Development and performance evaluation of a tandem mass spectrometry assay for 4 adrenal steroids.
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BACKGROUND: Congenital adrenal hyperplasia is a group of autosomal recessive disorders caused by a deficiency of 1 of 4 enzymes required for the synthesis of glucocorticoids, mineralocorticoids, and sex hormones. Analysis of 11-deoxycortisol (11DC), 17-hydroxyprogesterone (17OHP), 17-hydroxypregnenolone (17OHPr), and pregnenolone (Pr) in blood allows detection of these enzyme defects. METHODS: The steroids were extracted from 200 microL of serum or plasma by solid-phase extraction, derivatized to form oximes, and extracted again with methyl t-butyl ether. Instrumental analysis was performed on an API 4000 tandem mass spectrometer with electrospray ionization in positive mode and multiple reaction-monitoring acquisition. RESULTS: The limits of detection were 0.025 microg/L for 11DC, 17OHP, and Pr and 0.10 microg/L for 17OHPr. The method was linear to 100 microg/L for 11DC, 17OHP, and Pr, respectively, and to 40 microg/L for 17OHPr. Within- and between-run (total) imprecision (CVs) were <7.1% and 11%, respectively. Reference intervals for children in Tanner stages 1 through 5 and adult males and females for 17OHP, 11DC, Pr, and 17OHPr were established. Prepared samples were stable for >72 h. CONCLUSIONS: The detection limit and selectivity of this method and its small sample volume requirement allow analysis of endogenous concentrations of adrenal steroids in serum or plasma from children and adults. The method thus has an important potential role in the evaluation of the status of 4 of the enzymes involved in adrenal steroid biosynthesis. (+info)
Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats.
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Pregnenolone (PREG) and dehydroepiandrosterone (DHEA) have been reported to improve memory in aged rodents. In brain, these neurosteroids are transformed predominantly into 7alpha-hydroxylated metabolites by the cytochrome P450-7B1 (CYP7B). The biological role of steroid B-ring hydroxylation is unclear. It has been proposed to generate bioactive derivatives that enhance cognition, immune, and other physiological processes. In support, 7alpha-hydroxylated DHEA increases the immune response in mice with greater potency than the parent steroid. Whether the memory-enhancing effects of PREG in rats is mediated via its 7alpha-hydroxylated metabolite 7alpha-hydroxyPREG is not known. We investigated this by treating memory-impaired aged rats (identified by their spatial memory performances in the Morris water maze task compared with young controls) with 7alpha-hydroxyPREG or PREG administered intracerebroventricularly using osmotic minipumps and then tested the rats during week 2 of steroid treatment in the eight-arm radial-arm version of the water maze (RAWM) that allows repeated assessment of learning. CYP7B bioactivity in hippocampal tissue (percentage conversion of [14C]DHEA to [14C]7alpha-hydroxyDHEA) was decreased selectively in memory-impaired aged rats compared with both young and memory-intact aged rats. 7alpha-hydroxyPREG (100 ng/h) but not PREG (100 ng/h) administration to memory-impaired aged rats for 11 d enhanced spatial memory retention (after a 30 min delay between an exposure trial 1 and test trial 2) in the RAWM. These data provide evidence for a biologically active enzyme product 7alpha-hydroxyPREG and suggests that reduced CYP7B function in the hippocampus of memory-impaired aged rats may, in part, be overcome by administration of 7alpha-hydroxyPREG. (+info)
Optimizing the use of 17P in pregnant managed Medicaid members.
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OBJECTIVE: To evaluate the effect of 17 alpha-hydroxyprogesterone caproate (17P) on reducing the rate of neonatal intensive care unit (NICU) admissions and premature births in a managed Medicaid population that has a history of preterm delivery. Specifically, to measure the effect of initiating 17P treatment during the recommended time frame of 16-21 weeks gestation versus after 21 weeks gestation. DESIGN: A 2004-2007 observational, causal comparative study reviewed birth outcomes in 104 pregnant women with a confirmed history of preterm delivery. Women whose 17P treatment was initiated during the recommended time frame of 16-21 weeks gestation were compared to those whose treatment was initiated after 21 weeks gestation. METHODOLOGY: Intervention included offering 17P as a benefit to pregnant women who had a history of preterm delivery and who were deemed to be appropriate candidates for this treatment by their physician. RESULTS: No significant changes in birth outcomes were noted when comparing those members whose treatment was initiated during the recommended time frame of 16-21 weeks versus those whose treatment began after 21 weeks gestation. Members who received therapy of at least five injections of 17P, as opposed to those receiving fewer than five injections, experienced a statistically significant reduction in NICU admissions and in preterm birth at fewer than 37 weeks and at fewer than 32 weeks. CONCLUSION: The number of injections and not the time frame, which had been indicated by previous research, the initiation of 17P therapy is the factor in reducing preterm birth and decreasing NICU admissions for pregnant women with a history of preterm birth in a managed Medicaid population. (+info)
Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.
