Corticosteroid side chain oxidations--3. Evidence for isomerization and direct oxidation reactions in the formation of steroid 21-oic acids by rabbit liver cytosol.

Isomerase complexes that oxidize the alpha-ketol side chain of deoxycorticosterone (DOC) to pregnolic and pregnenoic acids without the addition of cofactors have been purified from rabbit and hamster liver cytosols. The isomerase complex in rabbit liver cytosol was partially resolved from a NAD-dependent dehydrogenase that oxidized the glycol side chain of 20 beta-dihydro DOC to the 20 beta-hydroxy-21-oic acid. Corticosterone (B) and 20 beta-dihydro B were less active substrates than DOC or 20 beta-dihydro DOC with the corresponding preparations. The rabbit resembles other rodent and human species in that isomerization and oxidation at C21 is the major pathway of acid formation whereas direct oxidation at C21 may only be of significance with metabolites that have the glycol side chain.

Corticosteroid side chain oxidations--II. Metabolism of 20-dihydro steroids and evidence for steroid acid formation by direct oxidation at C-21.

Rabbits have been injected with 4-14C-labelled progesterone, deoxycorticosterone and corticosterone and the corresponding 20 beta-3H-reduced steroids (20-dihydro steroids) in order to compare the influence of oxidation at C-20 on the excretion of steroid acids. Both 20 beta-reduced progesterone and deoxycorticosterone were extensively oxidized at C-20 and metabolized to 20-oxo-21-oic acids devoid of tritium. A small proportion of the acidic metabolites of [20 beta-3H]dihydro deoxycorticosterone retained tritium. By contrast the majority of the metabolites of [20 beta-3H]dihydro corticosterone were tritiated and [11 beta,20 beta-3H]-dihydroxy-4-pregnene-3-one-21-oic acid was identified as a major acidic metabolite. These results indicate that the presence of a 11 beta-hydroxyl in 20 beta-dihydro corticosterone inhibits oxidation at C-20 and provides evidence for the direct oxidation of this corticosteroid at C-21 in this species.

Corticosteroid side-chain oxidations--I. Structural effects on the excreted isotope ratios of 4-14C- and 21-3H-labelled corticosteroid metabolites in rabbit urine.

The metabolic fates of 4-14C- and 21-3H-labelled corticosteroids have been investigated in the rabbit by analysis of the normalized isotope ratios of neutral and acidic metabolites excreted in the urine. Isotope ratios of excreted radioactivity declined in the order cortisol (F) greater than corticosterone (B) greater than 11-desoxycortisol (S) greater than deoxycorticosterone (DOC). Steroid acids, isolated in alumina fraction C, represented 19.0, 15.0, 9.7 and 2.7% of the doses of DOC, B, S and F, respectively, and the isotope ratios declined in the order F greater than B greater than S greater than DOC. HPLC of steroid acid methyl ester derivatives indicated generally low isotope ratios for DOC and S steroid acids, consistent with complete side-chain oxidation to 20-oxo-21-oic acids and/or 17-carboxylic acids. Several B metabolite methyl esters peaks also exhibited low isotope ratios, but both B and F metabolites gave methyl esters that retained significant tritium consistent with the presence of 20-hydroxysteroid acids. The 21-hydroxy-steroid metabolite fractions had isotope ratios of F = S greater than B greater than DOC. HPLC showed that 20-oxo (tetrahydro) metabolites of B and F had reduced isotope ratios unlike the C-20 reduced (hexahydro) metabolites of DOC and S. It may be concluded that the metabolic fate of the corticoid side-chain in the rabbit is dependent on the steroid structure and may result in the excretion of both 20-oxo and 20-hydroxysteroid acids.

A novel chromatographic resolution of 21-hydroxysteroid metabolites of progesterone.

21-Hydroxysteroid metabolites of both progesterone and deoxycorticosterone are excreted in rabbit urine and are eluted from an alumina adsorption column after 21-deoxysteroids. The separation is independent of polarity and dependent on the interaction of the 21-hydroxyl function with the adsorbent. The group separation of these steroids allowed further analysis by high performance liquid chromatography and revealed different proportions of metabolites. This is the first report of the excretion of 21-hydroxysteroid metabolites of progesterone in rabbit urine.

Metabolic fate of [3H]deoxycorticosterone and [14C]progesterone--I. Early tissue metabolites and analysis of 21-hydroxysteroids by high pressure liquid chromatography.

