THE EXTRACTION OF PLASMA 3-HYDROXY-17-OXO STEROID SULPHATES AND THE MEASUREMENT OF THE CONSTITUENT DEHYDROEPIANDROSTERONE SULPHATE AND ANDROSTERONE SULPHATE.
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ABSTRACT:
SUBMITTER: MCKENNA J
PROVIDER: S-EPMC1215184 | biostudies-other | 1965 Apr
REPOSITORIES: biostudies-other
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Project description:The enantiomer pair androsterone and ent-androsterone are positive allosteric modulators of ?-aminobutyric acid (GABA) type A receptors. Each enantiomer was shown to bind at the same receptor site. Binding orientations of the enantiomers at this site were deduced using enantiomer pairs containing OBn substituents at either C-7 or C-11. 11?-OBn-substituted steroids and 7?-OBn-substituted ent-steroids potently displace [(35)S]-tert-butylbicyclophosphorothionate, augment GABA currents, and anesthetize tadpoles. In contrast, 7?-OBn-substituted steroids and 11?-OBn-substituted ent-steroids have diminished actions. The results suggest that the binding orientations of the active analogues are inverted relative to each other with the 7?- and 11?-substituents similarly located on the edges of the molecules not in contact with the receptor surface. Analogue potentiation of the GABA current was abrogated by an ?(1) subunit Q241L mutation, indicating that the active analogues act at the same sites in ?(1)?(2)?(2L) receptors previously associated with positive neurosteroid modulation.
| S-EPMC3276733 | biostudies-literature
Project description:1. Dehydroepiandrosterone sulphate was hydrolysed by human placental steroid 3beta-sulphatase in H(2) (18)O. Equimolar amounts of dehydroepiandrosterone and sodium sulphate were similarly incubated as controls. After incubation, unconjugated steroid was extracted with ether and sulphate precipitated as barium sulphate. Both were analysed for (18)O-content by mass spectroscopy, the sulphate as carbon dioxide after initial pyrolysis with graphite. 2. In duplicate experiments, amounts of (18)O equivalent to 67% and 72% respectively of the theoretical content calculated for rupture of the O-S bond were present in the sulphate. As the enzyme preparation used was a microsomal preparation containing unenriched endogenous sulphate and phosphate, and as no incorporation of isotope was found in the steroid, it is concluded that the placental enzyme effects hydrolysis by rupture of the O-S bond.
| S-EPMC1184956 | biostudies-other
Project description:1. The 17beta-hydroxy steroid dehydrogenase was solubilized during haemolysis of erythrocytes and was isolated from the membrane-free haemolysate. Membrane preparations isolated in different ways did not contain 17beta-hydroxy steroid dehydrogenase activity. The 17beta-hydroxy steroid dehydrogenase activity in the haemolysate was concentrated by repeated ammonium sulphate precipitation and gel filtration on Sephadex G-150. The 17beta-hydroxy steroid dehydrogenase activity of the purified preparation per unit weight of protein was 350-3000 times higher than the activity of the crude erythrocyte haemolysate. The 20alpha-hydroxy steroid dehydrogenase activity was lost during this purification procedure. 2. The 17beta-hydroxy steroid dehydrogenase was NADP-dependent and had a pH optimum for conversion of testosterone between 8.5 and 10. For the molecular weight of the enzyme a value of 64000 was calculated from Sephadex chromatography results. 3. p-Chloromercuribenzoate inhibited the enzymic activity. The oxidative activity of the enzyme for the 17beta-hydroxyl group was only partly inhibited when a large excess of 17-oxo steroids was added. The catalysing activity of the enzyme was influenced by the NADP(+)/NADPH ratio. The oxidation of the 17beta-hydroxyl group in the presence of NADP(+) proceeded faster than the reduction of the 17-oxo group with NADPH. When both reduced and oxidized cofactors were present the oxidation of the 17beta-hydroxyl group was inhibited to a considerable extent. 4. The enzyme had a broad substrate specificity and not only catalysed the conversion of androstanes with a 17beta-hydroxyl group, or 17-oxo group, but also the conversion oestradiolleft arrow over right arrowoestrone. In addition the steroid conjugates dehydroepiandrosterone sulphate and oestrone sulphate were also converted. There were no indications that more than one 17beta-hydroxy steroid dehydrogenase was present in the partially purified preparation.
