Intraspecies individuality for the metabolism of steroids

A variety of regulatory factors contribute to differences in the rates of 6 beta-hydroxylation, 16 alpha-hydroxylation and 21-hydroxylation of progesterone as catalysed by liver microsomes prepared from individual rabbits. It is likely that the 6 beta-hydroxylation of progesterone is catalysed primarily by cytochrome P-450 3c, an enzyme that exhibits allosteric activation by alpha-napthoflavone, and by a form of P-450 3b, 6 beta+, that is expressed in some rabbits in an autosomal dominant manner. The mechanism of activation for P-450 3c appears to reflect an effector mediated increase of the affinity of the enzyme for substrate as judged by substrate binding studies. A second form of P-450 3b, 6 beta-, catalyses a major portion of hepatic progesterone 16 alpha-hydroxylation and exhibits activation by a variety of C21 steroids of which 5 beta-pregnane-3 beta,20 alpha-diol is the most efficacious. P-450 1, which catalyses the 21-hydroxylation of progesterone, is expressed at 10-fold higher levels in the 21H phenotype than the 21L phenotype, and the former is inherited as an autosomal dominant characteristic. A cDNA encoding a P-450 1-related gene product exhibits a predicted amino acid sequence that is 95% homologous to that of P-450 1. The P-450 1-related gene product is expressed in liver to a similar degree in both 21H and 21L rabbits.     

The metabolism of antifertility steroids. The in virto metabolism of chlormadinone acetate

The isolation and identification of the major in vitro metabolites of chlormadinone acetate are reported. Human, rabbit, and rat liver incubations were employed in the isolation and identification, and male rat liver preparations were used to study the effect of phenobarbital stimulation on the formation of these metabolites. The metabolic pathway was influenced by the microsomal stimulating property of phenobarbital. 2alpha-hydroxychlormadinone was the major metabolite from the incubation of phenobarbital-stimulated rabbit and male rat liver. 17alpha-acetoxy-6-chloro-3beta-hydroxypregna-4,6-diene-20-one was the major metabolite from the incubation of unstimulated human and male rat liver. It was concluded that the 2alpha-hydroxychlormadinone acetate was demostrated only as a consequence of phenobarbital treatment.     

Effect of ovarian steroids on the metabolism of noradrenaline in rabbit uterus

The metabolism of (-)-3H-noradrenaline was examined in uterine slices from ovariectomized rabbits which were either untreated or treated with 17 beta-oestradiol, alone or in combination with progesterone. 17 beta-oestradiol caused uterine enlargement which was not accompanied by a change in the formation per g of O-methylated metabolites (3H-NMN, 3H-VMA, 3H-MOPEG). Accumulation of unchanged 3H-noradrenaline and the formation of deaminated catechols (3H-DOMA and 3H-DOPEG) were decreased per g tissue, but increased per uterine horn. Progesterone produced further enlargement of the oestrogen-dominated uteri which was accompanied by (a) a decrease in deaminated catechol formation and (b) an increase in 3H-NMN formation per unit mass of tissue. In all uteri (control and hormone-treated), cocaine inhibited the formation of deaminated catechols, but not that of the O-methylated metabolites. It is suggested, therefore, that, per unit of uterine mass, the neuronal deamination of (-)-3H-noradrenaline is decreased by 17 beta-oestradiol and further decreased by progesterone, and that these changes reflect failure of the intraneuronal deaminating system in the whole uterus to increase in proportion to the increase in uterine mass. Since other agents which decreased the deamination of (-)-3H-noradrenaline (cocaine and nialamide) did not affect 3H-NMN formation in oestrogen-dominated uteri, it is suggested that stimulation of 3H-NMN formation represents a direct effect of progesterone on the extraneuronal O-methylation of noradrenaline.     

Studies related to the metabolism of anabolic steroids in the horse: testosterone

1. After intramuscular administration of [4-¹⁴C]testosterone to two cross-bred gelded horses, 45% of the radioactivity was excreted in urine in 96 h. Small amounts of urinary activity could still be detected at 200 h.

2. Neutral metabolites obtained after both enzyme and acid hydrolysis of urine samples have been investigated by g.l.c.-mass spectrometry.

