Metabolism of androstenedione by guinea-pig peritoneal macrophages: synthesis of testosterone and 5α-reduced metabolites

Androstenedione metabolizing enzymes present in guinea-pig peritoneal macrophages were investigated using tritium-labeled androstenedionc as the substrate. We found that the metabolites of [³H]-androstenedione produced by these macrophages were testosterone. 5α-androstane-3,17-dione, isoandrosterone, androsterone, 5α-dihydrotestosterone, 5α-androstane-3α,17β-diol and 5α-androstane-3β,17β-diol. The rates of metabolite formation remained linear as a function of time of incubation for approximately 30 min and with macrophage number up to 2 × 10⁷ cells per ml. The formation of these metabolites is indicative that the following androstcnedione metabolizing enzymes are present in guinea-pig peritoneal macrophages: 5α-reductase, 3α-hydroxystcroid oxidoreductasc, 3β-hydroxysteroid oxidoreductase and 17β-hydroxystcroid oxidoreductasc. It is possible, therefore, that the macrophage, in vivo. may play a role in the metabolism of blood-borne androstcnedione to potent androgens. These hormones are important in the regulation of many biological processes, possibly including the activity of the macrophage itself.     

The metabolism of androgens in rat preputial glands

The metabolism of testosterone, androstenedione and dehydroepiandrosterone in the rat preputial gland has been studied. A high activity of 5α-reductase is present as shown by the formation of 17β hydroxy-5α-androstan-3-one and 5α-androstan-3, 17-dione as the major products from testosterone and androstenedione respectively. Other enzyme activities are present including 17β-hydroxy steroid dehydrogenase, but the amounts of testosterone and 17β-hydroxy-5α-androstan-3-one formed from androstenedione and dehydroepiandrosterone are low. The main product of dehydroepiandrosterone metabolism was androstenedione indicating a high level of 3β-hydroxy steroid dehydrogenase 4-5 isomerase activity. The metabolism was compared with that in rat skin where it was found that the extent of metabolism was much less. The possible significance of the various products formed and of differences between skin and preputial gland metabolism is discussed. Some differences were noted between the metabolism of androgens by rat skin and preputial gland and the metabolism of androgens by human skin.     

Metabolism of testosterone in human,rat and rabbit fetal lungs and in rabbit neonatal lung in vitro

• 1.1. Metabolism of 4-¹⁴C-testosterone was investigated in human, rat and rabbit fetal lung subcellular fractions and also in rabbit neonatal lungs. Androst-4-ene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and both 5α- and 5β-androstane-3α,17β-diols were identified as metabolites of testosterone.
• 2.2. The microsomal fraction produced mainly 5α-reduced epimers while the cytosol incubations resulted in 5β-reduced metabolites.
• 3.3. No conjugation was found.
• 4.4. The amounts of polar metabolites in the microsomal incubations and the amounts of dihydroxy-lated metabolites in the soluble fraction incubations were statistically significantly greater in the neonatal rabbit lung incubations compared with those of fetal lungs.

     

The metabolism of testosterone by human,rabbit and rat kidney

• 1.1. The metabolism of testosterone by the homogenate, microsomal and soluble fractions of human, rabbit and rat kidneys was investigated. Human kidneys were obtained from patients who underwent surgery for cancer, and the metabolism of testosterone was investigated using the intact part and in some cases also the cancerous part.
• 2.2. Testosterone was metabolized by the homogenate and microsomal preparation of male and female human and male rat kidneys to androstenedione (4-androstene-3,17-dione) and to a lesser extent to monohydroxymonoketosteroids, dihydroxysteroids and to more polar metabolites. The main metabolites in the soluble fraction were dihydroxysteroids of the 5β-series.
• 3.3. The rabbit kidneys differed from human and rat kidneys by showing a much greater rate of testosterone metabolism and by producing epitestosterone—via androstenedione—as a major metabolite. The formation of epitestosterone was especially marked in the homogenate of rabbit kidney.

