of 10-350 mg N/L nitrate, 17-500 mg N/L nitrite, 0.02-0.8 mg N/L aqueous NO, and 19-191 mg N/L aqueous nitrous oxide. Simultaneous methane production and N-oxide reduction was observed in 10 and 30 mg N/L nitrate and 0.02 mg N/L aqueous NO-amended cultures. However, addition of N-oxide resulted in immediate cessation of methanogenesis in all other cultures. Methanogenesis completely recovered subsequent to the complete reduction of N-oxides to nitrogen gas in all N-oxide-amended cultures, with the exception of the 500 mg N/L nitrite- and 0.8 mg N/L aqueous NO-amended cultures. Partial recovery of methanogenesis was observed in the 500 mg N/L nitrite-amended culture in contrast to complete inhibition of methanogenesis in the 0.8 mg N/L aqueous NO-amended culture. Accumulation of volatile fatty acids was observed in both cultures at the end of the incubation period. Among all N-oxides, NO exerted the most and nitrate exerted the least inhibitory effect on the fermentative/methanogenic consortia. The effect of multiple additions of nitrate (300 mg N/L) on the same methanogenic culture was also investigated. Long-term exposure of the methanogenic culture to nitrate resulted in an increase of N-oxide reduction rates and decrease of methane production rates, which was attributed to changes in the microbial community structure due to nitrate addition.     

Gonadotropin inhibition during chronic stress: role of the adrenal gland

The effect of adrenalectomy, metyrapone and dexamethasone treatments on gonadotropin response to chronic stress were studied. Adult male rats were submitted to chronic restraint (6 h daily over 4 days). At the end of the last stress period animals were decapitated and trunk blood was collected. Chronic restraint evoked a decrease in plasma LH and to a lesser degree in plasma FSH in the intact rat. Adrenalectomy did not prevent the LH reduction induced by stress and magnified the inhibitory effect of restraint on FSH secretion. Administration of the corticosterone synthesis blocker metyrapone increased the inhibitory effect of restraint on plasma LH and to a lesser degree on plasma FSH. Dexamethasone treatment did not significantly modify plasma gonadotropin levels in adrenalectomized unstressed rats, but this treatment totally blocked plasma LH and FSH reduction after chronic restraint. These results indicate that plasma LH and FSH reduction during chronic restraint is not due to the increase in glucocorticoid secretion, but seems to be mediated by the increase of the hypothalamic-pituitary components of the adrenal axis.     

Feprazone: an inducer of the P450 II B family of proteins in the rat

The ability of feprazone to induce the hepatic microsomal mixed-function oxidases was investigated in the rat, with emphasis being placed on the nature of the cytochrome P-450 family induced. Treatment with feprazone enhanced the p-hydroxylation of aniline and the dealkylations of benzphetamine and pentoxyresorufin but had no effect on the O-deethylation of ethoxyresorufin. The same treatment had no major effect on total cytochrome P-450 levels but increased the spectral interaction of metyrapone with reduced cytochrome P-450. Immunoblots employing monospecific polyclonal antibodies revealed that feprazone induces the apoprotein levels of the P450 II B, but not of the P450 I, family. It is concluded that feprazone is an inducer of the rat hepatic mixed-function oxidase system showing selectivity toward the P450 II B family.     

The genotype dependent presence of pyrrolizidine alkaloids as tertiary amine in Jacobaea vulgaris

Secondary metabolites such as pyrrolizidine alkaloids (PAs) play a crucial part in plant defense. PAs can occur in plants in two forms: tertiary amine (free base) and N-oxide. PA extraction and detection are of great importance for the understanding of the role of PAs as plant defense compounds, as the tertiary PA form is known for its stronger influence on several generalist insects, whereas the N-oxide form is claimed to be less deterrent. We measured PA N-oxides and their reduced tertiary amines by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We show that the occurrence of tertiary PAs is not an artifact of the extraction and detection method. We found up to 50% of tertiary PAs in shoots of Jacobine - chemotype plants of Jacobaea vulgaris. Jacobine and its derivatives (jacoline, jaconine, jacozine and dehydrojaconine) may occur for more than 20% in reduced form in the shoots and more than 10% in the roots. For 22 PAs detected in F(2) hybrids (J. vulgaris × Jacobaea aquatica), we calculate the tertiary amine percentage (TA%=the tertiary amine concentration/(tertiary amine concentration+the corresponding N-oxide concentration) × 100). We found that the TA% for various PAs was genotype-dependent. Furthermore, TA% for the different PAs were correlated and the highest correlations occurred between PAs which share high structural similarity.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