Rabbits injected with mixtures of [3H]deoxycorticosterone and [14C]progesterone had significant levels of both 3H and 14C in several tissues and fluids extracted 10-45 min later. The distribution of radioactivity between 21-deoxysteroid, 21-hydroxysteroid, steroid acid and steroid glucuronide fractions was determined by alumina adsorption chromatography. Steroid acids derived from both steroids accumulated in the liver, kidney and urine, but were quantitatively less significant in the bile, duodenum, uterus, spleen and lung and were detected in the blood for the first time. Different 21-hydroxysteroid profiles were detected in the tissue and fluid extracts by reverse and straight phase high pressure liquid chromatography. [3H]Deoxycorticosterone accumulated in the kidney, lung, spleen and uterus, whereas tetra and hexahydro reduced metabolites predominated in the liver, bile and duodenum. By contrast, [14C]progesterone was metabolised to more polar 21-hydroxylated metabolites which were detected in the liver, kidney and urine. These results show the influence of a steroid 21-hydroxyl function, when administered, as opposed to being formed in in vivo, on the metabolic fate and excretory pathways of 21-hydroxysteroids by the rabbit.

Ketoconazole inhibition of progesterone oxidation by the rabbit.

Ketoconazole, a known cytochrome P-450 inhibitor, inhibited both progesterone ring hydroxylation and side-chain oxidation to steroidal acids. Progesterone 21 6 beta- and 16 alpha-hydroxylase activities of rabbit liver microsomes were inhibited 50% by ketoconazole at concentrations between 10(-5) and 10(-4) M. Steroid acid formation was similarly inhibited at a 10(-5) M concentration. Ketoconazole administration to rabbits produced a significant reduction in the urinary excretion of acidic metabolites of [3H]deoxycorticosterone and [14C]progesterone by approximately 50 and 75% respectively. The differential effect of ketoconazole in vivo may indicate that more than one acidic metabolite pathway may be operative.

Progesterone hydroxylation and side chain oxidation to acidic metabolites by the pig.

The anesthetised pig excreted acidic metabolites of progesterone in the bile and urine. Liver microsomes hydroxylated progesterone at the C-21, 6 beta- and 16 alpha-position and were inhibited by Ketoconazole. The pig exhibited intraspecies variability in progesterone 21- and 6 beta-hydroxylases. Liver, adrenal and to a lesser extent kidney microsomes oxidised the alpha-ketol side-chain to a C-21-oic acid. The liver reaction gave high affinity, low Km kinetics and a Vmax of 28.6 pmol acids mm-1 mg microsomal protein-1. Both carbon monoxide and Ketoconazole were inhibitory. These results implicate the cytochrome P-450 system with the ring and side-chain oxidations of progesterone. The pig resembled the rabbit in its metabolism of progesterone and is the second species in which a microsomal alpha-ketol oxidase has been implicated in the biosynthesis of steroidal acids.

Primary and secondary progesterone hydroxylation by the rabbit liver microsomal cytochromes P-450 system.

Progesterone metabolism by the rabbit liver microsomal cytochromes P-450 system has been investigated with reverse phase (C18) high performance liquid chromatography. Time-course studies indicated that 6 beta-OHDOC accumulated as a secondary metabolite produced by the sequential 6 beta- and 21-hydroxylation of progesterone. When 21-hydroxylation occurred first, DOC accumulated and 6-hydroxylation was reduced. 16-OHP was resistant to secondary metabolism at both the 6- and 21-positions. The variable rates of 21-hydroxylase activity between different rabbits was further influenced by the nature of C-6-function in the order of 6-oxo greater than 6 beta-OH greater than 6 beta-OH greater than 6 alpha-OH greater than 6-H greater than 6 beta-acetoxy. These results indicate the potential interaction of the microsomal cytochromes P-450 and/or products in the sequential hydroxylation of a single steroid substrate at multiple sites.

Multiplicity of progesterone 21-hydroxylase activities associated with constitutive forms of rabbit liver cytochrome P-450.

Constitutive cytochromes P-450 have been solubilized from untreated outbred New Zealand White rabbit liver microsomes. Gradient phosphate buffer elution of DEAE-cellulose columns partially resolved six P-450 fractions. Progesterone 21-hydroxylase activity was reconstituted with several fractions and inhibited by an antibody towards P-450 Form 1. One fraction (LM3b) preferentially catalysed the 6 beta- and 16 alpha-hydroxylation of progesterone. SDS-PAGE indicated the presence of proteins with mobilities closely related to Form 1 in several fractions that were separated from this isozyme by DEAE-cellulose chromatography. These results suggest that several constitutive P-450 fractions may contribute to the regiospecific 21-hydroxylation of progesterone.