     

C-19-steroid 7α-hydroxylation by rat testes. isolation and identification of: 7α, 17β-dihydroxy-5α-androstan-3-one, 5α-androstan-3α,7α,17β-triol and 5α-androstane-3β,7α, 17β-triol

From incubations of testosterone with rat testicular homogenates in the presence of a NADPH-generating system, the following 7α-hydroxylated metabolites could be isolated and identified: 7α,17β-dihydroxy-4-androsten-3-one (7α-hydroxy-testosterone), 7α-17β-dihydroxy-5α-androstan-3-one (7α-hydroxy-Dht), 5α-androstan-3α,7α,17β-triol (7α-hydroxy-3α-A'DIOL) and 5α-androstane-3β,7α,l7β-triol (7α-hydroxy-3β-A'DIOL). To our knowledge this is the first demonstration of the formation of 5α-reduced-7α-hydroxylated metabolites of testosterone in the male gonad. These 5α-reduced-7α-hydroxylated metabolites could also be isolated after incubations of 5α-androstane-3α,17β-diol (3α-A'D10L) with testicular homogenates in the presence of a NADPH-generating system.Measured as the sum of 7α-hydroxy-testosterone, 7α-hydroxy-Dht. 7α-hydroxy-3α-A'DIOL and 7α-hydroxy-3β-A'DIOL formed using testosterone as substrate, total 7α-hydroxylase activity was six times higher in testes of mature rats than in testes from animals 23 days old. With 3α-A'DIOL as substrate total 7α-hydroxylase in the mature testis was about three times greater than in the sexually immature testis.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

Biotransformation of testosterone and other androgens by suspension cultures of Nicotiana tabacum “Bright yellow”

It has been shown that the cultured cells of Nicotiana tabacum “Bright Yellow” are capable of transforming testosterone to Δ⁴-androstene-3, 17-dione, 5α-androstan-17β-ol-3-one, 5α-androstane-3β, 17β-diol, its dipalmitate and 3- and 17-monoglucosides, epiandrosterone, its palmitate and glucoside, testosterone glucoside. 5α-Androstane-3β, 17β-diol dipalmitate and 3- and 17-monoglucosides, epiandrosterone palmitate and glucoside, and testosterone glucoside have been found for the first time as metabolites of testosterone in plant systems. Δ⁴-Androstene-3,17-dione was converted to testosterone. 5α-Androstan-17β-ol-3-one, which has been recognized as an active form of testosterone in mammals, was also detected. It has also been demonstrated that [4-¹⁴C]testosterone is actively incorporated in these transformations.     

Nuclear metabolism of benzo(a)pyrene and of (±)-trans-7,8-dihydroxy-7,8,-dihydrobenzo(a)pyrene: Comparative chromatographic analysis of alkylated DNA

Rat liver nuclei were incubated with [¹⁴C]benzo(a)pyrene (BP) or [³H](±)-trans-7,8-dihydrodiol of BP (³H-BP-7,8-diol) in the presence of a NADPH-generating system. The nuclei were able to form from BP the 9,10-, 4,5- and 7,8-dihydrodiols, the 3,6- and 1,6-quinones as well as the 3- and 9-phenols. The total nuclear metabolism was stimulated 11-fold by prior administration to the rats of 3-methylcholanthrene (3MC). BP-7,8-dihydrodiol formation, under these circumstances, was enhanced 29-fold. The rat liver nuclei were also able to form from [³H]BP-7,8-diol, (±)-7β,8α-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 1), (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro BP (diol epoxide 2), as well as three unknown metabolites. Diol epoxides 1 and 2 represented 23 and 65% of the total metabolites produced during the control nuclear incubation. Pretreatment of the rats with 3MC resulted in 4-fold increase in nuclear metabolic activity. Under the latter circumstances, the diol epoxides 1 and 2 represented 43 and 38%, respectively, of the total nuclear metabolites. Incubation of liver nuclei with labeled BP or BP-7,8-diol in the presence of NADPH resulted in alkylation of DNA. The alkylated deoxyribonucleosides were separated by Sephadex LH-20 chromatography. Two peaks of radioactivity were noted after incubation with the parent polycyclic hydrocarbon while only one peak was seen after incubation with the diol derivative. These results emphasize the importance of nuclei in the metabolism of BP and in the subsequent alkylation of DNA, reactions which may be related to mutagenesis or carcinogenesis.     

