Metabolism of Corticosterone in the Isolated Perfused Rat Liver

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The 3/?-hydroxy-A5-steroid oxidoreductase inhibitor 2ol-cyano-4,4,17ol-trimethyl-5-androsten-1715-01-3-0ne was administered intramuscularly to germfree and conventional, male and female, rats. Under the experimental conditions used, no steroids were found in faeces and urine from the male rats, but a large number of steroids were identified in excreta from female rats. The following 3/?-hydroxy-A6-steroids, none of which is present in normal rats, appeared in urine and faeces from germfree and conventional cyanoketone treated female rats (chiefly as monosulphates) : 5-androstene-38, 17p-dio1, 38, 7a(and 7/?)-dihydroxy-5-androsten-17-one, 5-androstene-38, 7a(and 7/?), 17/?-trioI, 38, 16a-dihydroxy-5-androsten-l7-one, 38, 17/?-dihydroxy-5-androsten-l6-one, 3&hydroxy-5,16-pregnadien-20-one, 3/?-hydroxy-l7ol-pregn-5-en-2O-one, 3/?-hydroxy-5-pregnen-20-one, 3/?,7a (and 78)-dihydroxy-5-pregnenen-20-one, 38, 17ol-dihydroxy-5-pregnen-20-one, 38, 16~-dihydroxy-5-pregnen-2O-one, 5-pregnene-3j3, 1601, 2Oa-triol, 5-pregnene-3/?,17a,20a-triol and 38,2l-dihydroxy-5-pregnen-20-one. In addition, several saturated steroids were identified which were identical with steroids excreted by normal germfree and conventional female rats.3/?,16a-Dihydroxy-5-pregnen-20-one normally accounts for less than 1 O/ , , of the total amount of 3/?,16a-dihydroxy-5-pregnen-20-one and 3a,16a-dihydroxy-5olI-pregnan-20-one found in urine. This percentage was found to be about 70-80 in urine from cyanoketone treated germfree as well as conventional female rats. This figure is in good agreement with the extent of inhibition (75 -85 of adrenal and gonadal (testicular and ovarian) 3/?-hydroxy-A5-steroid oxidoreductase found in experiments in vitro. It was established that the formation of saturated steroids from 3/?-hydroxy-A5-steroids in cyanoketone treated rats involved 3-0x0-steroids as intermediates since [3a-3H,4-1*C Jpregnenolone was converted into saturated steroids with loss of 3H. The hepatic S/?-hydroxy-A5-~teroid oxidoreductase active on C2, steroids was unaffected by treatment in vivo or in dtro with the cyanoketone.3/%Hydroxy-A5-steroid oxidoreductase is a key enzyme in the biosynthesis of all active steroid hormones of the C,, and C, series. A genetic deficiency of this enzyme has been described in some human cases with adrenal cortical hyperplasia [1,2]. A similar condition with a marked hypertrophy of the adrenals is obtained in rats treated with a steroidal cyanoketone, 2a-cyano-4,4,17ol-trimethyl-5-androsten-l7,S-ol-3-one, which is a substrate analogue that Trivial and Xystematic Names and Unusual Abbreviations. inhibits the mammalian 3/?-hydroxy-A6-steroid oxidoreductase in the adrenal and gonads [3]. The effect of this 3/3-hydroxy-A5-steroid oxidoreductase inhibitor on the biosynthesis and metabolism of C,, and C,, steroids has been almost exclusively studied in vitro, and no information is available on how the pattern of C,, and C,, steroids excreted in urine and faeces is affected by blocking the 3,9-hydroxy-A5-steroid oxidoreductase...

