Stanley Ulick

Adrenocortical factors in hypertension

Low renin essential hypertension and the syndrome of mineralocorticoid excess have two features in common, low plasma renin activity and volume-sensitive hypertension. The proposal that both disorders share a common mechanism--because of the ability of agents that inhibit or antagonize the adrenocortical secretion to lower blood pressure in the low renin hypertensive group--appears to be based on a circular argument. The beneficial effect of removal or neutralization of the adrenocortical contribution only constitutes evidence for volume-dependency or sensitivity, which is how the low renin group is defined. Any measure that blocks a component of the normal homeostatic chain for the maintenance of extracellular and intravascular volume including the adrenal cortex would be expected to have a beneficial effect in volume-sensitive hypertension. Evidence for an adrenal factor in low renin hypertension must rest on the isolation of an active substance that reproduces the effect when readministered. 18-Hydroxy-11-deoxycorticosterone (18-OH-DOC) does not meet these criteria. It is not significantly increased in experimental hypertension and, although its overproduction in unselected low renin essential hypertensive patients remains controversial, the magnitude of the reported elevations is insufficient in relation to the low biologic activity of the steroid to account for a significant effect. Apart from its increase in the 17alpha-hydroxylase defect, 18-OH-DOC is increased in primary aldosteronism and may also be an indicator of a histologic variant of the aldosteronoma. On the basis of a large body of evidence showing parallelism between the 11beta- and 18-hydroxylase functions of the fasciculata zone, we have proposed that both enzymic functions are functionally related and may involve the same enzyme protein and catalytic site. According to this view, the secretion of 18-OH-DOC would have no special significance of its own but would be an obligatory consequence of the secretion of fasciculata zone corticosterone.

Congenitally defective aldosterone biosynthesis in humans: The involvement of point mutations of the P-450C18 gene (CYP11B2) in CMO II deficient patients

The gene for steroid 18-hydroxylase (P-450C18) has been recently assigned to encode corticosterone methyl oxidases Type I and Type II which were previously postulated to catalyze the final two steps in the biosynthesis of aldosterone in humans. Molecular genetic analysis of the P-450C18 gene is three patients from three different families affected with CMO II deficiency has indicated that a point mutation of CGG----TGG (181Arg----Trp) in exon 3 and one of GTG----GCG (386Val----Ala) in exon 7 occur exclusively in the gene of the patients. Analysis of PCR products by restriction enzymes (HapII and HphI) has indicated that the patients are homozygous and the unaffected parent is heterozygous for both mutations, in accordance with the established concept that CMO II deficiency is inherited in an autosomal recessive manner. These data clearly provide the molecular genetic basis for the characteristic biochemical phenotype of CMO II clinical variants.

A new form of the syndrome of apparent mineralocorticoid excess.

The syndrome of apparent mineralocorticoid excess combines the features of unexplained but spironolactone-correctable mineralocorticoid excess in association with a decreased rate of oxidation of cortisol to cortisone. No relationship was initially implied between the pathogenesis of the disorder and the metabolic disturbance as expressed by an elevated cortisol:cortisone metabolite ratio but the ratio itself has served as a biochemical marker for the disorder. Cortisol has been suggested as the mineralocorticoid in a setting of enhanced sensitivity to the steroid as a result of the incomplete oxidative metabolism of cortisol by the kidney. We present evidence that diminished conversion of cortisol to cortisone is not an obligatory mechanism in the syndrome of apparent mineralocorticoid excess. A form of the disorder is described, designated the Type 2 variant, in which all features are preserved except that the cortisol:cortisone metabolite ratio is normal. An essential feature of both variants, however, is a decrease in the cortisol metabolic clearance rate. These findings require a more generalized definition of the syndrome of apparent mineralocorticoid excess to include other deficient mechanisms of metabolic inactivation of cortisol.

Structure and mechanism of formation of the two forms of 18-hydroxy-11-deoxycorticosterone

Abstract 18-Hydroxy-11-deoxycorticosterone (18-OH-DOC) has been observed to exist in two interconvertible forms of markedly different chromatographic mobility. The structure of these forms has now been established. The more polar form is the cyclic hemiketal and the less polar is a mixed ketal at C-20 derived by reaction with an alcoholic solvent. This unusually facile ketalization is catalyzed by traces of acidic impurities present in most commercial sources of reagent grade methanol or ethanol, but can be abolished by removal or neutralization of these impurities.

[45] Isolation and synthesis of the major metabolites of aldosterone and 18-hydroxycorticosterone

Publisher Summary Aldosterone and 18-hydroxycorticosterone are conveniently considered together because both steroids are secreted by the glomerulosa zone of the mammalian adrenal cortex in response to the same stimulating factors. The 18-hydroxysteroid is normally secreted at a 2–3 times greater rate, except in an inborn error of aldosterone biosynthesis due to a defect in the terminal oxidase mechanism in which there is marked overproduction of 18-hydroxycorticosterone relative to aldosterone. The metabolism of aldosterone in vivo has been studied best in humans. With few exceptions, little is known about metabolism in other species. The major metabolites in human urine are a tetrahydro derivative coniugated with glucuronic acid at C-3 and a pH 1 hydrolyzable conjugate, which is probably an aldosterone C-18 glucuronoside. Procedures for the isolation of aldosterone from this conjugate in urine and subsequent measurement are extensively reviewed. Several minor urinary metabolites are also isolated following the administration of large amounts of aldosterone to human subjects, but their production from endogenous aldosterone at normal rates of secretion is not reported.

Molecular genetic studies on the biosynthesis of aldosterone in humans.

