The steroid sulphatase of Patella vulgata

Ab, Roy

doi:10.1016/0006-3002(54)90078-5

Cited by 22 publications

(10 citation statements)

References 13 publications

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“…Ester sulfates were determined in each fraction by the Vlitos reaction, as modified by Crepy (3,6,7). After the addition of methylene blue to the aqueous extract, the methylene blue-steroid sulfate complex was extracted with chloroform, and the absorbance at 650 m~uwas compared with that obtained with amounts ranging between 5 and 40 Ag of sodium dehydroepiandrosterone sulfate.…”

Section: Methodsmentioning

confidence: 99%

IDENTIFICATION OF 3β,21-Dihydroxy-5-Pregnene-20-One DISULFATE IN HUMAN URINE

Pasqualini¹,

Jayle²

1962

J. Clin. Invest.

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The chromatographic procedure described by Crepy, Jayle and Meslin (1) separates urinary steroid conjugates into two fractions, the less polar comprising the ester sulfates and the more polar, the glucuronides. In the application of this method to extracts of urines from normal and pathological subjects, it was observed (2) that the early glucuronide eluates contained a small fraction that gave a positive Vlitos reaction (3), characteristic of ester sulfates. This fraction was designated S11, to distinguish it from the major sulfate fraction, Si.The existence of this fraction was confirmed in a previous publication (4), and it was shown that it contained a steroid of low polarity that reduced blue tetrazolium. The purpose of the present investigation was to isolate and identify this conjugate.EXPERIMENTAL METHOD Biological material. Normal subjects were given two intramuscular inj ections each of 40 U of ACTH-zinc phosphate at 12-hour intervals. Urine was collected for 24 hours after the second injection and approximately 10 L of urine was used for extraction.Extraction of the free and conjugated steroids. Solid ammonium sulfate was added to the urine to give a 50 per cent (wt/vol) solution, which was extracted three times at pH 7 with an equal volume of ether: ethanol (3: 1, vol/vol) (5).Chromatography. A. Separation of ester sulfate and glucuronide fractions. The dry urine extract was dissolved in 100 ml of water and the free corticosteroids were extracted with dichloromethane. The conjugated corticosteroids corresponding to 1 L of urine were then chromatographed on several columns of 30 g of alumina.The columns were eluted with the following solvents: 1) 200 ml of butanol: water (98: 2); 2) 250 ml of butanol: water (94: 6); 3) 250 ml of butanol: water (90: 10); 4) 300 ml of butanol: 0.1 per cent ammonium hydroxide (85: 15). Ester sulfates were determined in each fraction by the Vlitos reaction, as modified by Crepy (3,6,7). After the addition of methylene blue to the aqueous extract, the methylene blue-steroid sulfate complex was extracted with chloroform, and the absorbance at 650 m~uwas compared with that obtained with amounts ranging between 5 and 40 Ag of sodium dehydroepiandrosterone sulfate. Glucuronides were determined by the Tollens reaction (8). B. Separation of fraction S11. The first eluates obtained with butanol-ammonium hydroxide contained glucuronides but also gave a positive Vlitos reaction, suggesting the presence of a small quantity of polar ester sulfates. These eluates were pooled, evaporated to dryness, and rechromatographed on several columns containing 20 g of alumina. Elution was carried out as follows: 1) 100 ml of ethanol: water (96: 4); 2) 100 ml of ethanol: water (90: Paper chromatography of ester sulfates. The Si, fraction, after removal of the glucuronides, and the synthetic steroid sulfate 1 were chromatographed in the two following solvent systems: 1) butyl acetate: toluene: n-butanol/ 4 N ammonium hydroxide: methanol (60: 30: 10/50: 50) (9); 2) butyl acetate: n-butanol/4 N amm...

