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Mitsuhiro Okamoto - One of the best experts on this subject based on the ideXlab platform.
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dahl s salt resistant normotensive rat has mutations in cytochrome p450 11 beta but the salt sensitive hypertensive rat does not
Journal of Biological Chemistry, 1993Co-Authors: Naomichi Matsukawa, Yasuki Nonaka, Jitsuo Higaki, Masahiro Nagano, Hiroshi Mikami, Toshio Ogihara, Mitsuhiro OkamotoAbstract:Abstract Molecular cloning of cytochrome P450(11 beta) cDNAs from the adrenal glands of Dahl's salt-sensitive hypertensive (DS) and salt-resistant normotensive (DR) rats was performed using a combined technique of the first strand cDNA synthesis by reverse transcriptase followed by polymerase chain reaction. The cDNA sequence of P450(11 beta)-DS was identical to that of wild type P450(11 beta). In contrast, the clone obtained from the DR rat contained six nucleotide substitutions causing five amino acid alterations (Arg-127-->Cys, Val-351-->Ala, Val-381-->Leu, Ile-384-->Leu, and Val-443-->Met). When the two cDNAs were expressed in COS-7 cells and steroid conversion rates of the transformed cells were determined, a ratio of 18-hydroxylation to 11 beta-hydroxylation of 11-Deoxycorticosterone by P450(11 beta)-DS-expressed cells was 0.58, whereas that by P450(11 beta)-DR-expressed cells was 0.23. Plasma levels of 18-hydroxy-11-Deoxycorticosterone and corticosterone (the 11 beta-hydroxylation product of 11-Deoxycorticosterone) in DS and DR rats well reflected the steroidogenic activities of the two P450s. These results suggest that the characteristic plasma steroid level of the DR rat is caused by the mutations in P450(11 beta) gene and may act to maintain the normotensive blood pressure in this rat strain during sodium loading.
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Functional expression of the cDNAs encoding rat 11β-hydroxylase [cytochrome P450(11β)] and aldosterone synthase [cytochrome P450(11β, aldo)]
European journal of biochemistry, 1991Co-Authors: Yasuki Nonaka, Mitsuhiro OkamotoAbstract:Expression plasmids containing two cDNAs of a rat cytochrome P450(11β) family, pcP450(11β)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21–26] and pcP450(11β, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245–252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11β) and P450(11β, aldo) were determined by using 11-Deoxycorticosterone, corticosterone, 18-hydroxy-11-Deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-Deoxycorticosterone as a substrate. Cytochrome P450(11β) catalyzed 11β-, 18-and 19-hydroxylations of 11-Deoxycorticosterone and 19-oxidation or 19-hydroxy-11-Deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11β, aldo) catalyzed 11β- and 18-hydroxylations of 11-Deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-Deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-Deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-Deoxycorticosterone was catalyzed by cytochrome P450(11β, aldo).
Yasuki Nonaka - One of the best experts on this subject based on the ideXlab platform.
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dahl s salt resistant normotensive rat has mutations in cytochrome p450 11 beta but the salt sensitive hypertensive rat does not
Journal of Biological Chemistry, 1993Co-Authors: Naomichi Matsukawa, Yasuki Nonaka, Jitsuo Higaki, Masahiro Nagano, Hiroshi Mikami, Toshio Ogihara, Mitsuhiro OkamotoAbstract:Abstract Molecular cloning of cytochrome P450(11 beta) cDNAs from the adrenal glands of Dahl's salt-sensitive hypertensive (DS) and salt-resistant normotensive (DR) rats was performed using a combined technique of the first strand cDNA synthesis by reverse transcriptase followed by polymerase chain reaction. The cDNA sequence of P450(11 beta)-DS was identical to that of wild type P450(11 beta). In contrast, the clone obtained from the DR rat contained six nucleotide substitutions causing five amino acid alterations (Arg-127-->Cys, Val-351-->Ala, Val-381-->Leu, Ile-384-->Leu, and Val-443-->Met). When the two cDNAs were expressed in COS-7 cells and steroid conversion rates of the transformed cells were determined, a ratio of 18-hydroxylation to 11 beta-hydroxylation of 11-Deoxycorticosterone by P450(11 beta)-DS-expressed cells was 0.58, whereas that by P450(11 beta)-DR-expressed cells was 0.23. Plasma levels of 18-hydroxy-11-Deoxycorticosterone and corticosterone (the 11 beta-hydroxylation product of 11-Deoxycorticosterone) in DS and DR rats well reflected the steroidogenic activities of the two P450s. These results suggest that the characteristic plasma steroid level of the DR rat is caused by the mutations in P450(11 beta) gene and may act to maintain the normotensive blood pressure in this rat strain during sodium loading.