Biliary metabolites of testosterone and testosterone glucosiduronate in the rat

A mixture of testosterone-4-¹⁴C and testosterone-1,2-³H-17-glucosiduronate was intraperitoneally administered into male and female rats with bile fistulas. Biliary metabolites were separated and purififd by a combination of column chromatography, enzymic hydrolysis or solvolysis of the conjugate fractions and identification of the liberated aglycones. The injected steroids were extensively metabolized and excreted predominantly in the blue. 5β-Androstane-3α, 17β-diol was found principally in monoglucosiduronate fraction and was produced preferentially from the injected conjugate in both sexes. Very marked sex differences from the injected conjugate in both sexes. Very marked sex differences were observed in the following metabolites: Androsterone was present only in the female as monoglucosidironate, which was preferentially derived from testosterone. 5α-Androstane-3α,17β-diol was identified in both monoglucosiduronate and diconjugate fractions of the female, which was formed significanrly more from the conjugate than testosterone. These findings provide evidence that testosterone glucosiduronate could be converted directly into 5α-steroids as well as 5β-ones $\text{in}$$\text{vivo}$. In marked contrast, the major portion of testosterone was metabolized to polar steroids in the male.     

Metabolism of prostaglandin D2 in isolated rat lung: the stereospecific formation of 9α, 11β-prostaglandin F2 from prostaglandin D2

The metabolic transformation of exogenous prostaglandin D₂ was investigated in isolated perfused rat lung. Dose-dependent formation (2–150 ng) of 9α,11β-prostaglandin F₂, corresponding to about 0.1% of the perfused dose of prostaglandinD₂, was observed by specific radioimmunoassay both in the perfusate and in lung tissue after a 5-min perfusion. To investigate the reason for this low conversion ratio, we analyzed the metabolites of tritium-labeled 9α,11β-prostaglandin F₂ and prostaglandin D₂ by boric acid-impregnated TLC and HPLC. By 5 min after the start of perfusion, 9α,11β-prostaglandin F₂ disappeared completely from the perfusate and the major product formed remained unchanged during the remainder of the 30-min perfusion. The major product was separated by TLC and identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ by GC/MS. In contrast, pulmonary breakdown of prostaglandin D₂ was slow and two major metabolites in the perfusate increased with time, each representing 56% and 11% of the total radioactivity at the end of the perfusion. The major product (56%) was identified as 13,14-dihydro-15-ketoprostaglandin D₂ and the minor one (11%) was tentatively identified as 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂ based on the results from radioimmunoassays, TLC, HPLC, and the time course of pulmonary breakdown. These results demonstrate that the metabolism of prostaglandin D₂ in rat lung involves at least two pathways, one by 15-hydroxyprostaglandin dehydrogenase and the other by 11-ketoreductase, and that the 9α,11β-prostaglandin F₂ formed is rapidly metabolized to 13,14-dihydro-15-keto-9α,11β-prostaglandin F₂.     

Identification of drostanolone and 17-methyldrostanolone metabolites produced by cryopreserved human hepatocytes

Methyldrostanolone (2α,17α-dimethyl-17β-hydroxy-5α-androstan-3-one) was synthesized from drostanolone (17β-hydroxy-2α-methyl-5α-androstan-3-one) and identified in commercial products. Cultures of cryopreserved human hepatocytes were used to study the biotransformation of drostanolone and its 17-methylated derivative. For both steroids, the common 3α- (major) and 3β-reduced metabolites were identified by GC-MS analysis of the extracted culture medium and the stereochemistry confirmed by incubation with 3α-hydroxysteroid dehydrogenase. Structures corresponding to hydroxylated metabolites in C-12 (minor) and C-16 were proposed for other metabolites based upon the evaluation of the mass spectra of the pertrimethylsilyl (TMS-d₀ and TMS-d₉) derivatives. Finally, on the basis of the GC-MS and ¹H NMR data and through chemical synthesis of the 17-methylated model compounds, structures could be proposed for metabolites hydroxylated in C-2. All the metabolites extracted from hepatocyte culture medium were present although in different relative amounts in urines collected following the administration to a human volunteer, therefore confirming the suitability of the cryopreserved hepatocytes to generate characteristic metabolites and study biotransformation of new steroids.     

