cortodoxone and 18-hydroxycortisol

In glucocorticoid-remediable aldosteronism (GRA), there is a large interfamily variation of phenotype. We report three subjects with GRA in a single family (parents, two brothers and two sisters), of whom only one (proband) displayed classical features of the mineralocorticoid excess. The proband was a man found to be hypertensive and hypokalaemic at the age of 24 years. Plasma renin activity was suppressed and plasma aldosterone was repeatedly elevated. Blood pressure and aldosterone levels normalized within 5 days of dexamethasone therapy. The presence of a chimaeric CYP11B1/CYP11B2 gene was demonstrated by long-PCR and Southern blotting (crossover site at the end of intron 3) in the proband, in the younger sister (sibling 1) and in the father. In these patients, sequencing of the chimaeric portion of CYP11B1 did not reveal any mutation, while sequencing of the chimaeric portion of CYP11B2 showed a V386A polymorphism in exon 7, known to cause only a minimal impairment of enzymatic activity. Sibling 1 was normotensive, normokalaemic and had normal PRA and aldosterone. The father had normal blood pressure and potassium, low-normal PRA and normal aldosterone. All three subjects had elevated levels of urinary 18-hydroxycortisol and 18-oxocortisol. Baseline 11-deoxycorticosterone (DOC), corticosterone (B) and aldosterone were high in the proband and normal in the father and sibling 1; 11-deoxycortisol (S) and cortisol (F) were normal. ACTH induced a normal increase of B, DOC, S and F, and an excessive aldosterone increase in all three patients. Abnormalities in the chimaeric portions of CYB11B1 or CYP11B2 genes did not account for the phenotypic disparity of the different members in a single GRA family. Altered regulation of the chimaeric gene may be responsible for differences in its activity.

Topics: Adult; Aged; Aldosterone; Cortodoxone; Cytochrome P-450 CYP11B2; Dexamethasone; Female; Genotype; Glucocorticoids; Humans; Hydrocortisone; Hyperaldosteronism; Hypertension; Male; Middle Aged; Pedigree; Phenotype; Renin; Steroid 11-beta-Hydroxylase

CYP11B1 (11beta-hydroxylase) and CYP11B2 (aldosterone synthase) are 93% identical mitochondrial enzymes that both catalyze 11beta-hydroxylation of steroid hormones. CYP11B2 has the additional 18-hydroxylase and 18-oxidase activities required for conversion of 11-deoxycorticosterone to aldosterone. These two additional C18 conversions can be catalyzed by CYP11B1 if serine-288 and valine-320 are replaced by the corresponding CYP11B2 residues, glycine and alanine. Here we show that such a hybrid enzyme also catalyzes conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol, and 18-oxocortisol. These latter two steroids are present at elevated levels in individuals with glucocorticoid suppressible hyperaldosteronism (GSH) and some forms of primary aldosteronism. Their production by the recombinant CYP11B enzyme is enhanced by substitution of further amino acids encoded in exons 4, 5, and 6 of CYP11B2. A converted CYP11B1 gene, containing these exons from CYP11B2, would be regulated like CYP11B1, yet encode an enzyme with the activities of CYP11B2, thus causing GSH or essential hypertension. In a sample of 103 low renin hypertensive patients, 218 patients with primary aldosteronism, and 90 normotensive individuals, we found a high level of conversion of CYP11B genes and four cases of GSH caused by unequal crossing over but no gene conversions of the type expected to cause GSH.

Topics: Case-Control Studies; Catalysis; Cortodoxone; Cytochrome P-450 CYP11B2; Gene Conversion; Genetic Code; Genetic Testing; Humans; Hydrocortisone; Hyperaldosteronism; Hypertension; Renin; Steroid 11-beta-Hydroxylase

Incubation of 11-deoxycortisol with a cytochrome P-450(11 beta)-reconstituted system yielded, in addition to cortisol, several new steroid products. In this study, structures of the three steroid products were elucidated. Retention time of the first product (Peak 2 substance) coincided with that of authentic 18-hydroxycortisol on reverse phase HPLC. To further confirm the chemical identity of this product, the purified sample was subjected to 1H-NMR analysis. The spectrum was essentially identical to that of 18-hydroxycortisol. The retention time of the second product (Peak 3 substance) did not coincide with those of commonly occurring steroids. The one- and two-dimension 1H-NMR spectra provided strong evidence for its structure of 19-hydroxy-11-deoxycortisol. The retention time of the third product (Peak 4 substance) did not coincide with those of commonly occurring steroids. The 1H-NMR spectrum showed the presence of signals of 19-CH3 and 18-CH2 protons. There was also evidence that this product is not hydroxylated at the 11-position. Further analysis of the COSY spectra identified its structure as 18-hydroxy-11-deoxycortisol. From these results, we conclude that bovine P-450(11 beta) can catalyze the hydroxylation of 11-deoxycortisol at 11 beta-, 18- and 19-positions and produce cortisol, 18-hydroxy-11-deoxycortisol, 18-hydroxycortisol and 19-hydroxy-11-deoxycortisol.

Topics: Adrenal Cortex; Animals; Catalysis; Cattle; Chromatography, High Pressure Liquid; Cortodoxone; Cytochrome P-450 Enzyme System; Hydrocortisone; Hydroxylation; Magnetic Resonance Spectroscopy