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Functional expression of the cDNAs encoding rat 11β-hydroxylase [cytochrome P450(11β)] and aldosterone synthase [cytochrome P450(11β, aldo)]
European journal of biochemistry, 1991Co-Authors: Yasuki Nonaka, Mitsuhiro OkamotoAbstract:Expression plasmids containing two cDNAs of a rat cytochrome P450(11β) family, pcP450(11β)-62 [Nonaka, Y., Matsukawa, N., Morohashi, K., Omura, T., Ogihara, T., Teraoka, H. & Okamoto, M. (1989) FEBS Lett. 255, 21–26] and pcP450(11β, aldo)-46 [Matsukawa, N., Nonaka, Y., Ying, Z., Higaki, J., Ogihara, T. & Okamoto, M. (1990) Biochem. Biophys. Res. Commun. 169, 245–252], were constructed and introduced into COS-7 cells by electroporation. Enzymatic activities of the expressed cytochromes P450(11β) and P450(11β, aldo) were determined by using 11-Deoxycorticosterone, corticosterone, 18-hydroxy-11-Deoxycorticosterone, 18-hydroxycorticosterone, or 19-hydroxy-11-Deoxycorticosterone as a substrate. Cytochrome P450(11β) catalyzed 11β-, 18-and 19-hydroxylations of 11-Deoxycorticosterone and 19-oxidation or 19-hydroxy-11-Deoxycorticosterone at substantial rates, 18-hydroxylation of corticosterone at a very low rate, but no aldosterone production. Cytochrome P450(11β, aldo) catalyzed 11β- and 18-hydroxylations of 11-Deoxycorticosterone, 18-hydroxylation of corticosterone and aldosterone production from 11-Deoxycorticosterone or corticosterone. But neither 19-hydroxylation of 11-Deoxycorticosterone nor 19-oxidation of 19-hydroxy-11-Deoxycorticosterone was catalyzed by cytochrome P450(11β, aldo).
Eduardo N. Cozza - One of the best experts on this subject based on the ideXlab platform.
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Inhibition of aldosterone production in rat adrenal mitochondria by 18-ethynyl-11-Deoxycorticosterone: a simple model for kinetic interpretation of mechanism-based inhibitors.
Journal of biochemistry, 2001Co-Authors: Laura Matković, Celso E. Gomez-sanchez, Eduardo N. CozzaAbstract:A simple mathematical model for studying mechanism-based inhibitors (MBIs) is presented. The mathematical equations are deduced for an experimental protocol consisting of a first incubation of the enzyme in the presence of MBI followed by a washing protocol to eliminate free MBI. Finally enzyme activity (initial velocity) is measured with specific substrate. The representation of the final equation obtained is a straight line, and the MBI-specific association constant of velocity (k) can be calculated from its slope. The mathematical model was then challenged with the effect of 18-ethynyl-11-Deoxycorticosterone (18-EtDOC) as an MBI on aldosterone biosynthesis from 11-Deoxycorticosterone (DOC) in rat adrenal mitochondria. The last step of the mitochondrial biosynthesis of aldosterone consists of the conversion of DOC into corticosterone (B) or 18-hydroxy-11-Deoxycorticosterone (18-OHDOC), and both steroids can then be transformed into aldosterone. The k (mM(-1) x min(-1)) values obtained for 18-EtDOC were: 451 +/- 36 for DOC to aldosterone; 177 +/- 16 for B to aldosterone; 175 +/- 15 for 18-OHDOC to aldosterone; and 2.7 +/- 0.2 for DOC to B. These results show that this MBI practically does not affect the metabolism of DOC to B in our enzyme preparation and that conversions of B and 18-OHDOC into aldosterone are catalyzed by the same enzyme.
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Cortisol: a tool to study aldosterone biosynthesis in rats.
Endocrine research, 1995Co-Authors: Laura Matković, Celso E. Gomez-sanchez, Carlos P. Lantos, Eduardo N. CozzaAbstract:Corticosterone (B) and 18-hydroxy-11-Deoxycorticosterone (18OHDOC) but not 11-Deoxycorticosterone (DOC) displaced cortisol (F) specifically bound to rat adrenal mitochondria. F competitively inhibited aldosterone formation from B, 18OHB and 18OHDOC but did not inhibit conversions of DOC to B or 18OHDOC. High concentrations of DOC increased its conversion to 18OHDOC rather than B.
