Development of Methods to Quantify Free and Conjugated Steroids in Fatty Matrices by HPLC–MS/MS

ABSTRACT After homogenization of testicular tissue from stallions aged 1, 2 and 5 years, the unconjugated and conjugated steroids were isolated by a combined solvent–solid extraction procedure. The conjugates were further separated into glucuronides and sulphates by chromatography using Sephadex LH-20. After enzyme hydrolysis and solvolysis of the respective conjugate classes, the three extracts, unconjugated steroids, aglycones and solvolysed sulphates, were purified by chromatography using Kieselgel 60H columns. Five fractions were resolved from each extract; an aliquot of each fraction was derivatized to form the methoxime-trimethylsilyl ethers and the steroids were identified by combined gas chromatography–mass spectrometry. The results have shown that in stallion testes (1) steroidogenesis proceeds by both the 4-ene and the 5-ene pathways, (2) age-linked changes occur in both unconjugated and sulphoconjugated steroid fractions and (3) 19-hydroxy androgens and the 19-nor (C18) neutral steroids (19-norandrostenedione and 19-nortestosterone) are detected only in the unconjugated fraction whereas oestrone, the isomers of oestradiol and of 5(10)-oestrene-3,17-diol are the only steroids detected in the sulphoconjugate fraction. It is suggested that the unconjugated 19-oxygenated androgens present in stallion testes are converted to 19-nor neutral steroids by a reverse aldol reaction and a mechanism showing the putative intermediates in their formation is illustrated. Journal of Endocrinology (1989) 120, 223–229

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Simultaneous Separation of Free and Conjugated Steroids by Micellar Electrokinetic Chromatography and Its Clinical Application.

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.18.1614 ◽

1995 ◽

Vol 18 (11) ◽

pp. 1614-1616 ◽

Cited By ~ 11

Author(s):

Yoshiharu KOBAYASHI ◽

Jun MATSUI ◽

Fukuko WATANABE

Keyword(s):

Clinical Application ◽

Micellar Electrokinetic Chromatography ◽

Electrokinetic Chromatography ◽

Simultaneous Separation ◽

Conjugated Steroids

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Stabilization of coenzyme binding by conjugated steroids and carboxylic acids

Life Sciences ◽

10.1016/0024-3205(66)90307-9 ◽

1966 ◽

Vol 5 (9) ◽

pp. 837-844 ◽

Cited By ~ 6

Author(s):

S. Singer ◽

J. Ford ◽

L. Schirch ◽

M. Mason

Keyword(s):

Carboxylic Acids ◽

Coenzyme Binding ◽

Conjugated Steroids

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A CHROMATOGRAPHIC INVESTIGATION OF Δ4-3-KETOSTEROIDS AND α-KETOLIC STEROIDS IN HUMAN URINE

Journal of Endocrinology ◽

10.1677/joe.0.0090401 ◽

1953 ◽

Vol 9 (4) ◽

pp. 401-417 ◽

Cited By ~ 37

Author(s):

CONSTANCE de COURCY ◽

I. E. BUSH ◽

C. H. GRAY ◽

J. BARBARA LUNNON

Keyword(s):

Paper Chromatography ◽

Human Urine ◽

The Other ◽

Additional Material ◽

The Third ◽

Neutral Ph ◽

Chromatographic Investigation ◽

Final Fraction ◽

Conjugated Steroids

The Δ4-3-ketosteroids and α-ketolic steroids present in extracts prepared by five different procedures from urine have been examined by paper chromatography. The first extract, which consisted of those steroids extractable with chloroform at neutral pH contained cortisone and cortisol* and one to four unknown Δ4-3-ketosteroids. The second extract, representing steroids extractable immediately after acidification to pH 1 contained very little additional material. The third fraction consisted of extra steroids liberated after standing for 24 hr with acid; this rarely contained any detectable steroid material. A fraction which contained chloroform-soluble conjugated steroids was found to include small quantities of cortisone and cortisol and the other Δ4-3-ketosteroids. The final fraction contained large quantities of steroids liberated by hydrolysis with β-glucuronidase; these were mainly α-ketolic steroids including pregnane-3α: 17α: 21-triol-11: 20-dione, together with other unknown Δ4-3-ketosteroids. The distribution of the steroids in the various fractions is discussed and suggestions are made concerning the possible structure of some of the unknown steroids.

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Metabolism of oestrone and oestradiol-17beta to conjugated steroids by the accessory sex glands of the male pig

Journal of Endocrinology ◽

10.1677/joe.0.1630049 ◽

1999 ◽

Vol 163 (1) ◽

pp. 49-53 ◽

Cited By ~ 9

Author(s):

JI Raeside ◽

HL Christie ◽

RL Renaud

Keyword(s):

Synergistic Effect ◽

Culture Medium ◽

Seminal Vesicles ◽

Enzyme Hydrolysis ◽

Accessory Sex Glands ◽

Tissue Extracts ◽

In Series ◽

The Media ◽

Testosterone Metabolites ◽

Conjugated Steroids

Oestrogens are secreted in large amounts by boar testes and are known to have a synergistic effect with testosterone on the production of large volumes of seminal plasma. Thus, oestrogens play a role in regulating the large accessory sex glands in the boar. Since testosterone metabolites (e.g. 5alpha-dihydrotestosterone) account for much of its action in target tissues we have looked at the metabolism of oestrogens in the accessory sex glands of the male pig. Tissues from seminal vesicles and bulbourethral glands of 6-week-old castrate and intact males, and 12-week-old castrate animals, were incubated with (3)H-labelled oestrone and oestradiol-17beta. Aliquots of spent culture medium and of methanolic tissue extracts were taken to measure radioactivity, prior to separation of unconjugated and conjugated steroids on Waters C(18) Sep-Pak cartridges. About one-third of the radioactivity appeared as conjugates in the media from both glands with each oestrogen. Subsequently, sulphoconjugated steroids and glucuronidates were recovered in series from C(18) cartridges after solvolysis and enzyme hydrolysis respectively. Furthermore, about one-third of the conjugated fraction in each case remained unhydrolysed after these treatments. In conclusion, it is clear that a study of the actions of oestrogens on these glands must consider the dynamics of metabolism of the oestrogens presented to them by the testes and would include conjugation of steroids by the glands themselves.

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