Hormonal diagnosis of 21-hydroxylase deficiency in plasma and urine of neonates using benchtop gas chromatography-mass spectrometry.
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We aimed at measuring the first plasma concentrations of 17-hydroxyprogesterone (17OH-P) determined by benchtop isotope dilution/gas chromatography-mass spectrometry (ID/GC-MS) in term neonates with or without 21-hydroxylase deficiency. Plasma samples from normal cord blood specimens (n=30), unaffected neonates (n=38) and neonatal patients with classical 21-hydroxylase deficiency (eight salt-wasters, three simple virilizers) were analyzed. Steroid profiling of random urinary specimens by GC-MS served as a confirmatory test for 21-hydroxylase deficiency. 17OH-P (nmol/l) in cord blood plasma lay between 11.66 and 75.92 (median 24.74). It declined shortly after birth. In the first 8 days of life, the time that screening for 21-hydroxylase deficiency is performed, 17OH-P ranged between undetected levels and an upper limit of 22.87 (median 4.11). Thereafter (days 9-28) its concentrations lay between 2.18 and 20.30 (median 6.22). Except one simple virilizer, all other patients with 21-hydroxylase deficiency had clearly elevated plasma 17OH-P at the time that screening for 21-hydroxylase deficiency would be performed. We suggest ID/GC-MS, which provides the highest specificity in steroid analysis, for checking suspicious concentrations of 17OH-P in neonates and underscore the potential of urinary steroid profiling by GC-MS as a rapid, non-invasive and non-selective confirmatory test for congenital adrenal hyperplasia. (+info)
Induction of delta-aminolevulinic acid synthetase in isolated rat liver cells by steroids.
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The role of steroids in regulation of delta-aminolevulinic acid synthetase has been studied in isolated rat liver cell suspensions under conditions previously shown to support inducation of the enzyme by drugs. Addition of a variety of C-19 and C-21 steroids to cell suspensions resulted, after 4 to 6 hours of incubation, in 2- to 5-fold increase in the activity of delta-aminolevulinic acid synthetase as measured in liver cell homogenates. The increase was prevented by cycloheximide. The most active steroid inducers tested were pregnene or pregnane derivatives with keto or hydroxyl groups at C-3 and C-20; in particular a beta-hydroxyl group at C-20 enhanced activity. These C-21 steroids at optimal initial concentrations caused 3- to 5-fold induction over 4 hours. A number of C-19 androstene and androstane compounds caused 2- to 3-fold inducation over the same period. Hydrocortisone had no effect. For a variety of androstane and pregnane derivatives, inducation by 5alphaH steroids was as great as or greater than that by 5betaH compounds, in contrast to previous findings in chick embryo liver. Induction of delta-aminolevulinic acid synthetase by steroids in isolated liver cells was shown to be subject to feedback repression by hemin. (+info)
Sweet pregnane glycosides from Telosma procumbens.
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An intensely sweet polyoxypregnane glycoside, telosmoside A15 (15), was isolated from an Asian Asclepiadaceae plant, Telosma procumbens, collected in Vietnam. This is the first time a sweet pregnane glycoside has been found, and its sweetness intensity is 1000 times greater than that of sucrose. From the same plant, 17 other new glycosides were isolated, having the same aglycone; they are named telosmosides A1-A14 (1-14) and A16-A18 (16-18). Some of these glycosides are also sweet, but others are tasteless or bitter. Chemical structures of the 18 glycosides were determined, and the structure-taste relationship was discussed. (+info)
Evaluation of new pregnane derivatives as 5alpha-reductase inhibitor.