Section: Extraction and Purification Of Faecal 8teroichmentioning

confidence: 99%

Metabolism of Steroids in Germfree and Conventional Rats Treated with a 3β‐Hydroxy‐δ⁵‐Steroid Oxidoreductase Inhibitor

Björkhem

Gustafsson

1970

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“…At present, the data may be reconciled with the hypothesis that pregnane-3,11,20,2l-tetrols are primary metabolites in male rats whereas sulphates of 3,11,15,21-tetrahydroxypregnan-20-ones are primary metabolites in female rats [5]. In the conventional rats, steroid sulphates are hydrolyzed by microorganisms in the intestinal tract [16]. Steroids containing a 20-0x0 group may undergo 16 or 21-dehydroxylation whereas steroids with a 16a,20-or 20,21-dihydroxy structure are not susceptible to this type of microbial attack [6].…”

Section: Itmentioning

confidence: 52%

“…The main part of the radioactive steroids in urine from both germfree and conventional female rats was found in the monosulphate fraction and several of these steroids have been identified. No such conjugates were found in urine from the male rats [1,2]. The radioactive metabolites in the free steroid fraction were more polar in excreta from male than in those from female conventional rats [l].…”

mentioning

confidence: 99%

Steroids in Germfree and Conventional Rats

Eriksson¹

1971

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Gas chromatography-mass spectrometry and radio-gas chromatography were used to identify metabolites of [14C]corticosterone and [14C]pregnenolone in faeces and urine from germfree and conventional male rats. The metabolites were generally more polar than those in female rats and the main part of those which could be identified were found in the free steroid fraction. In the conventional male rats a number of pregnane-3,l I ,16,20,21-pentol isomers constituted the major corticosterone metabolites identified both in urine and in faeces. In addition 3n(and 3/3),20,3-dihydroxy-5n-pregnan-ll-one and 5a-pregnane-3a(and 3/3),1 1,3,20/3,21-tetrol were excreted in faeces. I n the germfree male rats, minor amounts of some C,,O, and C,,O, steroids were excreted as monosulphates in faeces. However, 5n-pregnane-3n(and 3/3),11/3,20/3,21-tetrols were the predominant steroids in this fraction. These compounds also constituted the major part of steroids identified in the disulphate fraction, which also contained 3a(and 3/3),11/3,21 -trihydroxy5n-pregnan-20-one. All radioactive steroids excreted in the urine of germfree male rats were unconjugated. Although none of these compounds could be fully identified evidence was obtained for the presence of several C,,O,, C,,04, C,lO,, C,,O, and C,,O, steroids.The findings are discussed in relation to previous information on the steroid metabolizing activities of the intestinal flora and sex differences in the metabolism of steroids in rats.When I4C-labelled pregnenolone and corticosterone were administered to germfree and conventional, male and female rats, the amount of radioactivity in faeces and urine and the nature of the metabolites excreted showed considerable differences between the four types of animals [l]. The main part of the radioactive steroids in urine from both germfree and conventional female rats was found in the monosulphate fraction and several of these steroids have been identified. No such conjugates were found in urine from the male rats [1,2]. The radioactive metabolites in the free steroid fraction were more polar in excreta from male than in those from female conventional rats [l]. Thus the steroid metabolites in faeces and urine from male rats appeared to be quite different from those previously identified in excreta from female rats [3-51. The present paper offers further proof of the differences in steroid metabolism between male and female rats, and describes the identification of a number of steroid metabolites in faeces and urine from conventional and germfree male rats.Systematic Names and Unusual Abbreviations. Corticosterone, 11~,21-dihydroxy-4-pregnene-3,20-dione; pregnenolone, 3p-hydroxy-5-pregnen-20-one; silyl, trimethylsilyl; oxime-silyl, 0-methyloxime trimethylsilyl. Retention times, t~. are calculated relative to 5a-cholestane. MATERIALS AND METHODS Reference Compounds[4-14C]Corticosterone (specific activity 56.7 mC/ mmole), and [4-14C]pregnenolone (specific activity 55.7 mC/mmole) were purchased from the Radiochemical Centre (Amersham, England). The...

