Kinetics and Mechanism of Solvolysis of Steroid Hydrogen Sulfates<sup>1</sup>

Burstein, Shlomo; Lieberman, Seymour

doi:10.1021/ja01552a054

Cited by 146 publications

(64 citation statements)

References 5 publications

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“…In contrast to enzyme preparations of different origin, solvolysis leads to a complete cleavage of steroid sulphates as was found with dehydroepiandrosterone sulphate as test compound. Using the well-known method ofBurstein &Lieberman (14), no formation of artefacts during solvolysis was noted; this agrees with reports by other authors (11,19,20,21). The use of internal radioactive standards allows the calculation of 100% recovery.…”

Section: Discussionsupporting

confidence: 89%

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

Külpmann¹,

Breuer²

1977

Clinical Chemistry and Laboratory Medicine

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Summary: An improved method for the simultaneous gas-chromatographic determination of individual 17-oxosteroids and of pregnanediol in the same sample of urine is described. The method involves the following steps. The urinary sample is chromatographed on Amberlite XAD-2. The fraction containing the steroid conjugates is evaporated; the residue is dissolved in -heptane and the organic phase extracted with water. The steroid glucuronides are hydrolysed by a bacterial ß-glucuronidase; subsequently, the steroid sulphates are solvolysed in ethyl acetate. Free 17-oxosteroids and pregnanediol are separated by two-dimensional thin-layer chromatography. The zone containing the steroids to be investigated is eluted; after evaporation, the residue is treated with N-methyl N-trimethyl silyl trifluoroacetamide. The reaction mixture is injected into a gas Chromatograph; the trimethyl silyl ethers of the individual 17-oxosteroids and of pregnanediol are quantitated by measuring the peak heights.The losses occurring during the working-up procedure are corrected for by measuring the recovery of radioactive dehydroepiandrosterone which is added as glucuronide and as sulphate to the urinary sample. The specificity of the method was established by mass fragmentography. Eine verbesserte Methode zur gleichzeitigen Bestimmung verschiedener 17-Oxosteroide und von Pregnandiol im Urin mit Hilfe der GaschromatographieZusammenfassung: Es wird eine verbesserte Methode zur gleichzeitigen Bestimmung verschiedener 17-Oxosteroide und von Pregnandiol in derselben Urinprobe mit Hilfe der Gaschromatographie angegeben. Das Verfahren umfaßt folgende Analysen-und Reaktionsschritte. Die Urinprobe wird an Amberlite XAD-2 chromatographiert. Die Fraktion, welche die Steroidkonjugate enthält, wird eingedampft und der Rückstand in rc-Heptan aufgenommen. Die Steroidkonjugate werden mit Wässer aus der organischen Phase extrahiert. Anschließend werden die Steroidglucuronide mit einer bakteriellen ß-Glucurdnidase hydrolysiert und die Steroidsulfate in Essigsäureethylester solvolysiert. Die freien 17-Oxosteroide und Pregnandiol werden durch Dünnschichtchromatographie von begleitenden Verunreinigungen befreit. Die steroidhaltende Zone des Dünnschichtchromatogramms wird eluiert und der Rückstand des Eluats mit N-Methyl-N-trimethylsilyl-trifluoracetamid behandelt. Ein aliquoter Teil der Reaktionsmischung wird in den Gaschromatographen injiziert; die quantitative Bestimmung der Trimethylsilyläther der verschiedenen 17-Oxosteroide und von Pregnandiol erfolgt durch Ausmessung der aufgezeichneten Peakhöhen. Die Korrektur von Aufarbeitungsverlusten erfolgt durch Ermittlung der Wiederfindung von radioaktivem Dehydroepiandrosteron, das als Glucuronid und Sulfat der Urinprobe zugesetzt wird. Die Spezifität der hier beschriebenen Methode wurde mit Hilfe der Massenfragmentographie überprüft und bestätigt.Introduction the procedure as modified by Norymberski et al. (1) in 1953 has been the most popular one. However, in certain The determination of 17-oxosteroids in urine ha...

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Section: Discussionsupporting

confidence: 89%

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

Külpmann¹,

Breuer²

1977

Clinical Chemistry and Laboratory Medicine

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“…The purity of [1,2,6,7- (17). The extracts were washed with water, filtered through Na2SO4, and dried.…”

Section: Methodsmentioning

confidence: 99%

Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.