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The objective of this study was to synthesize several new pregnane derivatives and evaluate them as antiandrogens. From the commercially available 16-dehydropregnenolone acetate (7), two new steroidal compounds were synthesized: 17alpha-hydroxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dion e (18) and 17alpha-acetoxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dion e (19). The 5alpha-reductase inhibitory effect of the new compounds 18 and 19 together with the previously synthesized intermediates 7, 8, 13, 16, and 17 was determined in three different models: gonadectomized hamster flank organs diameter size, incorporation of [1,2-(14)C]sodium acetate into lipids in flank organs and conversion of [3H]testosterone (T) to [3H]dihydrotestosterone (DHT) by Penicillium crustosum. The evaluation of these steroids was carried out with three different controls: one group was treated with vehicle, the second with T and the third group with T plus finasteride. The pharmacological results from this work demonstrated that T significantly increases the diameter of the pigmented spot on the flank organs (p<0.05) as well as the incorporation of labeled sodium acetate into lipids in gonadectomized hamster flank organs (from 0.125 to 0.255 nmol per gland). In this study we also observed that broth of Penicillium crustosum converted [3H]T to [3H]DHT in a manner comparable to that of the flank organs. All experiments indicated that finasteride as well as steroids 7, 8, 13, 16-19 reduced significantly the conversion of T to DHT in P. crustosum. These compounds also decrease the size of the pigmented spot in the flank organs as well as reducing the incorporation of radiolabeled sodium acetate into lipids; T and the control sample (treated with vehicle only) were used for comparison. Apparently the presence of the 4,6-diene-3,20-dione moiety and also the C-17 ester group produce a higher inhibitory effect on the parameters used. PPThe data from this study indicated also that the three models used for the pharmacological evaluation exhibited comparable results. (+info)
Steroid induction of delta-aminolevulinic acid synthase and porphyrins in liver. Structure-activity studies and the permissive effects of hormones on the induction process.
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Quantitative aspects and structure-activity relationships of the inducing effects of natural steroids on delta-aminolevulinic acid (ALA) synthase and porphyrins have been investigated in monolayer cultures of chick embryo liver cells maintained in a serum-free medium as well as in the chick embryo liver in ovo. Many 5 alpha and 5 beta metabolites of neutral C-19 and C-21 hormones and hormone precursors stimulated porphyrin formation and ALA-synthase induction in the cultured liver cells as we have previously described. In these inducing actions a number of 5 beta epimers (A:B cis) were found to be more potent than their corresponding 5 alpha epimers (A:B trans). The structure-activity relationship between 5 beta and 5 alpha steroid epimers with respect to ALA-synthase induction in culture was also found to prevail with respect to induction of this enzyme in chick embryo liver in ovo. Hemin in concentrations of 2 x 10(-7) M inhibited steroid induction of porphyrin formation, and CaMgEDTA enhanced the responsiveness of the cultured liver cells to steroids by approximately 10 times. The addition of insulin, or insulin plus hydrocortisone or insulin plus hydrocortisone plus triiodothyronine, was important for the maintenance of protein synthesis and essential for maximal expression of the ability of steroids to induce porphyrins and ALA-synthase in the "permissive" effect which insulin, hydrocortisone, and triiodothyronine exert on allylisopropylacetamide induction of porphyrins and ALA-synthase also extends to the induction process which is elicited by natural steroids. These findings also strongly suggest that the regulation of hepatic porphyrin-heme biosynthesis by endogenous as well as exogenous chemicals is significantly influenced by the internal hormonal milieu. (+info)
Effects on gamma-aminobutyric acid (GABA)(A) receptors of a neuroactive steroid that negatively modulates glutamate neurotransmission and augments GABA neurotransmission.