“…Evidence has been presented that these compounds are metabolites of 1601-hydroxylated steroids [1,4] and that a t least in the rat the conversion is carried out mainly by intestinal microorganisms in reactions involving the intermediate formation of A16-unsaturated steroids. I n a previous study from this laboratory, it was shown that 3P-hydroxy-5,16-pregnadien-20-one was efficiently converted into 3B-hydroxy-17a-pregn-5-en-20-one when incubated with caecal contents from rats under anaerobic conditions [i].…”

mentioning

confidence: 99%

Microbial Formation of 17α‐C₂₁ Steroids

Björkhem

Eriksson

Gustafsson

1971

Self Cite

After anaerobic incubation in deuterated water of 3p-hydroxy-5,16-pregnadien-2O-one with caecal contents from rats, 3@-hydroxy-l701-pregn-5-en-20-one with deuterium label in ring D was isolated. Enolization of this compound with alkali yielded 3p-hydroxy-i7p-pregn-5-en-20-one with about 0.4 atoms of deuterium in ring D. Incubation of this compound with rat liver microsomes fortified with NADPH yielded 3/?,16a-dihydroxy-l7/?-pregn-5-en-2O-one devoid of deuterium in ring D. The results show that the saturation of the Als-double bond, catalyzed by intestinal microorganisms, involves a trans addition of hydrogen to the 1601-and l7p-positions.C,,-steroids with a i'iol-side chain have been identified in faeces and urine from female rats [ l , 21 and in urine from humans [3,4]. Evidence has been presented that these compounds are metabolites of 1601-hydroxylated steroids [1,4] and that a t least in the rat the conversion is carried out mainly by intestinal microorganisms in reactions involving the intermediate formation of A16-unsaturated steroids. I n a previous study from this laboratory, it was shown that 3P-hydroxy-5,16-pregnadien-20-one was efficiently converted into 3B-hydroxy-17a-pregn-5-en-20-one when incubated with caecal contents from rats under anaerobic conditions [i]. The present work deals with t,he stereochemistry in the microbial saturation of the AlS double bond, and evidence is presented that the addition of hydrogens is trans diaxial . MATERIALS AND METHODSSteroids 3p-Hydroxy-5-pregnen-20-one and 3j3-hydroxy-5,i6-pregnadien-20-one were purchased from Ikapharm (Ramat-Gan, P. 0. B. 31, Israel). 3/3,16a-Dihydroxy-5-pregnen-20-one was kindly supplied by Dr. C. H. L. Shackleton. 3j3-Hydroxy-1701-pregn-5-en-20-one was prepared from the corresponding 17j3-epimer as described previously [5]. 3B-Hydroxy-17P-[4-14C]pregn-5-en-20-one (pregnenolone) was purchased from the Radiochemical Centre (Amersham, England) and had a specific radioactivity of I .S x lo8 counts x min-l x mg-l. Gas Chromatography-Mass SpectrometryAll steroids were analysed as silyl ethers. The gas chromatographic-mass spectrometric analyses were Unusual Abbreviations. Silyl, trimethylsilyl; pregnenolone, 38-hydroxy-5-pregnen-ZO-one.carried out as described previously [i] using 1.501, SE-30 as stationary phase. Incubation with Caecal MicroorganismsIncubations of 3B-hydroxy-5, i 6-pregnadien-20-one, with caecal contents from rats of the SpragueDawley strain were carried out as described previously [ l ] with the exception that the buffer medium was prepared in deuterated water (99.7O/, pure, obtained from Norsk Hydro). The final concentration of deuterated water in the incubation mixtures was about 90 OlO. Extraction of the incubation mixtures and further analyses were performed as described previously [I].Conversion of 3P-Hydroxy-17a-pregn-5-en-20-one into 3/?-Hydroxy-17p-pregn-5-en-ZO-one Deuterated 3p-hydroxy-i7a-pregn-5-en-20-one, isolated after incubation of 3fl-hydroxy-5,lB-pregnadien-20-one with caecal contents, was refluxed with 2010...