A Radioimmunoassay for Serum and Urine Aldosterone by Celite Column Chromatography

Chakmakjian, Zaven H.; Pryor, W. W.; Abraham, Guy E.

doi:10.1080/00032717408058743

Cited by 25 publications

(14 citation statements)

References 12 publications

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“…Thorough clinical evaluation of patients genotyped in this study suggests that most but not all of the phenotypic variability in 2 1-hydroxylase deficiency results from allelic variation in CYP2 1. (25). This metabolite ofaldosterone represents a constant percentage of secreted aldosterone (26,27).…”

Section: Introductionmentioning

confidence: 99%

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Speiser¹,

Dupont²,

Zhu³

et al. 1992

J. Clin. Invest.

532

401

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Genotyping for 10 mutations in the CYP21 gene was performed in 88 families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Southern blot analysis was used to detect CYP21 deletions or large gene conversions, and allele-specific hybridizations were performed with DNA amplified by the polymerase chain reaction to detect smaller mutations. Mutations were detected on 95% of chromosomes examined. The most common mutations were an A G change in the second intron affecting pre-mRNA splicing (26%), large deletions (21%), Ile-172 --Asn (16%), and Val-281 --Leu (11%). Patients were classified into three mutation groups based on degree of predicted enzymatic compromise. Mutation groups were correlated with clinical diagnosis and specific measures of in vivo 21-hydroxylase activity, such as 17-hydroxyprogesterone, aldosterone, and sodium balance. Mutation group A (no enzymatic activity) consisted principally of salt-wasting (severely affected) patients, group B (2% activity) of simple virilizing patients, and group C (10-20% activity) of nonclassic (mildly affected) patients, but each group contained patients with phenotypes either more or less severe than predicted. These data suggest that most but not all of the phenotypic variability in 21-hydroxylase deficiency results from allelic variation in CYP21. Accurate prenatal diagnosis should be possible in most cases using the described strategy. (J. Clin. Invest. 1992. 90:584-595.) Key words: steroid 21-hydroxylase deficiency-CYP21

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Section: Introductionmentioning

confidence: 99%

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Speiser¹,

Dupont²,

Zhu³

et al. 1992

J. Clin. Invest.

532

401

View full text Add to dashboard Cite

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“…Transfected cells were treated identically to those used for TLC assays, except that 1 nmol of nonradioactive deoxycorticosterone was used. After extraction with methylene chloride, steroids were resolved by Celite column chromatography and the concentrations of deoxycorticosterone, corticosterone, and aldosterone were determined by RIA as described (26,27).…”

mentioning

confidence: 99%

Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency.

Pascoe

Curnow

Slutsker

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

168

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Corticosterone methyloxidase H (CMO-II) deficiency is an autosomal recessive disorder of aldosterone biosynthesis, characterized by an elevated ratio of 18-hydroxycorticosterone to aldosterone in serum. It is genetically linked to the CYPiIB) and CYP11B2 genes that, respectively, encode two cytochrome P450 isozymes, P45OXIB1 and P450XIB2. Whereas P45OXIB1 only catalyzes hydroxylation at position 1113 of 11-deoxycorticosterone and 11-deoxycortisol, P450XIB2 catalyzes the synthesis of aldosterone from deoxycorticosterone, a process that successively requires hydroxylation at positions 1113 and 18 and oxidation at position 18. To determine the molecular genetic basis of CMO-II deficiency, seven kindreds of Iranian-Jewish origin were studied in which members suffered from CMO-Hl deficiency. No mutations were found in the CYP1IBl genes, but two candidate mutations, R181W and V386A, were found in the CYPJ1B2 genes. When these mutations were individually introduced into CYP11B2 cDNA and expressed in cultured cells, R181W reduced 18-hydroxylase and abolished 18-oxidase activities but left 1113-hydroxylase activity intact, whereas V386A caused a small but consistent reduction in the production of 18-hydroxycorticosterone. All individuals affected with CMO-ll deficiency were homozygous for both mutations, whereas eight asymptomatic subjects were homozygous for R181W alone and three were homozygous for V386A alone. These findings confirm that P450XIB2 is the major enzyme mediating oxidation at position 18 in the adrenal and suggest that a small amount of residual activity undetectable in in vitro assays is sufficient to synthesize normal amounts of aldosterone.

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“…Transfected cells were treated identically to those used for TLC assays, except that 1 nmol of nonradioactive deoxycorticosterone was used. Concentrations of corticosterone and aldosterone were determined on duplicate samples as described (22,23).…”

mentioning

confidence: 99%

Glucocorticoid-suppressible hyperaldosteronism results from hybrid genes created by unequal crossovers between CYP11B1 and CYP11B2.

Pascoe

Curnow

Slutsker

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

250

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Glucocorticoid-suppressible hyperaldosteronism (GSH) is an autosomal dint form of familial hypertension. The biochemical abnormalities seen in this disorder may be remedied by ami tion of dexamethse, implying that aldosterone synthesis is being abnormally regulated by corticotropin. The final three steps of aldosterone synthesis, 11(3 and 18-hydroxylation and 18-oxidation, are mediated by a cytochrome P450 in the zona glomerulo of the adrenal cortex termed CYP11B2. A related isozyme in the zona fascculata, CYPIlB1, is required for cortisol synthesis; this isozyme, which is normally expressed at much higher levels than CYP11B2, only has 1l-hydroxylase activity. These isozymes are encoded by genes on human chromosome 8q22. We have now studied four unrelated patients with GSH. We found that each patient has one chromosome that carries three CYPIIB genes instead of two. This has presumably been generated by unequal meiotic crossing-over. The extra gene is a hybrid with 5' regulatory and coding regions corresponding to CYPIIBi and 3' coding regions from CYPIIB2. The breakpoint is in intron 2 in two cases, intron 3 in one, and exon 4 in one. Cells transfected with hybrid cDNAs containing up to the first three exons of CYPIIBI synthesized aldosterone at levels near that of cells carrying normal CYPIIB2, but cells transfected with hybrids contining the first five or more exons of CYPIIBI could not synts detectable amounts of aldosterone. These data demonstrate that GSH is caused by expression of a gene that is regulated like CYPIIBI but that encodes a protein able to synthesize aldosterone.

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A Radioimmunoassay for Serum and Urine Aldosterone by Celite Column Chromatography

Cited by 25 publications

References 12 publications

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Mutations in the human CYP11B2 (aldosterone synthase) gene causing corticosterone methyloxidase II deficiency.

Glucocorticoid-suppressible hyperaldosteronism results from hybrid genes created by unequal crossovers between CYP11B1 and CYP11B2.

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