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Neurosteroids positively and negatively modulate gamma-aminobutyric acid (GABA)(A) receptors and glutamate receptors, which underlie most fast inhibition and excitation in the central nervous system. We report the identification of a neuroactive steroid, (3 alpha,5 beta)-20-oxo-pregnane-3-carboxylic acid (3 alpha 5 beta PC), with unique cellular actions. 3 alpha 5 beta PC positively modulates GABA(A) receptor function and negatively modulates N-methyl-D-aspartate (NMDA) receptor function, a combination that may be of particular clinical benefit. 3 alpha 5 beta PC promotes net GABA(A) potentiation at low steroid concentrations (<10 microM) and at negative membrane potentials. At higher concentrations, the steroid also blocks GABA receptors. Because this block would presumably counteract the NMDA receptor blocking actions of 3 alpha 5 beta PC, we characterize the GABA receptor block in some detail. Agonist concentration, depolarization, and high extracellular pH increase the block. The apparent pK for both potentiation and block was 6.4 to 6.9, substantially higher than expected from carboxylated steroid in an aqueous environment. Block is not dependent on the stereochemistry of the carboxylic acid at carbon 3 and is relatively insensitive to placement of the carboxylic acid at the opposite end of the steroid (carbon 24). Potentiation is critically dependent on the stereochemistry of the carboxylic acid group at carbon 3. Consistent with the pH dependence of potentiation, effects of the amide derivative (3 alpha,5 beta)-20-oxo-pregnane-3-carboxamide, suggest that the un-ionized form of 3 alpha 5 beta PC is important for potentiation, whereas the ionized form is probably responsible for block. Further refinement of the neuroactive steroid to promote GABA potentiation and NMDA receptor block and diminish GABA receptor block may lead to a clinically useful neuroactive steroid. (+info)
Specific deuterium labelling and computerized gas chromatography -- mass spectrometry in studies on the metabolism in vivo of a steroid sulphate in the rat.
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The metabolism of 3beta-hydroxy-5alpha-pregnan-20-one sulphate was studied in bile fistula rats and in isolated perfused livers. Computerized gas chromatography--mass spectrometry, in combination with specific deuterium-labelling, was employed to follow the metabolic transformations. Male animals excreted metabolites into bile more rapidly than females, a finding which could be correlated with the preferential formation of glucuronide conjugates in the male liver. The major metabolic pathway in male rats involved the steps: hydrolysis, 2alpha-hydroxylation, oxidoreduction at C-3 and glucuronide conjugation, yielding 2alpha, 3alpha-dihydroxy-5alpha-pregnan-20-one glucuronide as the major metabolite. Only traces of the injected steroid sulphate were detected in bile from male animals. In contrast, the administered compound was the major steroid excreted in bile of female rats, where the main metabolite was identified as 3beta,15beta-dihydroxy-5alpha-pregnan-20-one sulphate. A minor metabolite, 3beta,16alpha-dihydroxy-5alpha-pregnan-20-one, was found as a monosulphate in female rats and as both a disulphate and a glucuronide conjugate in male rats. The deuterium content of the sulphated 15beta-and 16alpha-hydroxylated metabolites was consistent with metabolic pathways involving direct hydroxylation of the injected steroid sulphate. The results obtained from the liver perfusions were essentially the same as those from the experiments with bile fistula animals. This indicates that all the observed metabolic reactions took place in the liver. (+info)
Synthesis and pharmacological evaluation of new 16-methyl pregnane derivatives.
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The pharmacological activity of several new pregnane derivatives 15-19 were determined on gonadectomized male hamster flank organs, seminal vesicles and in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors. Steroids 15-19 decreased the diameter of the pigmented spot in the flank organs as compared to the T treated animals; in this model, steroids 16 and 19 showed a higher activity than the commercially available finasteride 3. Injection of T increased the weight of the seminal vesicles. Compounds 15-19 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. The trienone 19 exhibited a considerably higher activity than finasteride. Steroids 15-19 inhibited the in vitro metabolism of [3H]T to [3H]DHT in seminal vesicles homogenates of gonadectomized male hamsters. Compounds 18 and 19 showed a much higher antiandrogenic effect than finasteride. This enhancement of the biological activity could probably be attributed to the coplanarity of the steroidal skeleton as previously observed by our group. The high antiandrogenic activity of the epoxy compound 16 is probably the result of the ring opening of the oxiran ring with the nucleophilic part of the enzyme 5alpha-reductase thus leading to a stable adduct with concomitant deactivation of this enzyme. (+info)