3. 5α-Androstane-3β, 17α-diol was found only in the enzyme-hydrolysable extract and testosterone only in the acid-hydrolysable extract. 5α-Androstane-3β, 17β-diol and 3β-hydroxy-5α-androstan-17-one were found predominantly in the acid-hydrolysable extract.     

Further studies on the inhibition of drug metabolism by pregnanolone and related steroids

Because of our previous findings that pregnanolone and related metabolites of progesterone inhibit drug demethylation and hydroxylation reactions in vitro, a variety of other steroids and naturally occurring substances were tested for inhibition of the O-demethylation of p-nitroanisole by 9000 g hepatic supernatants from adult male rats. Of some 58 steroids tested, none was more potent than pregnanolone. Other potent inhibitors, with the exception of norethindrome, had a similar structure (C₂₁) with hydroxyl or keto groups at C₃ and C₂₀. Inhibitory activity was not related to the endocrine activity of the steroid. Inhibition by a group of 11 steroids was generally greater with 9000 g supernatants from female rats, and was uniformly increased with supernatants from pregnant rats. By 10 days after delivery, inhibition returned to levels equivalent to those found with supernatants from virgin females. Cholesterol and related compounds, fatty acids and esters, ceramides, gangliosides, phosphatidyl inositol and other natural fatty acid conjugates and a variety of indoles and lipids had no inhibitory activity. Pregnanolone administration increased the duration of hexobarbital- and of barbital-induced sleep in rats, apparently due to an additive sedative effect. However, microsomes prepared from these animals exhibited lesser rates of demethylation, with kinetics compatible with uncompetitive inhibition. These results indicate that progesterone, its metabolites and structurally similar steroids are potent inhibitors of microsomal drug metabolism. Such inhibition may be responsible in part for the low rates of drug metabolism seen in the female, during pregnancy, and in the neonate.     

Studies related to the metabolism of anabolic steroids in the horse: testosterone.

1. After intramuscular administration of [4-14C]testosterone to two cross-bred gelded horses, 45% of the radioactivity was excreted in urine in 96 h. Small amounts of urinary activity could still be detected at 200 h. 2. Neutral metabolites obtained after both enzyme and acid hydrolysis of urine samples have been investigated by g.l.c.-mass spectrometry. 3. 5 alpha-Androstane-3 beta, 17 alpha-diol was found only in the enzyme-hydrolysable extract and testosterone only in the acid-hydrolysable extract. 5 alpha-Androstane-3 beta, 17 beta-diol and 3 beta-hydroxy-5 alpha-androstan-17-one were found predominantly in the acid-hydrolysable extract.     

Structural modifications in contraceptive steroids altering their metabolism and toxicity

Norethisterone and, to a lesser extent, d-norgestrel are metabolically activated by rat liver microsomal enzymes to intermediates which are capable of irreversibly binding to proteins. This microsomal activation in vitro depends on presence of NADPH and is inhibited by glutathione. Irreversible binding of metabolites of progesterone, nortestosterone acetate and cyproterone acetate is very low, compared to that of norethisterone metabolites. Phenol as a reference compound shows quantitatively a similar binding behaviour as norethisterone. Norethisterone-4,5-epoxide, a microsomal metabolite of norethisterone, binds non-enzymatically to albumin, at a rate of 380 pmol/mg albumin per hour (at 37°). The corresponding rate for norgestrel-4,5-epoxide, 42 pmol/mg albumin per hour, indicates a considerably lower reactivity of norgestrel-epoxide. The non-SH-proteins concanavalin A and bovine -globulin do not react with either norethisterone-epoxide or norgestrel-epoxide. Also, DNA and RNA show no binding reaction. Thus, the requirements for irreversible protein binding of the 19-nortestosterone progestagens norethisterone and norgestrel are similar to those found for oestrogens which, when activated by rat liver microsomes, only bind to proteins with SH-groups, not to DNA or RNA.Presented at the Symposium Influence of Metabolic Activations and Inactivations on Toxic Effects held at the 18th Spring Meeting of the Deutsche Pharmakologische Gesellschaft, Section Toxicology, D-6500 Mainz, March 15, 1977     

Influence of sex and oral contraceptive steroids on paracetamol metabolism.