C19-steroid 5β-reductase and 3- and 17-oxidoreductases of adult male hamster kidney

Male hamster kidney cytosol exhibited strong 5β-reductase activity. Incubation of cytosol with [4-¹⁴C]-testosterone at pH 6.7 yielded 5β-DHT with minor quantities of 5β-androstane-3α,17β-diol and 5β-androstane-3β,17β-diol. Incubation with [4-¹⁴C]-androstendione yielded 5β-androstanedione and smaller quantities of testosterone, 5β-DHT, 3α-hydroxy-5β-androstan-17-one, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α,17β-diol. The two major metabolites were progressively increased with increase in the concentration of the respective substrates but the other metabolites showed very little change. The metabolism of the respective substrates was progressively decreased with changes in pH of the incubation mixture from 6.0–7.5 accompanied by a parallel decrease in the formation of the respective major metabolites. NADPH was much more effective than NADH as coenzyme. The microsomes exhibited a trace of 5β-reductase activity only with NADPH and androstenedione.The kidney homogenate at pH 10.1 effectively converted [4-¹⁴C]-testosterone to [4-¹⁴C]-androstenedione. The dehydrogenase activity was present in the cytosol and microsomes. NAD⁺ was more effective than NADP⁺ in the cytosol and the reverse was indicated for the microsomes. Spectrophotometric assay revealed not only NADP⁺-linked Hβ-dehydrogenase activity but also a lower 3α-dehydrogenase activity but no detectable 3β- or 17α-dehydrogenase activity. NAD⁺-linked activity was not explored because of the interference by the very high endogenous NAD⁺-reduetase activity.     

Isotope ratio mass spectrometry analysis of the oxidation products of the main and minor metabolites of hydrocortisone and cortisone for antidoping controls

Metabolites of hydrocortisone (HC) and cortisone (C), namely tetrahydrocortisol (THF), tetrahydrocortisone (THE), allo-THF, allo-THE for the main metabolites and 11-hydroxyandrosterone, 11-hydoxyetiocholanolone, 11-ketoandrosterone, and 11-ketoetiocholanolone for the minor metabolites, as well as the two main metabolites of testosterone, androsterone and etiocholanolone, were separated from each other using HPLC fractionation of urine extracts. An isotopic ratio mass spectrometry (IRMS) analysis determined the absolute δ¹³C values of 5α-androstanetrione (5α-AT) and 5β-androstanetrione (5β-AT) as the oxidation products (ox-products) of the HC and C metabolites and as target compounds (TCs). We also performed IRMS analysis of 5α-androstanedione (5α-AD) and 5β-androstanedione (5β-AD) as the ox-products of etiocholanolone and androsterone and as endogenous reference compounds (ERCs). Urine samples came from two male volunteers treated with a single 10-mg oral dose and a single 100-mg intramuscular dose of HC hemisuccinate, a male volunteer treated with a single 25-mg oral dose of C acetate, and a control group of 30 drug-free athletes. The mean −3SD of δ¹³C depletion values from the controls were −1.46, −1.98, −1.78 and −2.42 for 5β-AT-5β-AD, 5α-AT-5β-AD, 5β-AT-5α-AD and 5α-AT-5α-AD, respectively, indicating −3‰ as a safe cut-off value for differentiating the pharmaceutical from the natural form. In the main metabolite fraction, δ¹³C depletion values peaked around −5‰ and −9‰ after oral and intramuscular administration of HC, respectively, and around −6‰ after oral administration of C. In comparison, less impressive results were obtained when IRMS analysis focused on the ox-products of the minor metabolites.     

Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.     

