Reduction Of Microsomal Cytochrome P-450 Research Articles

Eplerenone relieves spironolactone-induced painful gynaecomastia in a patient with primary aldosteronism

Sir, Eplerenone is the second oral aldosterone antagonist available for the treatment of essential hypertension and congestive heart failure. Spironolactone, the first aldosterone antagonist, although effective for the above conditions, has progestational and antiandrogenic adverse effects due to its non-specific binding to various steroid receptors. We report the case of a 54-year-old Caucasian man, who was admitted to hospital because of severe hypertension and hypokalaemia. The plasma aldosterone/plasma renin activity ratio was well above the cut-off level of 30 ng/dl/ng ml � 1 h � 1 . The acute intravascular volume expansion with the intravenous administration of isotonic saline showed autonomous aldosterone production. The aforementioned results were considered diagnostic of primary aldosteronism. A computerized tomography scan with fine cuts (2.5–3 mm) showed bilateral adrenal gland hyperplasia. Therefore, the patient was started on spironolactone 100 mg t.d.s., and in the following days normokalaemia was restored and blood pressure was well-controlled. Two months later, the patient presented with bilateral painful gynaecomastia. We switched spironolactone to eplerenone 25 mg t.d.s., and the patient was completely relieved within 1 month, while his blood pressure remained well-controlled and serum potassium was normal. Spironolactone is the drug of choice for the treatment of primary aldosteronism, but presents sexual side effects, such as impotence, painful gynaecomastia and menstrual disturbances in pre-menopausal women. Several mechanisms have been proposed for these side effects, such as a dosedependent reduction of microsomal cytochrome P-450, alterations of the testosterone–estrogen ratio, a fall in plasma testosterone or a significant increase in its metabolic clearance, and the peripheral conversion of testosterone to estradiol or increased serum levels of estrone and estradiol [1]. Gynaecomastia may be quite remarkable and its occurrence is dose- and time-dependent. Eplerenone is a highly selective aldosterone antagonist. Spironolactone is approximately 40-fold more potent than eplerenone in blocking aldosterone activation of mineralocorticoid receptor. However, the selectivity of eplerenone is significantly greater than spironolactone at androgen, progesterone and glucocorticoid receptors. Eplerenone is 370-fold less potent at blocking dihydrotestosteroneactivation of androgen receptors compared with spironolactone [2]. Two large clinical studies have evaluated the efficacy and tolerability of these aldosterone antagonists. The Randomized Aldactone Evaluation Study (RALES) found that the administration of spironolactone 12.5–50 mg/day,

担癌マウス肝におけるヘム代謝とチトクロームP-450の代謝回転

Hepatic heme metabolism and in vitro translation of poly(A) + RNA from livers were studied to elucidate the mechanism of the reduction of microsomal cytochrome P-450 in tumor-bearing mouse livers. Hepatic delta-aminolevulinic acid synthase activity in male C57BL/6N mice (0.203 nmol/mg protein/h) at 8 days after the transplantation of Ehrlich ascites tumor cells was the same level as that of normal livers (0.206 nmol/mg protein/h). On the other hand, hepatic heme oxygenase activity of tumor-bearing mice (0.482 nmol/mg protein/10 min) had increased 8 days following i. p. transplantation of tumor cells when compared with that of normal mouse livers (0.296 nmol/mg protein/10 min). SDS-gel electrophoresis of in vitro translation products of poly(A) + RNA extracted from membrane-bound polysomes of the livers from tumor-bearing mice showed no significant differences from that of normal controls in the region of cytochrome P-450, that is, the molecular weight region of Mr = 47,000-60,000, although microsomal protein content in this region estimated by Coomassie-blue staining was reduced. These results suggest that the decrease of heme biosynthesis or the translation of cytochrome P-450 mRNA played a very small part in the reduction of microsomal cytochrome P-450 in tumor-bearing mice.

Loss of hepatic monooxygenase activities, glutathione, and ‘green pigment’ formation after the administration of vinyl-cyclooctane to mice

Vinylcyclooctane, when administered to mice at 500 mg/kg, produced reduction of microsomal cytochrome P-450, heme, aminopyrine- N-demethylase and ethoxycoumarin- O-deethylase activities with respect to control values; furthermore the hepatic reduced glutathione level was depleted suggesting that glutathione is involved in the vinylcyclooctane metabolism. The reduction of cytochrome P-450 and monooxygenase activities was accompanied by the formation of abnormal ‘green pigments’.

Kinetic investigation of rat liver microsomal electron transport from NADH to cytochrome P-450.