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When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. (+info)
Steroidogenesis in interstitial cells and microsomal fraction of immature pig testes.
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Testicular interstitial cells (greater than 90% viable) obtained from 6-day-old and 3-6-week-old piglets were capable of producing dehydroepiandrosterone (DHEA, 5-10 ng/500,000 cells) and responded to hCG (60 mi.u./ml), dibutyryl-cAMP (1 mmol/l) and cholera toxin (5 ng/ml) with a 2-3-fold increase in DHEA. Aminoglutethimide (100 mumol/l) abolished the response. Testosterone was produced in comparatively minor quantities (less than 0.3 ng/500,000 cells) and was unaffected by stimulation or inhibition. When cells from both age groups were incubated with [14C]- or [3H]-pregnenolone (360 and 3.0 nmol/l), 17-hydroxypregnenolone (15%) and DHEA (5-10%) were the major metabolites on the androgen pathway and 5,16-androstadien-3 beta-ol (andien-beta, 5-10%) and 4,16-androstadien-3-one (dienone, 5-10%) on the 16-androstene pathway. Stimulation and inhibition of endogenous steroidogenesis did not alter the metabolism of exogenous pregnenolone, the same metabolites being found in the same proportions at similar times. Microsomal enzyme activities accurately reflected the metabolic profile of pregnenolone metabolism seen in intact cells, with low activities for 17 beta-HSD, 3 beta-HSD-isomerase, and 16-ene-5 alpha-reductase being observed. Since steroidogenic capacity, enzyme complement and pregnenolone metabolism were the same in testes from both age groups, the differences in Leydig cell activity observed in vivo would not appear to be consequences of changes in steroidogenic enzymes or responsiveness to gonadotrophin stimulation. The lack of effect of stimulation and inhibition of steroidogenesis on the cellular metabolism of exogenous pregnenolone suggests that the endogenous and exogenous supplies of pregnenolone are metabolized by different populations of enzymes. The relative magnitudes of these populations indicate that most of the steroidogenic enzymes in the interstitial cells are not involved in the normal response to trophic stimulation. (+info)
Successful treatment of a metastatic hormone-producing adrenal cancer by a combination of mitotane, tegafur and surgical resection.
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A 34-year-old man had a huge hormone-producing adrenal cancer with multiple lung metastases, direct liver invasion and a tumor thrombus in the inferior vena cava. Initial treatment was mitotane alone. The dose of mitotane was 2 g/day initially and gradually increased to 15 g/day in combination with 600 mg of tegafur per day. During the initial phase of chemotherapy, the serum mitotane level was relatively low (2.9-4.6 micrograms/ml) and the pulmonary metastases tended to grow in size in spite of a gradual decline in urinary 17-KS and 17-OHCS and a regression of the primary tumor. A seemingly marked increase in serum mitotane (20.5-34.5 micrograms/ml) was coincident with the addition of tegafur. Rapid and consistent regression of the primary tumor occurred. This excellent response to the chemotherapy made the primary tumor with liver invasion and the metastases resectable. The adverse effect of mitotane, central nervous toxicity, appeared to be serum mitotane level dependent. The present results, together with previous reports in the literature, seem to recommend the following therapeutic approaches to advanced adrenal cancer: monitoring of the serum level may be useful in predicting the efficacy as well as the occurrence of side effects of mitotane, surgical treatment of the lesions should be performed whenever possible, even though it may be only palliative, and the combination of mitotane and tegafur is a choice of chemotherapy which should be evaluated in future studies. (+info)
Light-dependent and circadian clock-regulated activation of sterol regulatory element-binding protein, X-box-binding protein 1, and heat shock factor pathways.
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