A change in the steroid metabolic pathway in human testes showing deteriorated spermatogenesis

During androgen biosynthesis, the human testes normally produce only small quantities of Δ⁴-C₂₁ steroids as these are products of the Δ⁴-pathway and healthy human testes preferentially use the Δ⁵-pathway. However, the Δ⁴-C₂₁ steroid progesterone accumulates in the thickened lamina propria of the seminiferous tubules in testes with deteriorated spermatogenesis. The objectives of this study were to analyse the pregnenolone metabolites in testes with deteriorated spermatogenesis and to establish whether the androgen biosynthesis pathway changes in this condition. Biopsied or orchiectomised testicular samples were obtained from patients with varicocele, non-obstructive azoospermia, obstructive azoospermia, testicular cancer, and cryptorchidism. The samples were segregated into spermatogenesis related Johnsen’s score groups: Low-JS (< 5.0) and High-JS (> 7.8). Higher levels of progesterone and 17α-hydroxyprogesterone were metabolised under in vitro conversion in the Low-JS testes than the High-JS testes when cell-free homogenates from each group were separately incubated with ¹⁴C-labelled pregnenolone. Nevertheless, the serum hormone levels did not differ between groups. Two novel pregnenolone metabolites 5β-pregnan-3β-ol-20-one and 5α-pregnan-3α, 21diol-20-one were identified from in vitro conversion in Low-JS testes and by recrystallisation. Immunohistochemistry revealed the higher βHSD expression in the Low-JS than the High-JS testes. However, the CYP17A1 expression levels did not differ between groups. Infertile testes increase the relative βHSD levels in their Leydig cells and synthesised testosterone from pregnenolone via the Δ⁴- rather than the Δ⁵-pathway. A new insight into a change of metabolites in Low-JS testes will be relevant to understand the mechanism of the deteriorated spermatogenesis under the normal range of testosterone level.     

Testosterone metabolism by isolated human axillary corynebacterium spp.: A gaschromatographic mass-spectrometric study

Transformations of [4-¹⁴C]testosterone have been studied in Corynebacterium spp. isolated from the axillae of men. Metabolites have been separated by TLC and capillary gas chromatography and have been identified by gas chromatography-mass spectrometry (GC-MS). The introduction of a clean-up step using Florisil columns, prior to TLC, removed Tween-80 which co-extracted from the medium with the metabolites. This procedure greatly improved TLC resolution.Testosterone was converted enzymically to 5α- and 5β-DHT, identification being assisted by the inclusion of [3,4-¹³C]testosterone in some incubations. Other metabolites formed enzymically were 4-androstene-3,17-dione, 5β-androstane-3,17-dione, 3β-hydroxy-5β-androstan-17-one and 5β-androstane-3α.l7α-diol. Some spontaneous breakdown of [¹⁴C]testosterone occurred giving rise to 5α(β)-DHT, androstanediol and a monohydroxy-diketo-androstene, the latter being reduced enzymically to 2 monohydroxy-diketo-androstanes. Under the conditions used, no clear evidence has been obtained for the formation of 5α-androst-16-en-3-one, an odorous steroid that occurs in the axillae of men; the possible reasons why we were unable to prove the biosynthesis of this compound are discussed.     

N-oxidation of imipramine by isolated perfused rat and rabbit lung

Pulmonary uptake and metabolism of imipramine (IMP) was investigated in isolated perfused rat (IPrL) and rabbit (IPRL) lung preparations. Perfusate containing ¹⁴C-IMP (1.2 μmole/g lung) was recirculated through the pulmonary artery in artificially ventilated lungs. The radioactivity in the perfusate declined rapidly and about 80% of the dose was taken up by the lungs within 10 minutes in both IPrL and IPRL preparations. A steady-state was apparently reached thereafter in the IPRL, while a portion of the radiolabel effluxed into the perfusate of the IPrLs, thus reducing the net lung content to 54% of added IMP by 60 minutes. After 60 minutes perfusion, metabolites of IMP accounted for the major radioactivity (80%) in the perfusate, while the lung contained mainly (83%) the unchanged parent compound. The principal metabolite was identified as IMP-N-oxide (IMP-NO) which was found in the perfusate after 5 minutes of perfusion. Only 3% of the added IMP was metabolized by IPRL in 60 minutes. SKF-525A, an inhibitor of cytochrome P-450-mediated monooxygenase system, did not inhibit but enhanced the metabolism of IMP by IPrL to IMP-NO. IMP was principally metabolized to IMP-NO by incubations of 9,000 g supernatant fractions of rat lungs to a significantly higher extent than similar rabbit lung preparations. Including SKF-525A significantly accelerated the metabolism of IMP to IMP-NO in accordance with the perfusion experiments. These results suggest that in contradiction to publishedd reports, IMP is appreciably metabolized by the rat lung $\text{via}$ N-oxidation by non-cytochrome P-450 pathway and the metabolite formed in the lung is released into the circulation indicating its low affinity for the lung tissue.     