Corpéchot¹,

Röbel²,

Axelson³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

562

324

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Dehydroepiandrosterone (3f3-hydroxy-5-androsten-17-one, I) sulfate (Ia) has been characterized in the anterior and the posterior parts of the brain of adult male rats. Its level (1.58 ± 0.14 and 4.89 ± 1.06 ng/g, mean ± SD, in anterior and posterior brain, respectively) largely exceeded that of I in brain (0.42 ± 0.10 and 0.12 ± 0.03 ng/g in anterior and posterior brain, respectively) and of Ia in plasma (0.26 ± 0.13 ng/ml). Brain Ia level did not seem to depend on adrenal secretion; it was unchanged after administration of corticotropin or dexamethasone for 3 days, and no meaningful change occurred in brain 15 days after adrenalectomy plus orchiectomy, compared with sham-operated controls. In contrast, stress conditions prevailing 2 days after adrenalectomy plus orchiectomy or after the corresponding sham operation resulted in a significantly increased concentration of Ia in the brain. Changes of Ia level in brain occurred irrespective of changes in corresponding plasma samples. It is proposed that Ia formation or accumulation (or both) in the rat brain depends on in situ mechanisms unrelated to the peripheral endocrine gland system. Dehydroepiandrosterone (3,3hydroxy-5-androsten-17-one, I) sulfate (Ia) is below detection limit in the plasma of most adult mammals (1); the exceptions are man and the highest nonhuman primates (1-3). It is a major secretory product ofhuman adrenals (4-7), and its concentration in adult plasma is larger than that of any other steroid. Although Ia is the main precursor of placental estrogens (8-10) and is occasionally converted into active androgens in peripheral tissues (11,12) was added to 2 ml of plasma or <1 g of tissue, and then 5 ml ofacetone/ethanol (1:1) was added. Tissues were homogenized in acetone/ethanol (1:1) with a Teflon/glass homogenizer and sonicated with a Branson Jl sonifier equipped with a minitip at a 100-W setting for 10 sec. The suspensions were kept at 390C overnight and centrifuged at 1000 x g for 10 min. The supernatant was saved, and the pellet was extracted with 4 ml of methanol/chloroform (1:1) with continuous shaking at room temperature for 30 min. The extract was centrifuged, and the two supernatants were combined and taken to dryness. The residue was dissolved in 4 ml of methanol/chloroform (1:1)/10 mM NaCl and deposited on a Sephadex LH-20 column (10 X 445 mm) equilibrated and developed in the same solvent system (15). The first 50 ml to run off contained unconjugated I, Ia was eluted in the next 75 ml. It was completely separated from free I and from I conjugated to fatty acids (16) 4704The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…This residue was chromatographed on a 250-g celite column in system 3, where the tritiated carrier is eluted in hbv 4-5. Those fractions containing 3H (0.34 X 106 dpm) were combined and evaporated, and the residue was solvolyzed by treatment with 100 ml of H4furan containing 0.09 ml of 70% perchloric acid (8). After the sample remained for 2 days at room temperature, 3 ml of NH40H in 10 ml of methanol was added to the H4furan solution.…”

Section: Methodsmentioning

confidence: 99%

Detection in bovine adrenal cortex of a lipoidal substance that yields pregnenolone upon treatment with alkali.

Hochberg¹,

Bandy²,

Ponticorvo³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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Bovine adrenal cortical tissue contains a lipoidal derivative of pregnenolone (3#-hydroxypregn-en-20-one) from which.the free steroid can be liberated by treatment with alkali. Evidence for the presence of such an entity comes from examination of a nonpolar extract of tissue from which pregnenolone and its sulfate had been removed by chromatography. Treatment of the nonpolar fraction with alkali followed by exhaustive chromatographic analysis led to the detection of pregnenolone. The steroid was identified by both gas chromatography/mass spectrometry and double isotope procedures. Quantitative analysis indicated that the three forms of pregnenolone are present in bovine adrenal cortical tissue in the following amounts (jtg/kg): lipoidal derivative, 290; free steroid, 435; and sulfate, 65. Because the only known metabolic function of pregnenolone is to serve as a precursor of the steroid hormones, these findings have far-'reaching implications for steroid hormone biochemistry. This paper describes experiments that indicate that there is present'in bovine adrenal cortices a lipoidal substance that yields pregnenolone (3fl-hydroxy-pregn-5-en-20-one) upon treatment with alkali. Adrenals have long been known to contain pregnenolone and pregnenolone sulfate, but we now present conclusive evidence for the existence of a nonpolar steroidal derivative which, upon alkaline treatment, leads to the liberation of pregnenolone. This C21-steroid, as is well known, serves as a common precursor for all the steroid hormones. Although it is not now possible to assess what significance the lipoidal derivative of pregnenolone has, its existence is noteworthy and accounts for the fact that this report is being made before its exact structure has been determined.We were led to search for such a lipoidal form of pregnenolone by the recent observation (1) that acyl esters of cholesterol, as well as some inorganic esters of that sterol, such as the nitrate or phosphate, can serve as substrates for the cholesterol sidechain cleavage enzyme present in adrenal mitochondria. When cholesterol nitrate, cholesterol acetate, or cholesterol caproate was used as substrate for this enzyme, the corresponding pregnenolone ester was found to be the product. These results suggested that the side-chain cleavage enzyme was unable to distinguish between cholesterol derivatives that differ from each other by the presence of various carboxylic and inorganic acid esters at C-3.These findings recalled some observations that were made almost a decade ago and whose full significance still remains unexplained. During a search for intermediates that might be involved in the conversion of cholesterol into pregnenolone, we reported the isolation of (20S)-20-hydroxycholesterol from adrenal extracts (2). In that study, fractions that were assumed to contain the fatty acid esters of cholesterol were processed in a manner that would permit us to explore the possibility that an esterified form of (20S)-20-hydroxycholesterol was also present in these extracts....

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Kinetics and Mechanism of Solvolysis of Steroid Hydrogen Sulfates¹

Cited by 146 publications

References 5 publications

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.

Detection in bovine adrenal cortex of a lipoidal substance that yields pregnenolone upon treatment with alkali.

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Kinetics and Mechanism of Solvolysis of Steroid Hydrogen Sulfates1

Cited by 146 publications

References 5 publications

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

An Improved Method for the Simultaneous Determination of Individual 17-Oxosteroids and of Pregnanediol in Urine by Gas-Liquid Chromatography

Characterization and measurement of dehydroepiandrosterone sulfate in rat brain.

Detection in bovine adrenal cortex of a lipoidal substance that yields pregnenolone upon treatment with alkali.

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Kinetics and Mechanism of Solvolysis of Steroid Hydrogen Sulfates¹