Metabolism of testosterone by preparations from the rat testis

Seminiferous tubules isolated from immature and adult rats were incubated with [¹⁴C] testosterone. Androstanediol and androstenedione were the major metabolites; dihydrotestosterone and androsterone were produced in lesser amounts. Cell suspensions of spermatocytes prepared from tubules of immature rats formed dihydrotestosterone as the major metabolite of testosterone. Smaller amounts of androstanediol were formed and no androsterone was detectable. The results show that spermatocytes in common with androgen responsive tissues have the capacity to metabolize testosterone to 5α-reduced products.     

Metabolism of testosterone in hypothalamus of male rat.

Metabolism of testosterone in the hypothalamus of the male adult rat was studied in vitro. The whole homogenate of the hypothalamus in the presence of NADPH₂ generating system converted testosterone mainly to dihydrotestosterone and 5α-androstan-3α-17β-diol. The reduction of testosterone to dihydrotestosterone was irreversible, while that of dihydrotestosterone to 5α-androstan-3α-17β-diol was reversible. Michaelis constant for the 5α-reductase in the hypothalamus was 7.4 × 10⁻⁷ M.The administration of testosterone propionate caused no significant change of the 5α-reductase activity in the hypothalamus, in contrast to the marked induction of 5α-reductase activity in the prostate. Furthermore, the 5α-reductase appeared to be widely distributed in the subcellular paniculate components in the hypothalamus, being located mainly in the nuclear fraction in the prostate.These results suggest that there is some difference in the characteristics of the metabolism of testosterone in the hypothalamus and in the prostate.     

Metabolism of rat bone proteoglycans in vivo.

Former evaluations of the role of proteoglycans in mineralization have neglected to address the possibility that the metabolism of proteoglycans may be of significance in this regard. This problem was studied by using radiolabeling in vivo of rat calvaria with [35Sulphate for 2-72 h and a sequential extraction procedure to yield two pools of newly synthesized proteoglycans: one obtained from non-mineralized tissue by extraction with guanidinium chloride (GdmCl) and another obtained only after demineralization with EDTA. Total radioactivity in calvaria was maximal after 12 h of incorporation, but by 36 h had declined to a level that was about 55-65% of maximum. Radioactivity in the GdmCl extract declined steadily after 12 h, whereas that in the EDTA extract remained constant until 36 h, when it began to increase. Each extract contained a minor proteoglycan that eluted at the void volume (Vo) of a Sepharose CL-6B column. Unlike in the EDTA extract, this proteoglycan gradually disappeared from the GdmCl extract. Each extract also contained a major, smaller proteoglycan, with a Kav. of 0.24 and 0.36 in the GdmCl and EDTA extracts respectively. Papain digestion of each extract yielded glycosaminoglycan chains with Kav. values of 0.32 and 0.50 on CL-6B in the GdmCl and EDTA extracts respectively. Digestion of each extract with chondroitinase ABC and chondroitinase AC showed that the glycosaminoglycans were of similar disaccharide composition, with about 85% being 4-sulphated and the remainder 6-sulphated and/or iduronic acid-containing. These data suggest that about 45% of the newly synthesized proteoglycans are removed from the tissue during the course of mineralization.     

Metabolism of testosterone sulfate in the rat: analysis of biliary metabolites.

Following intraperitoneal injection of a mixture of testosterone-7-3-H-17-sulfate and testosterone-4-14-C into male and female rats with bile fistulas, biliary metabolites were separated and purified 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 bile. The major portion of the 3H was excreted in the disulfate fraction in both sexes. Solvolysis of the disulfate revealed the sex-specific aglycone pattern: 5alpha-Androstane-3beta,17beta-diol was the major metabolite in the male rat, whereas 5alpha-androstane-3alpha,17beta-diol and polar steroids were found in the female. In marked contrast, testosterone was metabolized in a different way than testosterone sulfate. 14-C radioactivity was distributed in monoglucosiduronate, monosulfate, and diconjugate fractions. Analysis of the aglycones showed that polar steroids were the main metabolites in the male. In the female, testosterone was metabolized to polar steroids, androsterone, and 5alpha-androstane-3alpha,17beta-diol.     

