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A. P. WADE
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Sub-Department ofEndocrine Pathology, University of Liverpool, The Liverpool Clinic, Liverpool, L7 7DE

(Received 17 October 1974)

Although mild methods of hydrolysis are available, hot acid hydrolysis is frequently used to hydrolyse oestriol conjugates before estimation of the free steroid. Assessment of losses arising from hydrolysis, extraction and other manipulations is possible, using radioactive oestriol. Tritiated oestriol is preferable to 14C-labelled oestriol because of the high specific activity of tritium-labelled compounds. For radioimmunoassay, compounds of very high specific activity have been prepared, labelled in the 2,4 positions, with or without additional radioactivity at positions 6,7 or 6,9.

We have found that oestriol labelled in the 2,4 positions is unsuitable for following procedural losses if hot acid hydrolysis is used.

Tritiated steroids (Radiochemical Centre, Amersham) were checked for radiochemical purity by thin-layer chromatography, followed by scanning in a radiochromatogram scanner. No evidence of impurity was found. The steroid (either 20 000

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G. S. WILKINSON
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A. P. WADE
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Since Baulieu (1962) suggested that dehydroepiandrosterone sulphate (DHAS) was a natural adrenal secretory product, several endocrine tissues have been shown to form steroid sulphates. These can be metabolized and still preserve their conjugated form (Calvin & Lieberman, 1964). This report presents evidence for such transformations by polycystic ovarian tissue.

Homogenized tissue, in 3 ml. Krebs—Ringer improved bicarbonate buffer, was incubated with 25 μc [7α-3H]DHAS (Radiochemical Centre, Amersham, 605 mc/m-mole) for 2 hr. at 37°, in 95%O25%CO2. Neutral, phenolic and conjugate fractions were obtained after adding carrier steroids to the incubation mixture (Fahmy, Griffiths, Turnbull & Symington, 1968). Complete extraction of free steroids was made before conjugate extraction.

The neutral fraction (45% of recovered radioactivity) was chromatographed on

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silica gel (TLC) (acetone: methylene chloride, 1:9). Areas were eluted corresponding in position to the added steroids (Δ4-androstenedione, DHA, testosterone, Δ5-androstenediol, 16α-hydroxy DHA

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R. W. H. EDWARDS
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A. P. WADE
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During the analysis of human urines for their steroid content, a substance was encountered which could not be identified with a known steroid. It was noticed in both laboratories; as a contaminant of the pregnanediol fraction from the urine of a female child with hirsutism (A.P.W.) and on two-dimensional paper chromatograms of urine extracts from five cases of congenital adrenal hyperplasia (R.W.H. E.). Acetylation and chromatography of the pregnanediol fraction showed that this substance was not pregnanediol.

The substance has since been found in almost all urines examined, childrens' and adults', in amounts ranging from a few micrograms to several milligrams/24 hr., but as yet the excretion cannot be related to age, sex, or physiological state. Generally it is excreted as the glucuronide, but is sometimes unconjugated.

Isolation was achieved by ethyl acetate extraction following enzyme hydrolysis of extracted urinary glucuronides, separation into ketones and non-ketones with Girard's reagent, and

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OLIVE W. SMITH
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A. P. WADE
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F. M. DEAN
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SUMMARY

A Pettenkofer and sulphuric acid chromogen, excreted as a glucuronide and found in the fractions of urine that also contain hydroxylated Δ5-3β-hydroxysteroids and pregnanediol, was identified as p-menth-1-ene-8,9-diol (uroterpenol). Pettenkofer chromogenicity in a compound of this nature has not previously been reported. The recovery of uroterpenol in urine after ingestion of limonene showed that it was a metabolic product of this unhydroxylated monoterpene. Although experiments on its excretion by normal subjects indicated that it was largely, if not entirely, of dietary origin, there was evidence that its rate of excretion in women was influenced by endocrine factors. Hormonal effects on the formation and/or excretion of glucuronides in general are suggested. Care is needed to ensure that dietary constituents and their metabolites, which are excreted as glucuronides and exhibit colour reactions commonly used in the estimation of urinary steroids, are not confused with hormonal metabolites.

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A. P. WADE
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G. S. WILKINSON
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J. C. DAVIS
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T. N. A. JEFFCOATE
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SUMMARY

[4-14C]Testosterone, [4-14C]androstenedione and [4-14C]oestrone were incubated with testicular tissue obtained from an 18-yr.-old patient with the complete form of the testicular feminization syndrome.

Considerable biosynthesis of testosterone from androstenedione occurred, but metabolism of testosterone by the tissue was minimal. The small phenolic fraction from these incubations did not contain any recognizable oestrogens.

Metabolism of oestrone was almost complete, less than 4% being recovered as unchanged oestrone. Of eight areas of radioactivity found during chromatography, five were shown to be associated with oestrone, 2-methoxyoestrone, oestradiol-17β, 2-hydroxyoestradiol-17β, and 2-methoxy-oestradiol-17β. Chromatographic evidence suggested that an oxo-oestrone and a compound more polar than oestriol were present. No oestriol was found.

The results confirm those of other workers to the effect that testosterone is the major metabolite of androstenedione in feminizing testes. Incubation of the testes with oestrone showed them also to possess the enzyme systems necessary for hydroxylation at position 2 and the subsequent methylation of this group.

Urinary steroid measurements before and after removal of the testes showed these organs to be actively secreting.

Attempts to demonstrate oestrogenic activity in the urine additional to that accounted for by chemical estimation were unsuccessful.

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P Fu Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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P-J Shen Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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C-X Zhao Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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D J Scott Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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C S Samuel Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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J D Wade Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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G W Tregear Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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R A D Bathgate Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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A L Gundlach Howard Florey Institute,
Department of Biochemistry and Molecular Biology,
Department of Anatomy and Cell Biology, The University of Melbourne, Victoria 3010, Australia

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Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8, or RXFP2) is a member of the type C leucine-rich repeat-containing G protein-coupled receptor family, and its endogenous ligand is insulin-like peptide-3 (INSL3). Although LGR8 expression has been demonstrated in various human tissues, including testis, ovary, brain and kidney, the precise roles of this receptor in many of these tissues are unknown. In an effort to better understand INSL3–LGR8 systems in the rat, we cloned the full-length Lgr8 cDNA and investigated the presence and cellular localization of Lgr8 mRNA expression in adult and developing rat kidney. On the basis of these findings, we investigated the presence and distribution of renal 125I-labelled human INSL3-binding sites and the nature of INSL3–LGR8 signalling in cultured renal cells. Thus, using in situ hybridization histochemistry, cells expressing Lgr8 mRNA were observed in glomeruli of renal cortex from adult rats and were tentatively identified as mesangial cells. Quantitative, real-time PCR analysis of the developmental profile of Lgr8 mRNA expression in kidney revealed highest relative levels at late stage gestation (embryonic day 18), with a sharp decrease after birth and lowest levels in the adult. During development, silver grains associated with Lgr8 mRNA hybridization were observed overlying putative mesangial cells in mature glomeruli, with little or no signal associated with less-mature glomeruli. In adult and developing kidney, specific 125I-INSL3-binding sites were associated with glomeruli throughout the renal cortex. In primary cultures of glomerular cells, synthetic human INSL3 specifically and dose-dependently inhibited cell proliferation over a 48 h period, further suggesting the presence of functional LGR8 (receptors) on these cells (mesangial and others). These findings suggest INSL3–LGR8 signalling may be involved in the genesis and/or developmental maturation of renal glomeruli and possibly in regulating mesangial cell density in adult rat kidney.

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