The identification and characterization of the c19o3 steroid metabolites of 5α-androstane-3β, 17β-diol produced by the canine prostate: 5α-androstane-3β, 6α, 17β-triol and 5α-androstane-3β, 7α, 17β-triol

This study has identified the polar metabolites of 5α-androstane-3β, 17β-diol(3β-diol) produced by the canine prostate. The major metabolite is 5α-androstane-3β, 7α, 17β-triol (7α-triol) accounting for approximately 80% of the total polar metabolites of 3β-diol. The remaining 20% is accounted for exclusively by another triol, 5α-androstane-3β, 6α, 17β-triol(6α-triol). This study has also characterized two enzymatic hydroxylases responsible for respective triol formation: 5α-androstane-3β, 17β-diol 6α-hydroxylase (6α-hydroxylase) and 5α-androstane-3β, 17β-diol 7α-hydroxylase (7α-hydroxylase). Both of these irreversible hydroxylases are located in the particulate fraction of the prostate and can utilize either NADH or NADPH as cofactor. Several $\text{in vitro}$ steroid inhibitors of these hydroxylases were identified including cholesterol, estradiol and diethylstilbestrol. Neither of the hydroxylases were found to be decreased by castration (3 months) when expressed as activity/DNA. Using a variety of C₁₉ androstane substrates, 6α- and 7α-triol were found to be major components of the total 3β-hydroxy-5α-androstane metabolites produced by the canine prostate.     

A rapid,sensitive method for simultaneous measurements of tissue levels of oxygenated derivatives of cholesterol

A mass spectrometric procedure which utilizes multiple selected ion monitoring (SIM) for measuring the tissue levels of cholest-5-en-3β,7α-diol, cholest-5-en-3β,7β-diol, cholest-5-en-3β,25-diol, and cholest-5-en-3β-ol-7-one is described. Trimethylsilyl ethers (TMS) of sterols in a lipid extract are analyzed directly by focusing the ions at $\text{m}\text{e}$ 546, 472, and 443. Endogenous cholesterol serves as an internal standard and its concentration is determined by gas chromatography. The sensitivity of this method has allowed measurement of 2 ng of oxygenated sterol which corresponded to the amount present in 1 mg of rat liver.     

Androgen metabolism by rat epididymis. 1. Metabolic conversion of 3 H-testosterone in vivo

The epididymis of adult rats metabolize ³H-testosterone by experiments $\text{in vivo}$. Thirty minutes after the injection of 100 μCi ³H-testosterone, some 10 per cent of the total radioactivity of the epididymis was found in the water-soluble fraction, whereas 90 per cent was found in the ether soluble fraction (free steroids). The free steroids were examined further and the following androgenic metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 8, 9%, $\text{androstendipne}$ (4-androstene-3, 17-dione, 2,7%,$\text{5α-A-dione}$ (5α-androstane-3, 17-dione) 6,5%, $\text{DHT}$ (17β-hydroxy-5α-androstan-3-one) 47, 2%, $\text{3β-diol}$ (5α-androstane-3β, 17β-diol) 4, 4%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 20, 8% and $\text{androsterone}$ (3α-hydroxy-5α-androstan-3-one) 3,4%. The relative amount of each metabolite is given in per cent of total radioactivity in the ether soluble fraction.     

Androgen metabolism by rat epididymis. 2. Metabolic conversion of 3H-testosterone in vitro

The epididymis of adult rats metabolizes ³H-testosterone by experiments $\text{in}$$\text{vitro}$. After incubation of slices from epididymal tissue for 2 hrs at 37°C, 8% of the total radioactivity was found in the water-soluble fraction, whereas 92% in the ether soluble fraction (free steroids). The free steroids were examined further and the following metabolites identified: $\text{testosterone}$ (17β-hydroxy-4-androsten-3-one) 10,4%, $\text{androstendione}$ (4-androstene-3,17-dione) 6,2%, $\text{5α-A-dione}$ (5α-androstane-3,17-dione) 7,3%, $\text{DHT}$ (17β-hydroxy-5α-androstane-3-one) 39,3%, $\text{3α-diol}$ (5α-androstane-3α,17β-diol) 22,7%, $\text{3β-diol}$ (5α-androstane-3β,17β-diol) 4,6% and androsterone(3α-hydroxy-5α-androstan-17-one) 8,9%. The relative amount of each metabolite is given in per cent of the total radioactivity in the ether soluble fraction. When segments (caput, corpus, cauda) of epididymis were incubated in the same way, differences in steroid metabolism were demonstrated. Characteristic for caput epididymidis was high formation of DHT (58,4%) and 3α-diol (23,5%). Corpus epididymidis showed lower formation of DHT (50,6%) and 3α-diol (12,7%), but an approximately 3 times higher formation of 5α-A-dione (12,0%) than caput (3,4%) and cauda (3,5%). Cauda epididymis showed the lowest formation of DHT (38,3%), whereas 3α-diol (29,1%) and androsterone (11,4%) formation were relatively high. The ratio between 17β-hydroxy metabolites (DHT and androstanediols) and 17-keto metabolites were much higher in the caput (8,8) than in the corpus (3,2) and cauda (3,6), indicating a higher 5α-reductase activity in this segment.     