Metabolism of testosterone and dihydrotestosterone in cultured rat hepatoma cells.

Steroid metabolism in hepatoma tissue culture (HTC) cells derived from a male rat was investigated. Steroids in ethanol were incubated with the cells for various lengths of time. Volume of ethanol never exceeded 1% of incubation volume. Thin-layer and paper chromatography were used. Incubation was with tritiated steroids. It was demonstrated that testosterone as well as dihydrotestosterone is transformed. The main enzyme activities detected were 5alpha-reduction and 3alpha-, 3beta, and 17beta-hydroxysteroid dehydrogenation. The pattern of metabolism was reproducible and varied with time, substrate concentration, and number of cells incubated. Some steroids interfered with androgen metabolism. 17beta-estradiol, 17-epitestosterone, and progesterone competed for the 17beta-hydroxyprogesterone dehydrogenase. it is concluded that 3beta and 17beta reduction in the HTC cells may be catalyzed by the same enzyme which might differ considerably from the 3beta-hydroxysteroid dehydrogenase assayed in intact liver cells. A hepatoma derived from a female rat also produced considerable amounts of 3beta-derivatives of testosterone.     

Adrenalectomy does not influence basal secretion of testosterone in rat in vivo

We have studied the effect of adrenalectomy on the testicular secretion of testosterone in the rat. In the acute period following adrenalectomy plasma testosterone levels were reduced but this was no different from those levels in appropriate sham-operated controls. This reduction in plasma testosterone levels is probably a result of direct effects of anaesthesia and surgical stress. Whilst studies on the late effect of adrenalectomy avoided this problem, plasma testosterone levels were normal in both adrenalectomised and sham-operated animals. Resetting of anterior pituitary-gonadal relationships may mask the absence of any contribution made by the adrenal gland to testicular steroidogenesis. In contrast to previous data we were unable to demonstrate that adrenalectomy influenced the secretion of testosterone in the male rat.     

Metabolism of [4-14C] testosterone in the rat uterus in vitro.

In view of the uterine action of androgens we have investigated in vitro the metabolism of [4-14C]-testosterone in uterine tissue of ovariectomized rats. After purification of the extracts on Amberlite XAD-2 the metabolites have been isolated by gel. Five metabolites were isolated and identified during these incubation studies: 4-androstene 3,17-dione, 17beta-hydroxy-5alpha-androstan-3-one, 5 alpha-androstane-3alpha17beta-diol, 4-androstene-3 beta, 17beta-diol and 4-androstene-3alpha, 17beta-diol. Furthermore, two polar C19O3-metabolites and one isopolar to 5 alpha-androstane-3, 17-dione have also been detected. The metabolites were characterized by radioactive gas chromatogrphy, and determination of the relative specific activity in the eluates of Sephadex column chromatography. The identification of allylic alcohols was complemented by their oxidation to 4-androstene-3,17-dione. The present data show that activity of 17beta,3alpha- and 3beta-hydroxysteroid-oxidoreductase and 5alpha-ring-reductase are involved in the metabolism of testosterone in vitro in the rat uterus. The very low 5 alpha-reductase activity under the experimental conditions used in this work explains the formation of allylalcohols as the principal metabolites of testosterone in the rat uterus.     

Metabolism of estrone sulfate in the pregnant sheep

The metabolism of ³H-estrone sulfate (³H-E₁S) in 4 pregnant sheep, two injected i.v. and two i.m., has been studied. Intravenously injected ³H-E₁S had a plasma half-life of approximately 8 min, and metabolic clearance rate of approximately 800 ml/min. Using this clearance rate and the previously published mean plasma concentration of E₁S, the estimated production rate of E₁S is between 8.8 nmol (3.3 μg) and 78.2 nmol (29.1 μg) per min from 2-day to 0-day before parturition.Intramuscularly injected ³H-E₁S disappeared from plasma linearly and was completely cleared well within 3 hours. In all cases, whether i.v. or i.m. injected, the main metabolite isolated was ³H-estradiol-17β-3-sulfate, with only a trace amount as ³H-estradiol-17β-3-sulfate.     