Paracetamol metabolism was investigated in eight healthy males, eight healthy females and eight healthy females receiving oral contraceptive steroids (OCS). Paracetamol clearance was 22% greater in males compared to the control female group. This difference was entirely due to increased activity of the glucuronidation pathway in males, there being no sex-related differences in the sulphation or oxidative metabolism of paracetamol. Paracetamol clearance in females using OCS was 49% greater than in the control females. Glucuronidation and oxidative metabolism were both induced in OCS users (by 78% and 36% respectively) but sulphation was not altered. Although sex-related differences in paracetamol metabolism are unlikely to be of clinical importance, induction of paracetamol metabolism by OCS may have clinical and toxicological consequences.     

Comparative metabolism of 17alpha-ethynyl steroids used in oral contraceptives.

The metabolism of mestranol, ethynylestradiol, norethynodrel, norethindrone, ethynodiol diacetate, lynestrenol, and norgestrel is reviewed. The estrogenic components of the oral contraceptives, mestranol or ethynylestradiol, have nearly identical metabolic pathways since mestranol is rapidly and almost completely converted to ethynylestradiol The major fraction of the drugs plus metabolites is excreted in the urine as conjugated materials. All of the 17beta-ethynyl progestins reviewed follow similar metabolic paths. For three of these, norethynodrel, ethynodiol diacetate and lynestrenol, a principal metabolite is norethindrone. Biotransformation to more polar metabolites and conjugation proceed rapidly for these three precursor drugs and norethindrone. Norgestrel follows metabolic paths similar to those of norethindrone. However, the ethyl moiety at the C-13 position appears to slow the metabolism of this steroid so that biotransformation to more polar metabolites and the conjugation of these steroids does not proceed as rapidly as that of the other progestins. The high progestational potency of norgestrel may be attributed to this slow rate of biotransformation. Some of the pharmacokinetic parameters derived from the research reports reviewed here are summarized. The compounds appear to be readily absorbed, and they and their metabolites are excreted to a greater extent in the urine than in the feces.     

Effects of the oestrous cycle and exogenous ovarian steroids on metabolism of beta-phenylethylamine in rat lung.

1 Metabolism of [14C]-beta-phenylethylamine (PEN), a substrate for monoamine oxidase-B (MAO-B), was measured in lung homogenates and in perfused lungs during the 4 day oestrous cycle of the rat. 2 Metabolism in vitro was high during met-oestrus and di-oestrus and low during pro-oestrus and oestrus; this variation in activity correlated with changes in Vmax of the enzyme without changes in Km. 3 PEN metabolism in lung homogenates was also altered by treatment of rats with 17 beta-oestradiol but not by progesterone treatment. 4 Metabolism of [14C]-PEN in perfused lungs was the same during either pro-oestrus or met-oestrus. Uptake of [14C]-PEN in perfused lung measured directly was also the same at these two stages. 5 These results demonstrate that in lungs MAO-B activity was affected by endogenous changes in steroid level but that such changes in enzymic activity were not reflected in the metabolic properties of whole lung.     

Influence of gender and oral contraceptive steroids on the metabolism of salicylic acid and acetylsalicylic acid.

Salicylic acid and acetylsalicylic acid (aspirin) disposition after an oral dose of aspirin, 900 mg (equivalent to 689.7 mg of salicylic acid) was studied in eight males, eight females and eight females receiving oral contraceptive steroids (OCS). Salicylic acid clearance was 61% higher in males compared to the control female group, an effect due largely to enhanced activity of the glycine conjugation pathway (salicyluric acid formation) in males. Salicylic acid clearance was 41% higher in OCS-users compared to the control female group due to increases in both the glycine and glucuronic acid conjugation pathways in the pill users. There was no difference in any salicylic acid disposition parameter between males and OCS-users. Area under the plasma concentration-time curve (AUC) and elimination half-life of aspirin was significantly greater and aspirin plasma hydrolysis rate was significantly lower in both female groups compared to males. There was no difference between OCS-users and the control female group in any of these parameters. Aspirin AUC and elimination half-life were significantly correlated with aspirin plasma hydrolysis rate. These data confirm the importance of hormonal factors in the regulation of drug conjugation reactions in humans and suggest that sex-related differences in salicylic acid and aspirin disposition may be of clinical importance.