Rates of progesterone oxidation by rabbit liver microsomes before and after phenobarbitone treatment.

Liver sections, removed from outbred NZW rabbits under anesthesia, were used to determine the rates of progesterone oxidation prior to assessment of the effects of phenobarbitone(PB)-treatment. Progesterone 21-hydroxylase exhibited the same high affinity, low Km kinetics before and after PB-treatment, whereas the Vmax was significantly reduced. PB-treatment did not affect progesterone 21-hydroxylation by the adrenal microsomes of two groups of PB-treated and untreated rabbits. 21-Hydroxylase activity was detected for the first time with spleen microsomes but the majority of spleens examined lacked activity. 6 beta-Hydroxylation by control liver microsomes showed consistently low affinity, high Km-values and was inhibited to a lesser, inconsistent degree than 21-hydroxylation by PB-treatment.

Lack of correlation between hepatic microsomal progesterone 21-hydroxylase activity and the excretion of acidic metabolites in rabbit urine.

Hepatic microsomes were prepared from liver sections removed from anesthetised New Zealand White rabbits. Two groups of rabbits, distinguished by different levels of progesterone 21-hydroxylase, were compared for in vitro and in vivo metabolism of [3H]deoxycorticosterone/[14C]progesterone mixtures. In vitro, microsomes with low 21-hydroxylase activity gave significantly lower yields of pregnenoic acid than those with higher 21-hydroxylase activities. In vivo, these differences were not evident from an examination of the 3H/14C ratios of acidic urinary metabolites.

Solubilisation and fractional precipitation of a steroid alpha-ketol oxidase.

An alpha-ketol oxidase that converts deoxycorticosterone to pregnenoic acid has been solubilised and fractionally precipitated with polyethylene glycol from rabbit liver microsomes. Maximal activity was obtained with the 12-14% fraction. Activity was linear with cytochrome P-450 concentrations up to 0.2 nmol and was inhibited with carbon monoxide.

Progesterone C21 hydroxylation and steroid carboxylic acid biosynthesis in the rabbit. In vitro studies with endocrine, metabolic, and potential target tissues.

Progesterone C21-hydroxylase activity has been demonstrated with rabbit kidney microsomes for the first time and the formation of 21-hydroxy-4-pregnen-3,20-dione (DOC) by rabbit liver and kidney microsomes has been quantitated. Considerable intraspecies variability in enzyme activity occurred with both tissues. The liver enzyme (Vmax = 1.28-38.0 nmol/mg protein per 30 min of incubation) was significantly more active than the kidney enzyme (Vmax = 0.028-0.28 nmol/mg protein per 30 min of incubation). Apparent KM values were 1.39 and 0.8 microM, respectively. Cytochrome c (10(-5)M), potassium ferricyanide (10(-3)M), and 2-methyl-1,2-di-3-pyridyl-1-propanone (metyrapone; 10(-3)M) were strongly inhibitory with both tissues, whereas the liver microsomal system was less sensitive than the kidney to CO-air (90:10 v/v) inhibition. Metabolism of [14C]DOC to 4-pregnen-3,20-dione-21-oic (pregnenoic) and 4-androsten-3-one-17 beta-carboxylic (etienic) acids by liver microsomes and adrenal and ovary homogenates was differentially affected by several factors. CO-air (90:10 v/v), cytochrome c (10(-5)M), and metyrapone (10(-3)M) inhibited pregnenoic acid synthesis to a greater extent than etienic acid. Sodium cyanide had a stimulatory effect on the synthesis of pregnenoic acid by the liver but less consistent effects with other tissues. These results suggest that one or more cytochrome P-450 systems may be involved in the oxidation of progesterone through to pregnenoic acid by rabbit tissues.

C-21- and 6-hydroxylation of progesterone by rabbit liver subcellular fractions.

In vitro C-21-hydroxylation of [3H]progesterone (P) has been demonstrated for the first time with rabbit liver microsomes and mitochondria. Deoxycorticosterone (DOC) was rigorously characterized as a metabolite of both mitochondrial and microsomal metabolism, whereas 6alpha-hydroxy DOC and 6alpha-hydroxy P were only identified as microsomal metabolites. 6beta-Hydroxy metabolites were also detected but were of less quantitative significance. Formation of 6alpha-hydroxy P and 6alpha-hydroxy DOC increased steadily between 5 and 120 min of incubation with the microsomal fraction, whereas DOC increased up to 30 min of incubation and then declined. Maximal yield of DOC was 25.9 and 22.5 pmol/mg protein with the mitochondrial and microsomal fractions, respectively.