Metabolism of testosterone in human lung in vitro

• 1.1. The metabolism of 4-¹⁴C-testosterone in human lung in vitro was investigated.
• 2.2. The metabolism was most pronounced in incubations of homogenated tissue, whereas it was rather restricted in the mitochondrial, microsomal and soluble fraction incubations.
• 3.3. The by far most prominent metabolite in all experiments was androst-4-ene-3,17-dione.
• 4.4. No slfate or glucuronide conjugation took place.

     

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 testosterone by human semen

Following the incubation of human sperm and seminal plasma with 13C2-labelled testosterone, the main metabolite, identified by gas chromatography-mass spectrometry (GC-MS), was 4-androstene-3,17-dione. In addition, 6 alpha- and 6 beta-hydroxytestosterone were identified. The more common metabolites of testosterone were not detected, and it is possible that the high substrate-tissue ratio influenced the result. Incubation of individual sperm and seminal plasma specimens with [14C]testosterone resulted in the identification, by specific activity measurements, of 4-androstene-3,17-dione in almost every specimen but with a widely varying conversion rate. Dihydrotestosterone, which on general grounds was considered a likely metabolite, could not be positively confirmed as such, although in some samples its presence was suspected. Gas chromatography-mass spectrometry was also used to identify steroids in sperm and seminal plasma extracts. Some, but not all the steroids identified as present in such extracts by other investigators, were found. During the course of this work C18 Sep-Pak cartridges were successfully used to prepare fractions suitable for SP-Sephadex and TEAP-Lipidex chromatography and subsequent analysis by GC-MS. Their use eliminated the need for purification steps otherwise necessary.     

Metabolism of lithocholic acid in the rat: formation of lithocholic acid 3-O-glucuronide in vivo

Milligram amounts of [3 beta-3H]lithocholic (3 alpha-hydroxy-5 beta-cholanoic) acid were administered by intravenous infusion to rats prepared with a biliary fistula. Analysis of sequential bile samples by thin-layer chromatography (TLC) demonstrated that lithocholic acid glucuronide was present in bile throughout the course of the experiments and that its secretion rate paralleled that of total isotope secretion. Initial confirmation of the identity of this metabolite was obtained by the recovery of labeled lithocholic acid after beta-glucuronidase hydrolysis of bile samples. For detailed analysis of biliary metabolites of [3H]lithocholic acid, pooled bile samples from infused rats were subjected to reversed-phase chromatography and four major labeled peaks were isolated. After complete deconjugation, the two major compounds in the combined first two peaks were identified as murideoxycholic (3 alpha, 6 beta-dihydroxy-5 beta-cholanoic) and beta-muricholic (3 alpha, 6 beta, 7 beta-trihydroxy-5 beta-cholanoic) acids and the third peak was identified as taurolithocholic acid. The major component of the fourth peak, after isolation, derivatization (to the methyl ester acetate), and purification by high pressure liquid chromatography (HPLC), was positively identified by proton nuclear magnetic resonance as lithocholic acid 3 alpha-O-(beta-D-glucuronide). These studies have shown, for the first time, that lithocholic acid glucuronide is a product of in vivo hepatic metabolism of lithocholic acid in the rat.     

The effect of cadmium administration in vivo on plasma testosterone and the ultrastructure of rat lateral prostate

Electron microscope microanalysis, atomic absorption analysis and ultrastructural survey were used to investigate the effects of parenteral cadmium administration on the lateral prostate of rats. Early fine structural changes in the epithelial cells of the prostatic tissue were associated with the detection of cadmium in the cellular organelles and alteration of the subcellular distribution of zinc. Involutionary changes appeared at later stages and differed from the usual castration effects. Basal cells did not regress with the altered physiological conditions but appeared to proliferate in the presence of cadmium. The observations are discussed in relation to the normal mechanisms which control the maintenance of the prostate gland.