NADH-dependent reduction of microsomal cytochrome P-450 has been analyzed kinetically by observing formation of the ferrous-carbonyl complex. Reduction is best described by two exponential equations with apparent first-order rate constants of 1.49 +/- 0.20 and 0.077 +/- 0.015 min-1. By either selective removal or inhibition of specific electron carriers, the kinetic data of minimally altered microsomes are further resolved into a third slow phase. Either addition of anti-cytochrome b5 immune globulin or reduction of cytochrome b5 with ascorbic acid markedly diminishes only the third phase. In reconstitution of purified flavoproteins, phospholipids, and a single isozymic form of cytochrome P-450, without cytochrome b5 only biphasic reduction of cytochrome P-450 is observed. Thus, microsomal cytochrome P-450 appears to be reduced via two independent pathways of electron transport from NADH; the biphasic reduction occurs via cytochrome P-450 reductase while the slower monophasic reduction occurs via cytochrome b5. Multiphasic reduction occurs via cytochrome b5. Multiphasic kinetics are not altered by in vivo inductions of different isozymes of cytochrome P-450. Accordingly, the rates of reduction appear to be an intrinsic property of the electron transport process and not directed by the heterogeneity of the isozymic mixture of ultimate electron acceptors.

Spectral Intermediates during the Reduction of Hepatic Microsomal Cytochrome P-450

The slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps: first reduction of cytochrome P-450 and then some changes in reduced state. The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423 nm and 429 nm, respectively, while that of azide had a peak at 445 nm and a trough at 404 nm. An essay method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducible pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425 nm.

Growth hormone modulation of liver drug metabolic enzyme activity in the rat—I: Effect of the hormone on the content and rate of reduction of microsomal cytochrome P-450

The liver metabolism of hexobarbital and aniline was decreased 48 hr after the first injection of growth hormone (GH) in adult male rats. The content and rate of reduction of hepatic microsomal cytochrome P-450 were lowered in these rats as compared with control animals. Liver NADPH-cytochrome c reductase showed a similar decrease in activity after GH treatment. The decrease in hexobarbital metabolism paralleled the change in cytochrome P-450 reductase activity as measured with or without addition of this drug substrate to a suspension of liver microsomes from GH-treated rats. The change in aniline metabolism approximated the extent and rate of cytochrome P-450 reduction after GH treatment only when cytochrome P-450 reductase activity was measured without addition of aniline. Injection of GH produced a parallel decrease in the metabolism of both drugs as compared with cytochrome c reductase activity. Differences in optimal requirements for drug substrates (hexobarbital or aniline) or NADPH for cytochrome P-450 reductase were not detected. Preincubation studies showed no differences in microsomal drug metabolic enzyme system stability in rats injected with GH. Inhibitors of this system in vitro were not demonstrated in liver from GH-treated rats. GH is presumed to affect the level of liver drug metabolism through mechanisms in vivo operative at the first stage transfer of reducing equivalents to cytochrome P-450. An additional effect of this hormone on the level or catalytic properties of the hemoprotein cytochrome P-450 may contribute to the decrease in aniline metabolism.

On the region of indifference for high pressure transtitutions in d-camphor (II–III) and phosphorus (I–II) : A.F. Gabrysh, H. Eyring & A. Van Hook (Insitute for the Study of Rate Processes and Department of Metallurgy, University of Utah, Salt Lake City, Utah, U.S.A.)

The reduction of both the membrane bound, rat liver microsomal and the soluble, bacterial cytochromes P-450 by sodium dithionite has been studied as a function of temperature (4–37º) and reductant concentration. The results indicate that in this temperature range (a) the reduction of the microsomal enzyme is a biphasic process; each phase is first order at a given dithionite concentration and the percentage of slower phase is relatively constant (70–80%) and independent of the presence or absence of exogenous substrate; (b) the reduction of cytochrome P-450cam is a first order process throughout the temperature range employed and independent of the presence or absence of d-camphor; (c) neither the fast or slow phase of reduction of microsomal cytochrome P-450 nor the single phase of cytochrome P-450cam reduction exhibits a break in the Arrhenius plot; (d) the presence of substrate results in a significant decrease in the rate constant for the dithionite dependent reduction; and (e) the reduction by dithionite does not appear to be a second order process. These findings are compared with previously obtained data from a study of the NADPH-dependent reduction of microsomal cytochrome P-450.

Rapid sorption processes on granular zeolite : W.W. Brand & Rudloff (Department of Chemistry, Illinois Institute of Technology, Chicago 16, Illinois, U.S.A.)

The reduction of both the membrane bound, rat liver microsomal and the soluble, bacterial cytochromes P-450 by sodium dithionite has been studied as a function of temperature (4–37º) and reductant concentration. The results indicate that in this temperature range (a) the reduction of the microsomal enzyme is a biphasic process; each phase is first order at a given dithionite concentration and the percentage of slower phase is relatively constant (70–80%) and independent of the presence or absence of exogenous substrate; (b) the reduction of cytochrome P-450cam is a first order process throughout the temperature range employed and independent of the presence or absence of d-camphor; (c) neither the fast or slow phase of reduction of microsomal cytochrome P-450 nor the single phase of cytochrome P-450cam reduction exhibits a break in the Arrhenius plot; (d) the presence of substrate results in a significant decrease in the rate constant for the dithionite dependent reduction; and (e) the reduction by dithionite does not appear to be a second order process. These findings are compared with previously obtained data from a study of the NADPH-dependent reduction of microsomal cytochrome P-450.