Reduction of lactoperoxidase-H2O2 compounds by ferrocyanide: indirect evidence of an apoprotein site for one of the two oxidizing equivalents

The titration by ferrocyanide and the localization of the oxidizing equivalents of lactoperoxidase "compound II" were studied as a function of pH. It was demonstrated that 1) whatever the pH, the structure of lactoperoxidase "compound II" was compatible with a Fe IV R degree state, 2) at acidic pH, ferrocyanide preferentially reduced the oxidizing equivalent localized on the heme iron to give an Fe III R degree compound, 3) at pH 4.2 only the Fe III R degree form was obtained after reduction of lactoperoxidase "compound II" with one mole of ferrocyanide and whereas at pH greater than 4.2, a mixture of both Fe III R degree and Fe IV R forms was present, 4) lowering the pH from 7.2 to 4.0 induced a transition of Fe IV R state to Fe III R degree state, but increasing the pH from 4.0 to 7.2 did not permit the formation of Fe IV R compound from Fe III R degree compound.     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

Relationship of urotensin I induced vasodilatory action in rat thoracic aorta to Ca2+ regulation

The relaxant effects of the synthetic fish neuropeptide urotensin I were examined in helical strips of rat aorta. In K+-depolarized aorta strips, urotensin I and verapamil competitively inhibited Ca2+-induced contractions. Urotensin I relaxed, in a concentration-dependent manner, the contraction produced by the Ca2+ ionophore A23187, whereas verapamil had no effect on this contraction, even at a concentration of 10(-5) M. In the absence and presence of extracellular Ca2+, urotensin I inhibited both components of the contractions elicited by norepinephrine or urotensin II, another fish neuropeptide. Verapamil reduced only the norepinephrine or urotensin II induced contraction in the presence of extracellular Ca2+, with little or no change in the contraction in Ca2+-free buffer. The urotensin I induced relaxation response in aortic strips contracted by 40 mM KCl was enhanced by pretreatment with papaverine or forskolin. Pretreatment with dibutyryl cAMP did not significantly alter the action of urotensin I. The presence or absence of endothelial cells did not change the response to urotensin I. These results suggest that urotensin I antagonizes the action and (or) mobilization of extracellular and intracellular Ca2+.     

Differences in the polymorphic forms of metallothionein

Metallothionein induced in rat liver by metal ions can be resolved into two forms, referred to as isoforms I and II. These polymorphic forms differ in several properties. Sequence analysis of the CNBr-digested thionein isoforms revealed seven differences in the first 25 amino acids between isoforms I and II of Cd-induced thionein. The sequence of the first 25 amino acids in isoform II of Cu-induced thionein was identical to that of isoform II of Cd-induced thionein, suggesting that the same polymorphic forms are induced with different metal ions. Isoform II of Cd,Zn-thionein exhibited a reproducibly greater Stokes radius (17 Å) by gel filtration compared to isoform I (16.2 Å), whereas isoform II of Cu-thionein eluted with a Stokes radius of 16.2 Å. The isoforms also differ in Zn-binding affinity. Isoform I of several preparations of Cd,Zn-thionein and Zn-thionein invariably reconstituted greater esterase activity in apo-carbonic anhydrase than did isoform II. In Cd,Zn-thionein samples, only the Zn²⁺ ions were transferred to apo-carbonic anhydrase. Similarity, the Zn²⁺ ions in isoform I were more reactive with EDTA compared to isoform II. The greater reactivity of isoform I with EDTA was evident kinetically from spectrophotometric assays as well as thermodynamically from equilibrium dialysis experiments. The significance of these differences is unclear, but it is conceivable that the dissimilarities in the conformational and Zn-binding affinity of rat liver metallothionein may correlate with a functional distinction.     

Participation of cytochrome P-450 in the steroid 17alpha-hydroxylase of testis microsomes.