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Section: Methodsmentioning

confidence: 99%

IDENTIFICATION OF 3β,21-Dihydroxy-5-Pregnene-20-One DISULFATE IN HUMAN URINE

Pasqualini¹,

Jayle²

1962

J. Clin. Invest.

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“…p-Nitrophenyl sulphate was measured by a method which involves the extraction of a sulphate ester-methylene blue complex into chloroform [Roy, 1956], except that deproteinization of the incubation medium was achieved by the addition of 10% w/v zinc sulphate (0-2 ml) followed by 05 mol/l sodium hydroxide (0-2 ml) and the optical density of the blue colour was read at a wavelength of 650 nm. Preliminary experiments showed that the rates of formation of formaldehyde, o-aminophenyl glucuronide and p-nitrophenyl sulphate increased linearly with increasing amounts of tissue up to about 10 mg dry weight, and that product formation was linear with time. The concentrations of aminopyrine (50 mmol/l) and o-aminophenol (0-23 mmol/l) were approximately eight times and twice the Km, respectively.…”

Section: Methodsmentioning

confidence: 99%

Activities of Mixed Function Oxidases, Udp‐glucuronyl Transferase and Sulphate Conjugation Enzymes in Galliformes and Anseriformes

Bartlet

Kirinya

1976

Exp Physiol

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The activities of certain drug metabolizing enzymes have been measured in liver and kidney slice preparations from domesticated birds. Aminopyrine demethylase activity was significantly lower in liver slices from the duck (Aylesbury x Pekin, KhakiCampbell) than from the rat (Wistar), and in the Aylesbury x Pekin duck lower than in the turkey (Triple 6 FLX), chicken (Brown Leghorn, Rhode Island Red x Light Sussex) and goose (Emden x Doulouse). The microsomal cytochrome P-450 was lower in duck liver (Aylesbury x Pekin) than in rat liver, and the aniline hydroxylase and aminopyrine demethylase activities in a 10,000 g supernatant fraction of liver were lower in duck preparations (Aylesbury x Pekin, Khaki-Campbell) than rat preparations. These observations suggest that the duck is likely to be susceptible to drugs which are metabolized by the cytochrome P-450 containing mono-oxygenases.UDP-Glucuronyl transferase activity was not detectable in liver and kidney slices from two mature geese. This observation was not the outcome of a deficiency of UDPglucuronic acid, rapid breakdown of glucuronide by ,B-glucuronidase or the presence of a substance inhibitory to UDP-glucuronyl transferase. Liver slices from geese, ducks (Aylesbury x Pekin) and chickens contained low UDP-glucuronyl transferase and high sulphate conjugation enzyme activities, whereas the reverse was found in Khaki-Campbell ducks. The activities of UDP-glucuronyl transferase and the sulphate conjugation enzymes were both relatively high in liver slices from the turkey and rat.The kidney contained lower enzyme activities than the liver except in the duck (Aylesbury x Pekin), in which low activities of aminopyrine demethylase and UDPglucuronyl transferase were present in slices of both organs.In liver slices from chickens and geese the activities of aminopyrine demethylase and the sulphate conjugation enzymes were similar in mature and immature birds, and the activity of UDP-glucuronyl transferase was considerably higher in chicks and goslings than in mature birds of the same species.In the chick the activities of aminopyrine demethylase, UDP-glucuronyl transferase and the sulphate conjugation enzymes were higher in the duodenum than the remainder of the alimentary tract. The activities of these enzymes in pieces of duodenum were as high as those in slices of liver.The inclusion of sulphate in the incubation medium produced a significant increase in the synthesis of p-nitrophenyl sulphate in liver slices and not kidney slices except those from the duck. The kidney slices seemed to produce sufficient sulphate for the reaction of the sulphate conjugation enzymes to proceed at the maximum rate, but the liver slices did not do so.Although domesticated birds sometimes exhibit great differences in susceptibility to foreign compounds, there is little information on the activities of enzymes which metabolize toxic substances in these species. In the present paper we describe experiments which compare the activities of mixed function

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“…It is excreted in small quantities (about 0.5 rag/day) in normal adult urine, where it occurs as a sulfate ester (Roy 1956). …”

Section: Introductionmentioning

confidence: 98%