| S-EPMC1178762 | biostudies-other
Project description:1. An enzyme from rat liver, catalysing 3alpha-hydroxy steroid-dependent NAD(P) transhydrogenation and NAD-linked and NADP-linked dehydrogenation of 3alpha-hydroxy steroids, has been purified 100-fold by chromatography on DEAE-cellulose and calcium phosphate gel. 2. No separation of these activities into different protein fractions has been achieved. 3. The properties of the enzyme in catalysing NAD-linked and NADP-linked dehydrogenation have been compared, with androsterone as substrate. Differences were found in pH optima, affinity for coenzyme and steroid, equilibrium constants and effects of salts. 4. NAD-linked dehydrogenation is inhibited by NADPH(2) but is protected from this inhibition by chloride, which alone is itself an inhibitor. 5. The relevance of these findings to the problem of the number of enzymes involved in catalysis of 3alpha-hydroxy steroid-dependent transhydrogenation is discussed.
| S-EPMC1207075 | biostudies-other
Project description:The sulphation of bile acids is an important pathway for the detoxification and elimination of bile acids during cholestatic liver disease. A dehydroepiandrosterone (DHEA) sulphotransferase has been purified from male and female human liver cytosol using DEAE-Sepharose CL-6B and adenosine 3',5'-diphosphate-agarose affinity chromatography [Falany, Vazquez & Kalb (1989) Biochem. J. 260, 641-646]. Results in the present paper show that the DHEA sulphotransferase, purified to homogeneity, is also reactive towards bile acids, including lithocholic acid and 6-hydroxylated bile acids, as well as 3-hydroxylated short-chain bile acids. The highest activity towards bile acids was observed with lithocholic acid (54.3 +/- 3.6 nmol/min per mg of protein); of the substrates tested, the lowest activity was detected with hyodeoxycholic acid (4.2 +/- 0.01 nmol/min per mg of protein). The apparent Km values for the enzyme are 1.5 +/- 0.31 microM for lithocholic acid and 4.2 +/- 0.73 microM for taurolithocholic acid. Lithocholic acid also competitively inhibits DHEA sulphation by the purified sulphotransferase (Ki 1.4 microM). No evidence was found for the formation of bile acid sulphates by sulphotransferases different from the DHEA sulphotransferase during purification work. The above results suggest that a single steroid sulphotransferase with broad specificity encompassing neutral steroids and bile acids exists in human liver.
| S-EPMC1149750 | biostudies-other
Project description:OBJECTIVE:Little is known about the relationships of dietary factors, physical activity and sedentary behaviour to dehydroepiandrosterone sulphate (DHEAS) and insulin-like growth factor-1 (IGF-1) concentrations among prepubertal children. Therefore, we studied the associations of these lifestyle factors with serum DHEAS and IGF-1 in children. DESIGN AND SUBJECTS:Cross-sectional analysis of a population sample of 431 prepubertal children aged 6-9 years. MEASUREMENTS:Assessment of dietary factors by food records and physical activity and sedentary behaviour by a combined heart rate and movement monitor and a questionnaire. Measurement of serum DHEAS and IGF-1. RESULTS:Consumption of low-fibre grain products (standardized regression coefficient ? = .118, P = .017) and intake of vegetable protein (? = .100, P = .045) was positively and consumption of sugar-sweetened beverages (? = -.117, P = .018) was inversely associated with DHEAS after adjustment for sex, age and body fat percentage. Energy intake (? = .160, P = .001) was positively associated with IGF-1 adjusting for sex, age and body fat percentage. Vigorous physical activity was inversely associated with DHEAS after adjustment for sex and age (? = -.120, P = .027), and total (? = -.137, P = .007), moderate (? = -.130, P = .012), vigorous (? = -.136, P = .011) and moderate to vigorous physical activity (? = -.160, P = .003) were inversely and total sedentary behaviour (? = .151, P = .003) was positively associated with IGF-1 adjusting for sex and age. None of physical activity measures was associated with DHEAS or IGF-1 after additional adjustment for body fat percentage. CONCLUSIONS:Lifestyle factors have weak and moderate associations with biochemical markers of adrenarche in prepubertal children. These associations indicate body fat independent and dependent influences of diet and physical activity, respectively.