Side chain functionalization of cholesterol in the biosynthesis of solasodine in Solanum laciniatum

(25R)-26-Amino-cholesterol-[7α-³H], (25R)-26-amino-5-cholestene-3β,16β-diol-[7α-³H] and (25R)-26-acetylamino-5-cholestene-3β,16β-diol-[7α-³H] administered to Solanum laciniatum were converted into solasodine. The results indicate that in the biosynthesis of solasodine the introduction of nitrogen occurs immediately after the hydroxylation at C-26 and before a further oxidation of the side chain of cholesterol. The next step after the amination at C-26 is not hydroxylation at the 16β-position but probably the functionalization of C-22.     

In vitro conversion of 5alpha-reduced metabolites of testosterone by the seminal vesicles, ventral prostate glands and testes of adult rats

In order to ascertain the ability of rat seminal vesicles, testes and ventral prostate glands to interconvert 5α-reduced androgens, these three organs were incubated with either tritiated 17β-hydroxy-5αandrostan-3-one (5α-dihydrotestosterone,DHT), 5α-androstane-3α, 17βdiol (3α-diol) or 5α-androstane-3β, 17β-diol (3β-diol). The incubation environment utilized (Krebs-Ringer bicarbonate glucose buffer) was selected because the histologic appearance of the tissue at the conclusion of the incubation was indistinguishable from tissue fixed immediately after sacrifice of the animal, thereby approximating the physiologic conditions as closely as possible. In incubations of rat seminal vesicles, ³H.-3β-diol was not metabolized while 26.7 ± 3.8% of ³H-3α-diol appeared as DHT and 17.2 ± 1.5% of ³H-DHT was metabolized to 3α-diol. A small amount (7.5 ± 0.8%) of ³H-DHT was, however, converted to 3β-diol. In incubations of rat testes, the major metabolite, regardless of substrate, was 3α-diol. The conversion of 75.7 ± 2.1% of ³H-3β-diol to 3α-diol has demonstrated, for the first time, that this steroid can be metabolized by the rat testis. Rat ventral prostate glands metabolized $\text{18.5 ± 2.5\%}\text{of}{}^3\text{H-3β-}\text{diol}$ to DHT and 61± 2.9% of ³H-3α-diol to DHT. When ³H-DHT served as the substrate, 83.2 ± 1.5% remained unmetabolized. The prostate glands are, therefore, capable of metabolizing 3β-diol to DHT.     

omega-Hydroxylase for 5beta-cholestane-3alpha,7alpha-diol in the biosynthesis of chenodeoxycholic acid.

The 5β-cholestane-3α,7α-diol 26-hydroxylase system, which is involved in the conversion of cholesterol to chenodeoxycholic acid, was studied in rat liver mitochondria. 26-Hydroxylase of 5β-cholestane-3α,7α-diol showed the following characteristics. (i) 5β-Cholestane-3α,7α-diol 26-hydroxylase requires electron donors similar to those required for 5β-cholestane-3α,7α,12α-triol 26-hydroxylase. (ii) Both enzyme activities are inhibited by similar inhibitors such as carbon monoxide and phenylisocyanide, but not by respiratory inhibitors such as rotenone, amytal, antimycin A, and cyanide. (iii) The presence of 5β-cholestane-3α,7α-12α-triol in the incubation mixture for 5β-cholestane-3α,7α-diol inhibits the latter activity in a competitive manner. (iv) The distribution patterns of both enzyme activities in submitochondrial fractions are similar. (v) The reconstituted enzyme system composed of partially purified cytochrome P-450 from rat liver mitochondrial inner membrane, NADPH-adrenodoxin reductase and adrenodoxin (both purified from bovine adrenocortical mitochondria), and NADPH showed 26-hydroxylation activity not only for 5β-cholestane-3α,7α-diol but also for 5β-cholestane-3α,7α,12α-triol; both activities were comparable.     

Rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihyorotestosterone in the male rat anterior pituitary: in vivo and in vitro studies

The $\text{in vivo}$ and $\text{in vitro}$ metabolism of $\text{(}{}^3\text{H}\text{)-5α-}\text{androstane}\text{-α, 17β-}\text{diol}$ by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5α-androstane-3α,17β-diol into 5α-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73 % of the recovered radioactivity were constituted by 5α-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5α-dihydrotestosterone was the main steroid recovered except from the 105,000 × g pellet. From $\text{in vivo}$ and $\text{in vitro}$ experiments it was concluded that the transformation of 5α-dihydrotestosterone into 5α-androstane-3α,17β-diol was a reversible process, and that this last steroid could exert its biological action mainly $\text{via}$ 5α-dihydrotestosterone.     

Species differences in biotransformation of 17α-cyanomethylestradiol 3-methyl ether

Following oral administration of 9,11- ³H-17α-cyano-methylestra-1,3,5(10)-triene-3,17-diol 3-methyl ether, urinary metabolites were studied in man, baboon, beagle dog, minipig and rat. The metabolite pattern revealed remarkable species differences, especially in quantitative respects. 17α-Cyanomethylestra-1,3,5(10)-triene-3,17-diol, 17α-cyanomethylestra-1,3,5(10)-triene-2,3,17-triol 2-methyl ether, 17α-hydroxymethylestra-1,3,5(10)-triene-3,17-diol and 17α-cyanomethylestra-1,3,5(10)-triene-3,16$\text{6}\text{5}$,17-triol were isolated as principal metabolites. In rat bile, a metabolite was tentatively identified as aγ-lactone of a 17α-carbozymethyl-16α-hydroxy compound.     

Incorporation of [1-14C]-acetate into testosterone,androstenediol, dehydroepiandrosterone and progestogens by ram testis following human chorionic gonadotropin administration

Testicular steroidogenesis in rams was examined by constant infusion (3 hr) of [1-¹⁴C]-acetate into the testicular artery of four conscious standing animals.The following steroids (in order of decreasing levels of [¹⁴C] labeling) were secreted by the testis and found in testicular tissue: testosterone, dehydroepiandrosterone, 3β-hydroxy-5-androsten-17-one, androstenediol, 5-androsten-3β,17β-diol and 17-hydroxy-4-pregnene-3,20-dione. In addition, [¹⁴C] labeling of 17,20α-dihydroxy-4-pregnen-3-one occurred in testicular tissue but not in blood. This $\text{in vivo}$ system with the conscious standing ram demonstrated an operative Δ⁵ steroidal pathway to testosterone. The physiological significance of 17,20α-dihydroxy-4-pregnen-3-one is not yet explained in this species.     

Metabolism and conjugation of [4-14C]progesterone by bovine liver and adipose tissues, in vitro

The ability of bovine liver and fat to metabolize progesterone and also to form glucuronide conjugates with these progestins $\text{in vitro}$ was investigated. Tissue supernatants were incubated with [4-¹⁴C] progesterone, UDP-glucuronic acid, and a NADPH generating system for 5 hr, at 37°C. Steroids were identified by thin-layer chromatography, high performance liquid chromatography, and recrystallization to a constant specific activity. The total original radioactivity which could not be removed by exhaustive ether extraction (presumptive conjugates) was 44.7 ± 14.2% in liver, 5.0 ± 3.6% in subcutaneous fat, and 3.7 ± 2.2% in kidney fat samples. Progestins identified in liver samples include 5β-pregnane-3α, 20α-diol (free and conjugate), 5β-pregnane-3α, 20β-diol (free and conjugate), 3α-hydroxy-5sB-pregnan-20-one (free and conjugate), 3β-hydroxy-5β-pregnan-20-one (free), 5β-pregnane-3, 20-dione (free), and progesterone (conjugate). Progestins identified in both the free and conjugate fractions of subcutaneous fat and kidney fat samples include progesterone, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-4-pregnen-3-one, and 20α-hydroxy-4-pregnen-3-one. Differences due to sex of bovine used were noted. These results confirm the ability of bovine liver to readily metabolize progesterone and form glucuronide conjugates of these compounds and suggest that adipose tissues take an active role in these actions in cattle.     