Corticosterone methyl oxidase Type I (CMO I) and II (CMO II) have been postulated to be the enzymes involved in the final two steps of aldosterone biosynthesis in humans. We have isolated human cDNAs for P450c11 and P450c18 as well as the corresponding genes, CYP11B1 and CYP11B2. Both protein products of these two genes as expressed in COS-7 cells exhibit steroid 11β-hydroxylase activity, but only P450c18, a product of CYP11B2, carried steroid 18-hydroxylase activity to form aldosterone. These results indicate that CYP11B2 encodes CMO, the actual catalytic function of which is retained by P450c18, a multifunctional enzyme. This conclusion is further supported by the finding that the P450c18 gene, CYP11B2, is mutated at several different loci in patients deficient in CMO I or II.

Inborn errors of aldosterone biosynthesis in humans

Corticosterone methyl oxidase (CMO) type I and type II deficiencies are inborn errors at the penultimate and ultimate steps in the biosynthesis of aldosterone in humans. Recently, steroid 18-hydroxylase (P450C18), or aldosterone synthase (P450aldo), was shown to be a multifunctional enzyme catalyzing these two steps of aldosterone biosynthesis, i.e., the conversion of corticosterone to 18-hydroxycorticosterone and the subsequent conversion of 18-hydroxycorticosterone to aldosterone. This observation suggests that CMO I and CMO II deficiencies are derived from two different mutations in the P450C18 gene (CYP11B2). To elucidate whether or not this is the case, we performed molecular genetic studies on CYP11B2 of both types of patients. Nucleotide sequence analysis has indicated that the gene of CMO I deficient patients is completely inactivated by a frameshift to form a stop codon due to a 5-bp nucleotide deletion in exon 1. Sequence analysis of CYP11B2 of CMO II deficient patients has revealed two point mutations, CGG-->TGG (Arg181-->Trp) in exon 3 and GTG-->GCG (Val386-->Ala) in exon 7. CYP11B1, the gene for steroid 11 beta-hydroxylase (P45011 beta) which was previously postulated to be the target for CMO II deficiency, is not impaired in these two types of patients. Expression studies using the corresponding mutant cDNAs have shown that CMO I deficient patients are null mutants with a complete lack of P450C18 whereas CMO II deficient patients are leaky mutants with an altered P450C18 activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and identification of an endogenous metabolite of 18-oxocortisol from human urine

The naturally occurring mineralocorticoid agonist, 18-oxocortisol, is secreted in increased amounts in two hypertensive syndromes. One is primary aldosteronism and the other a genetic disorder first described by Sutherland and co-workers in which aldosterone secretion is ACTH-dependent and the mode of inheritance is autosomal dominant. 18-Hydroxy and -oxocortisol are the components of the cortisol oxidation pathway which arise when cortisol becomes an alternate substrate for corticosterone methyl oxidase. This enzyme system normally resides in the glomerulosa zone of the mammalian adrenal cortex. In an effort to account for a larger fraction of 18-oxocortisol and provide a reliable index of its secretion and of the expression of the cortisol C-18 oxidation pathway, metabolites were sought in the urine of a patient with the ACTH-dependent autosomal dominant form of aldosteronism. Using a variant of the technique of reverse isotope dilution, a pool of [3H]-labeled urinary metabolites form a normal subject was mixed with the patients urine and subjected to customary methods of hydrolysis for urinary steroids. The radiolabeled glucuronide fraction was the most abundant and was subjected to repeated HPLC fractionation to yield the predominant component. The evidence from gas chromatography-mass spectrometry indicated that this metabolite was a tetrahydro derivative. The structure of the isolated tetrahydro 18-oxocortisol was confirmed by a biosynthesis of a reference standard from 18-oxocortisol and a 5 beta-pregnane reductase preparation.

Adrenocortical factors in hypertension—II the significance of 16-oxygenated C-19 steroids☆

Abstract One of the proposed steroid abnormalities in low renin hypertension, increased production of 16β-hydroxydehydroepiandrosterone, has been evaluated and not confirmed. Double isotopic and radioimmunoassay methods were developed and their specificity evaluated. Two isomeric 16-oxygenated C-19 steroids were also measured, the 16α-hydroxy epimer and the 16-keto,17β-hydroxy derivative, and no significant elevations were observed in hypertensive patients. Plasma levels of 16-keto-5-androstene-3β, 17β-diol were also measured and found to be elevated normally in late pregnancy. The proposal that the syndromes of mineralocorticoid excess and low renin essential hypertension share a common mechanism is based on certain similarities between the two disorders, and the ability of agents which inhibit or antagonize adrenocortical secretion to lower blood pressure in the low renin hypertensive group. However, the beneficial effect of the removal or neutralization of the adrenocortical contribution need only constitute evidence for volume dependency of blood pressure in the low renin group. Evidence for the existence of an adrenocortical factor in low renin hypertension must rest on the isolation of an active substance which reproduces the effect when readministered. This criterion has not yet been met.

Unusual steric course of aldosterone reduction in the bullfrog in vivo.

Abstract The major pathway of aldosterone metabolism in the bullfrog in vivo results in the reduction of ring A to yield the unusual isomer, 3β-hydroxy-5β-tetrahydroaldosterone (I). The metabolite was isolated from bullfrog urine following the injection of aldosterone. Identification was accomplished by infrared spectroscopic comparison of the etiolactone derivatives of metabolite and synthesized steroid. The 3β-hydroxy-5β-etiolactone (II) was prepared by catalytic hydrogenation of aldosterone etiolactone which also yielded the 3β,5α isomer (V) and smaller amounts of the 3α,5β isomer (IV).