The effect of metyrapone on the activity of the steroid 17alpha-hydroxylase from rat testis was evaluated. A competitive pattern of inhibition was observed after analysis of data using a least mean squares computer analysis. The substrate for the hydroxylase induced a Type I difference spectrum in an active suspension of Triton treated microsomes. The magnitude of this spectral change was dependent on steroid concentration and was diminished by metyrapone. The effect of metyrapone was abolished at infinite steroid concentration. These results confirm the participation of cytochrome P-450 as a reactant in the 17alpha-hydroxylase reaction.     

Metabolism of carcinogenic urethane to nitric oxide is involved in oxidative DNA damage

Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5'-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H(2)O(2) and Cu(I) in DNA damage. Free (*) OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H(2)O(2)-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[N-(dithiocarboxy)sarcosine](3), demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.     

Histochemical fiber typing and staining intensity in cat and rat muscles.

In the gastrocnemius muscle of cat and rat, staining for oxidative enzymes differentiated three fiber types (A,B,C) and staining for adenosine triphosphate at pH 9.4 differentiated two fiber types (I, II) with a reliability of 90% and 98%, respectively. In cat 96% and in rat 90% of the fibers were typed identically after staining for nicotinamide adenine dinucleotidelinked lactic dehydrogenase (LDH) and succinic dehydrogenase (SDH). When differentiated by staining for LDH, A and B fibers were of type I. IN RAT, 80-90% OF ALL FIBERS WERE OF TYPE 22, COMPPRISING A, B and C fibers. Type I fibers stained for LDH intensely as did C fibers of type II, but stained intermediately for SDH. The degree of staining was measured by photometry. When fibers were stained for LDH, histograms of density showed three peaks corresponding to A, B and C fibers in cat, but only two peaks corresponding to A and C fibers in rat, In cat and rat, the densities of A, B and C fibers belonged to different populations. In soleus muscle of cat and rat stained for LDH, menadione-linked alpha-glycerophosphate dehydrogenase and adenosine triphosphatase at pH 9.4, the degree of staining differed from thatin any type of fiber in gastrocnemius muscle     

Site-directed mutagenesis of dimethyl sulfoxide reductase from Rhodobacter capsulatus: characterization of a Y114 --> F mutant

A system for expressing site-directed mutants of the molybdenum enzyme dimethyl sulfoxide reductase from Rhodobacter capsulatus in the natural host was constructed. This system was used to generate and express dimethyl sulfoxide reductase with a Y114F mutation. The Y114F mutant had an increased k(cat) and increased K(m) toward both dimethyl sulfoxide and trimethylamine N-oxide compared to the native enzyme, and the value of k(cat)/K(m) was lower for both substrates in the mutant enzyme. The Y114F mutant, as isolated, was able to oxidize dimethyl sulfide with phenazine ethosulfate as the electron acceptor but with a lower k(cat) than that of the native enzyme. The pH optimum of dimethyl sulfide:acceptor oxidoreductase activity in the Y114F mutant was shown to be shifted by +1 pH unit compared to the native enzyme. The Y114F mutant did not form a pink complex with dimethyl sulfide, which is characteristic of the native enzyme. The mutant enzyme showed a large increase in the K(d) for DMS. Direct electrochemistry showed that the Mo(V)/Mo(IV) couple was unaffected by the Y114F mutant, but the midpoint potential of the Mo(VI)/Mo(V) couple was raised by about 50 mV. These data confirm that the Y114 residue plays a critical role in oxidation-reduction processes at the molybdenum active site and in oxygen atom transfer associated with sulfoxide reduction.     

Cytochrome P-450 ligands: metyrapone revisited

Expressing metyrapone interactions with ferrous cytochrome P-450 as ligand saturation by cytochrome, rather than the more conventional cytochrome saturation by ligand, an extinction coefficient of 68.5 +/- 1.8 mM-1 cm-1 for the metyrapone complex of dithionite-reduced rat hepatic microsomal cytochrome P-450 was derived. Utilizing this new extinction coefficient, the increased cytochrome P-450 present after phenobarbital induction was almost exclusively that which is able to both bind to metyrapone and form a metabolic-intermediate complex from norbenzphetamine. However, it was not the only subpopulation present in microsomes that was able to bind metyrapone, nor the only one capable of forming a metabolic intermediate complex from norbenzphetamine. Thus, neither technique alone can be used to quantitate the "phenobarbital-induced form" of cytochrome P-450.