Androgen metabolism by rat epididymis: 5. Metabolic conversion and nuclear binding after injection of 5α-androstane-3α,17β-diol, in vivo

The pattern of androgenic metabolites in blood, muscle, caput and cauda epididymidis has been investigated in functionally hepatectomized 24 hours castrated rats, 3 hours after the intra-muscular injection of 200 μCi of ³H -3α-diol. Identification of the radioactive metabolites showed only negligible differences between the epididymal regions. In both caput and cauda the main metabolite was DHT (17β-hydroxy-5α-androstane-3-one); 3α- and 3β-diol, androsterone (3α-hydroxy-5α-androstane-17-one), 5-A-dione (5α-androstane-3,17-dione), Δ¹⁶-3α-ol (5α-androst-l6-en-3α-ol), Δ¹⁶-3β-ol (5α-androst-l6-en-3α-ol) and Δ¹⁶-3-one (5α-androst-l6-en-3-one) were also present.$\text{Androsterone}$ and $\text{3α-diol}$ were the predominant metabolites in blood and muscle. No Δ¹⁶ compounds could be detected and in constrast to epididymis, more than 50% of the radioactivity was associated with polar compounds. From determination of total radioactivity, it was seen that retention by epididymis varied from two to four times that of muscle. Purification and identification of the radioactivity associated with the nuclear fraction demonstrated that DHT was the only nuclear bound androgen.It is suggested from these results that at least one effect of 3α-diol on the rat epididymis is exerted through its conversion to DHT.     

Role of sulfhydryl groups in catalytic activity of purified cholesterol 7α-hydroxylase system from rabbit and rat liver microsomes

Electrophoretically homogeneous preparations of cytochrome P-450 LM₄ from cholestyramine-treated rabbits catalyzed 7α-hydroxylation of cholesterol, 12α-hydroxylation of 5β-cholestane-3α,7α-diol and 25-hydroxylation of 5β-cholestane-3α,7α,12α-triol. Dithiothreitol, a disulfide reducing agent, specifically stimulated the cholesterol 7α-hydroxylase activity severalfold. The 7α-hydroxylase activity was much more sensitive to the sulfhydryl reagents p-chloromercuribenzoate, N-ethylmaleimide and iodoacetamide than the 12α- and 25-hydroxylase activities. Cholesterol 7α-hydroxylase activity, inactivated by these reagents, could be reactivated by treatment with dithiothreitol. Similar results were obtained with purified cytochrome P-450 from rat liver microsomes.The results indicate that sulfhydryl groups are more important for cholesterol 7α-hydroxylation than for other C₂₇-steroid hydroxylations.     

Sterol biosynthesis: Establishment of the structure of 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15β-ol

Previous studies have established that hydride reduction of 3β-benzoyloxy-5α-cholest-8(14)-en-15-one yields two epimers (at C-15) of 5α-cholest-8(14)-en-3β,15-diol which were designated as diol A and B. Efficient enzymatic conversion of both compounds to cholesterol was observed. To determine the absolute configuration of the 15-OH function in the two compounds, the 3β-p-bromobenzoyl ester of diol B was prepared from 3β-p-bromobenzoyloxy-5α-cholest-8(14)-en-15-one by reduction with sodium borohydride. Crystals of the derivative were found to belong to the space group P1, with unit cell parameters; $\text{a = 9.24}\text{A}\text{?}$, $\text{b = 12.61}\text{A}\text{?}$, $\text{c = 7.03}\text{A}\text{?}$, α = 93.05°, β = 100.27°, γ = 90.82°, and one molecule per unit cell. Least-squares refinement of the structure was carried out to final R value of 0.14. The configuration of the hydroxyl group at the 15 position of diol B has been determined to be β.     