| S-EPMC6195184 | biostudies-literature
Project description:The chemical syntheses of the 3-sulphates of 16 alpha-acetoxy-, 16 alpha-hydroxy- and 16 beta-hydroxy-dehydroepiandrosterone as their triethylammonium salts are described preparatory to studies of the metabolism of [7 alpha-3 H]dehydroepiandrosterone sulphate by homogenates of human foetal liver. Five main radioactive products of the incubation were separated by partition chromatography on a Celite column. Two were identified as 16 alpha-and 16 beta-hydroxydehydroepiandrosterone 3-sulphates by crystallization to constant specific radioactivity before and after solvolysis. The yields of the conversion to the two epimers were 24.4% (16 alpha) and 1.8% (16 beta).
| S-EPMC1163763 | biostudies-other
Project description:1. A method in use for the extraction of urinary steroid conjugates has been applied to study the recovery of synthetic steroid monoglucuronides from aqueous solution. 2. In the presence of dissolved ammonium sulphate (50g./100ml.), ether-ethanol (3:1, v/v, 3x0.5vol.) extracted the monoglucuronides of steroids of the C(18), C(19) and C(21) series, quantitatively at values pH2-9. 3. The hydrolysis of the synthetic steroid monoglucuronides by beta-glucuronidase (Patella vulgata) has been examined with reference to the pH value of the medium, enzyme concentration and substrate concentration. 4. The rate of hydrolysis of steroid monoglucuronides was dependent upon steroid structure and upon site of conjugation. 5. The rate of hydrolysis of the monoglucuronides decreased in the order C-3 (phenolic) >C-3beta>C-17beta>C-3alpha.
| S-EPMC1265194 | biostudies-other
Project description:The title compound, C(30)H(50)O(3), which was isolated from a marine endophytic fungus, is a new friedelan derivative. The mol-ecule contains five six-membered rings, which exhibit boat (ring A), distorted boat (ring B) and chair (rings C-E) conformations. The crystal structure is stabilized by inter-molecular O-H?O hydrogen bonds, which link neighbouring mol-ecules into 12-membered rings.
| S-EPMC2961169 | biostudies-other
Project description:The prevalence of nonalcoholic steatohepatitis (NASH) caused by estrogen deficiency increased sharply in recent decades and has become a major threat to liver health in postmenopausal women. There is no effective strategy to control the incidence and development of NASH. Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid with immune and metabolic regulatory properties, and its level markedly declines with increasing age in humans. Importantly, DHEA can convert into active sex hormones depending on the local needs of target tissues with little diffusion, which serves to avoid systemic side-effects from other tissues' exposure to estrogen. Here, we found that DHEA prevented the incidence and development of NASH, which is characterized by the reduction of hepatic steatosis, fibrosis, and inflammation in female mice fed with high-fat/high-cholesterol diets and effectively attenuated lipid accumulation, inflammatory response, and oxidative stress in palmitic acid-challenged hepatocytes. Mechanistically, in vitro and in vivo studies showed that the anti-NASH function of DHEA depended on its biotransformation into estrogen rather than androgen, and which up-regulates the expression of G protein-coupled estrogen receptor (GPR30), a non-classical estrogen receptor. The activation of GPR30-mediated AMP-activated protein kinase signaling is a necessary prerequisite for the alleviative effects of DHEA on NASH. Collectively, our data show the mechanisms of DHEA treatment and its effects on NASH that were previously overlooked; the data also show that GPR30 can be used as a target for treating lipid metabolism disorders and related diseases, such as NASH. Furthermore, these findings have the potential to help researchers develop new strategies for preventing NASH in postmenopausal women.
| S-EPMC8604675 | biostudies-literature