Bioactive microbial metabolites from glycyrrhetinic acid

Biotransformation of 18β-glycyrrhetinic acid, using Absidia pseudocylinderospora ATCC 24169, Gliocladium viride ATCC 10097 and Cunninghamella echinulata ATCC 8688a afforded seven metabolites, which were identified by different spectroscopic techniques (¹H, ¹³C NMR, DEPT, ¹H-¹H COSY, HMBC and HMQC). Three of these metabolites, viz. 15α-hydroxy-18α-glycyrrhetinic acid, 13β-hydroxy-7α,27-oxy-12-dihydro-18β-glycyrrhetinic acid and 1α-hydroxy-18β-glycyrrhetinic acid are new. The ¹³C NMR data and full assignment for the known metabolite 7β, 15α-dihydroxy-18β-glycyrrhetinic acid are described here for the first time. The major metabolites were evaluated for their hepatoprotective activity using different in vitro and in vivo models. These included protection against FeCl₃/ascorbic acid-induced lipid peroxidation of normal mice liver homogenate, induction of nitric oxide (NO) production in rat macrophages and in vivo hepatoprotection against CCl₄-induced hepatotoxicity in albino mice.     

Metabolism of progesterone in mouse vaginal tissue

The $\text{in vitro}$ and $\text{in vivo}$ metabolism of 1,2- ³H-progesterone was studied in estrogen-stimulated and control vaginae of ovariectomized mice. Employing two-dimensional thin-layer chromatography, gas-liquid chromatography and metabolite “trapping” techniques, the major and minor pathways for progesterone metabolism were determined $\text{in vitro}$ and shown to involve saturation of the Δ⁴-double bond to yield 5α-pregnane compounds and reduction of the C20 and C3 ketone groups to form 20α- and 3α- and 3β-hydroxy derivatives, respectively. The quantities of 20β-hydroxy metabolites and 5β-epimers that were detected were considered not to be significant. The major metabolites formed by untreated tissues following $\text{in vitro}$ incubation in the presence of both high (10^?6M) and low (10^?8M) progesterone concentrations were 3α-hydroxy-5α-pregnan-20-one and 5α-pregnane-3,20-dione. Although these two derivatives were also found in sizable quantities in estrogen-treated tissues, a marked increase (5-fold) in the rate of C20 ketone reduction at high progesterone concentrations (10^?6M) to yield 20α-hydroxy-4-pregnen-3-one was demonstrated. Following intravaginal administration of ³H-progesterone $\text{in vivo}$, only progesterone and 3α-hydroxy-5α-pregnan-20-one were retained in appreciable quantities through 2 hr, suggesting rapid loss of 20α-hydroxy-4-pregnen-3-one and the 5α-pregnanediols from this tissue under $\text{in vivo}$ conditions.     

Pulmonary uptake of PGE2 is inhibited by dipyridamole in rat isolated lungs

The metabolism of prostaglandin E₂ (PGE₂) is decreased by dipyridamole (20 μM) in rat isolated perfused lungs. The inhibition of the metabolism is reversible as the decreased metabolism returned to the control level when pulmonary infusion of dipyridamole was abolished. After pulmonary injection of ¹⁴C-PGE₂ (10 nmol) the radioactivity appeared more rapidly in the effluent when dipyridamole was infused into pulmonary circulation. Dipyridamole in vitro did not change the activity of 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) in the 100, 000 × g supernatant fraction of homogenized lungs. Thus, the decreased metabolism seems to be due to the inhibition of the uptake of PGE₂ into the lungs. When the rats were pretreated with dipyridamole in drinking water for one week the activity of 15-OH-PGDH in the 100, 000 × g supernatant fraction of the lungs was not changed significantly.