Carl-joachim Partsch - One of the best experts on this subject based on the ideXlab platform.
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Chromatographic system for the simultaneous measurement of plasma 18-hydroxy-11-Deoxycorticosterone and 18-hydroxycorticosterone by radioimmunoassay: reference data for neonates and infants and its application in aldosterone-synthase deficiency
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2003Co-Authors: Felix G. Riepe, Wolfgang G. Sippell, Nils Krone, Michael Peter, Carl-joachim PartschAbstract:Abstract A new chromatographic system for the steroid precursor separation and a sensitive radioimmunoassay system for the subsequent measurement of 18-hydroxy-11-Deoxycorticosterone and 18-hydroxycorticosterone has been developed. 18-Hydroxy-11-Deoxycorticosterone and 18-hydroxycorticosterone were extracted with methylene chloride and separated from cross-reacting steroids by Sephadex LH-20 column chromatography. Anti-18-hydroxy-11-Deoxycorticosterone and anti-18-hydroxycorticosterone antibodies raised in rabbits were used. The lower detection limit of the assay is 0.03 nmol/l and 0.128 nmol/l for 18-hydroxy-11-Deoxycorticosterone and 18-hydroxycorticosterone, respectively. Normal values for this assay in 128 healthy neonates and infants aged 0–5 months were established as a basis for the early hormonal diagnosis of aldosterone synthase deficiency types I and II. Its application for the diagnosis of aldosterone synthase deficiency is demonstrated in two patients with homozygous mutation/deletion in the encoding CYP11B2 gene.
Eric Pussard - One of the best experts on this subject based on the ideXlab platform.
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Multiplexed steroid profiling of gluco- and mineralocorticoids pathways using a liquid chromatography tandem mass spectrometry method
Journal of Steroid Biochemistry and Molecular Biology, 2017Co-Authors: Simon Travers, Laetitia Martinerie, Claire E. Bouvattier, Marc Lombes, Pascal Boileau, Eric PussardAbstract:Serum steroid assays are major tools in the clinical evaluation of adrenal disorders. The main adrenal steroids are routinely measured with immunoassays. However, chromatographic methods are known to offer better specificity. We report a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for simultaneous quantification of 15 adrenal steroids targeting the mineralo- and gluco-corticosteroid pathways. Serum steroids combined with deuterated internal standards were extracted using successive protein precipitation and solid phase extraction steps. Cortisol, cortisone, 11-deoxycortisol, 17-hydroxyprogesterone, 21-deoxycortisol, progesterone, 11-Deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, 18-hydroxycorticosterone, 18-hydroxy-11-Deoxycorticosterone, aldosterone, dehydroepiandrosterone sulfate, testosterone and androstenedione were resolved in fourteen minutes using a BEH C18 column coupled to a methanol-ammonium formate gradient. Detection was performed using multiple reaction monitoring quantitation. Routinely determined steroid levels by immunoassays were compared to those measured by LC–MS/MS. This method was applied to assess steroid profiles in congenital adrenal hyperplasia (CAH) patients with 21-hydroxylase deficiency. Low quantification limits depending on each steroid (ranging from 0.015 ng/mL for aldosterone to 20 ng/mL for DHEAS) are adapted to the clinical use. Recoveries of steroids range from 64% for 21-deoxycortisol to 101% for cortisol and are fully corrected by internal standards. A good linearity with R > 0.989 is obtained for each compound. The inter-day variation coefficients ranged from 4.7% for cortisol to 16.3% for 11-Deoxycorticosterone. The immunoassay for cortisol (Immulite 2000, Siemens) showed acceptable agreement with LC–MS/MS (bias +7.2%). However, Bland-Altman plots revealed large negative bias for aldosterone (−33.4%, AldoCT, CisBio international), for 17-hydroxyprogesterone at concentrations below 2 ng/mL (−74.1%, OHP-CT MP Biomedical), for androstenedione (−80.3%, RIA D4, Beckman Coulter) and for 11-deoxycortisol (−125.3%, Diasource Immunoassays). Finally, the analysis of samples from 21-hydroxylase defective patients demonstrated the potential usefulness of multiplexed steroid profiling for the diagnosis and/or monitoring of different forms of congenital adrenal hyperplasia. This LC–MS/MS method provides highly sensitive and specific assessments of mineralo- and glucocorticoids pathways from a small volume sample and is therefore a promising potent tool for clinical and experimental endocrine studies.