4-ethenylidene steroids as mechanism-based inactivators of 3β-hydroxysteroid dehydrogenases

The synthesis of 4-ethenylidene-5α-androstane-3β, 17β-diol ($\text{5}$) and of 4-ethenylidene-5α-androstane-3,17-dione ($\text{4}$) is described. Compound $\text{5}$ is a competitive inhibitor of solubilized bovine microsomal adrenal Δ⁵-3β-hydroxysteroid dehydrogenase, with Ki =2.7μM, and is converted by the enzyme to the corresponding 3-ketone. Compound $\text{4}$ shown to irreversibly inactivate the enzyme in a time-dependent manner ($\text{t}\text{1}\text{2}\text{=31}\text{min}\text{; 55μ}\text{M}\text{;}\text{pH}\text{=7.0}$). The substrate, dehydroepiandrosterone, protects against inactivation by compound $\text{4}$. In contrast, compound $\text{5}$ is not oxidized at the 3-position by the 3β-(and 17β)-hydroxysteroid dehydrogenase from $\text{P. testosteroni}$, but is oxidized at the 17-position. Nevertheless, the 4-ethenylidene-3,17-diketone ($\text{4}$) causes irreversible time-dependent inactivation ($\text{t}\text{1}\text{2}\text{=28}\text{min}\text{; 64μ}\text{M}\text{;}\text{pH}\text{=7.0}$) when incubated directly with this bacterial enzyme, acting as an affinity label.     

Formation of a 5 hydroxy sterol by Saccharomyces cerevisiae

The incorporation of [28 ¹⁴C] ergosta-7,24(28)-dien-3β-ol into ergosta-7,22-dien-3β,5α-diol by aerobically growing $\text{S.}\text{cerevisiae}$ has established its presence in this organism. This, coupled with previous work, is considered to be substantive evidence for the operation of a hydroxylation-dehydration mechanism in the introduction of Δ⁵ unsaturation in ergosterol biosynthesis in yeast.     

Structural requirement of sterol nucleus for the silkworm growth and development

Several cholesterol analogs structurally modified in nuclear substitutions were tested for sustaining the growth of the silkworm $\text{Bombyx mori}$. 5α-Cholest-7-en-3β-ol, 5,7-cholestadien-3β-ol and cholesteryl acetate can replace cholesterol as sterol source for $\text{B. mori.}$ Considerably good growth was also obained with 5α-cholest-14-en-3β-ol and 5α-cholesta-6,8(14)-dien-3β-ol. Other sterols tested were either partially effective or ineffective as nutrients.     

Sertoli cells from immature rats : in vitro stimulation of steroid metabolism by FSH.

Sertoli cells from 10 day old rats convert androstenedione to testosterone and 5α-androstane-3α,17β-diol, testosterone to 17β-hydroxy-5α-androstan-3-one and 5α-androstane-3α,17β-diol, and 17β-hydroxy-5α-androstan-3-one to 5α-andro-stane-3α,17β-diol after 72 hours $\text{in vitro}$. Conversions of androstenedione to testosterone and 5α-androstane-3α,17β-diol, and testosterone to 5α-androstane-3α,17β-diol were 2 to 3 times greater in FSH treated cultures. Steroid conversion was not stimulated significantly by LH or TSH. The results are interpreted as evidence that in young rats Sertoli steroid metabolism is stimulated by FSH, that Sertoli cells are an androgen target and that FSH may induce or facilitate Sertoli androgen responsiveness.     

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.     

Synthesis of 5α-Androstane-3α,16α,17β-triol from 3β-hydroxy-5-androsten-17-one

5α-Androstane-3α, 16α 17β-triol was synthesized from 3β-hy-droxy-5-androsten-17-one. The procedure Involved catalytic hydrogenation of 3β-hydroxy-5-androsten-17-one to 3β-hydroxy-5α-androstan-17-one. This was followed by conversion of the 3β-hydroxy group to 3α-benzoyloxy group by the Mitsunobu reaction. Further treatment with isopropenyl acetate yielded 5α-androsten-16-ene-3α, 17-diol 3-benzoate 17-acetate. This was then converted to 3α, 17-dihydroxy-5α-androstan-16-one 3-benzoate 17-acetate via the unstable epoxide intermediate after treatment with $\text{m}$-cloroperoxybenzoic acid. LiAlH₄ reduction of this compound formed 5α-androstane-3α, 16α, 17β-trlol. ¹H and ¹³C NMR of various steroids are presented to confirm the structure of this compound.