Aldosterone Glucuronide, an Important Biomarker: Synthesis and Structure Elucidation of Novel Isomers.

Aldosterone 1 is a mineralocorticoid, it has great influence on the blood pressure and its glucuronide is an important marker for the detection of several diseases. Here, we describe the chemical synthesis of different aldosterone-18- and 20-glucuronides. Reaction of trimethylsilyl 2,3,4-tri- acetyl-1-ß-glucuronic acid methyl ester 5 b and aldosterone diacetate 11 in the presence of TMSOTf gave the 18-α-glucuronide 9 a. The 18-ß-glucuronide 15 b and the 20-ß-glucuronide 16 b could be obtained by reaction of methyl 2,3,4-tri-O-isobutyryl-1α-glucuronate trichloroacetimidate 14 and aldosterone 21-acetate 8 in the presence of TMSOTf or BF3 ⋅OEt2 . Finally, reaction of aldosterone 21-acetate 8 and methyl 2,3,4-triacetyl-1α-glucuronate trichloroacetimidate 19 in the presence of TMSOTf gave the corresponding methyl 18-ß-triacetylglucuronate 9 b, which was transformed into the desired aldosterone-18-ß-glucuronide 3 by two enzyma- tic transformations.

A LC-MS/MS method for the simultaneous quantitative determination of aldosterone, its precursor 18-hydroxycorticosterone and its metabolite tetrahydroaldosterone in human urine.

Aldosterone (ALD), its precursor 18-hydroxycorticosterone (18-OHB) and its metabolite tetrahydroaldosterone (TH-ALD) are important biomarkers for the diagnosis of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is increasingly utilized in the detection of small molecules of hormones because it has advantages in terms of specificity and sensitivity. The objective of this study is to develop a new LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB, and TH-ALD in human urine and attempt to diagnose primary aldosteronism using different indicators. The urine samples were treated with a solid-phase extraction pretreatment technique and the three analytes were separated on a reversed-phase column and detected on a triple quadrupole mass spectrometer. The established method was validated according to CLSI C62-A standard guidelines. The calibration ranges from 25 pg/mL to 5000 pg/mL for aldosterone (free), 18-hydroxycorticosterone and tetrahydroaldosterone, and the lower limit of quantification for these three analytes was 25 pg/mL. The matrix effects and recoveries of these three analytes ranged from 85.1 % to 115 % and from 86.3 % to 114 %, respectively. The intra-day and inter-day precision ranged from 1.29 % to 6.78 % and from 1.77 % to 8.64 %, respectively. The performance of the method met the requirements of the guidelines. 40 clinical urine samples including 22 PA patients and 18 non-PA patients were detected, and the ROC curves of three diagnostic indicators were established. The area under the curve (AUC) of ALD (free) is the biggest, so ALD (free) was the best compound to be used as a diagnostic indicator in this study. When the cut-off point was taken as 141 ng/24-h, the sensitivity was 72.7 % and the specificity was 88.9 %. We developed and validated an LC-MS/MS method for the simultaneous quantification of ALD (free), 18-OHB and TH-ALD in human urine. Our study provides a reference for the use of new biomarkers for the diagnosis of primary aldosteronism.

Urinary sodium excretion is the main determinant of mineralocorticoid excretion rates in patients with chronic kidney disease.

BACKGROUND: Blockade of the mineralocorticoid receptor (MR) in patients with chronic kidney disease (CKD) improves surrogate cardiovascular outcomes, such as left ventricular mass. Animal models of renal disease support a pathological role of mineralocorticoids, in the context of a high sodium intake. We aimed to assess the regulation of mineralocorticoid biosynthesis in patients with CKD. METHODS: Seventy patients with CKD stages 3/4 and 30 patients with essential hypertension (EH) were recruited. Patients underwent detailed clinical phenotyping, drug history and biochemical assessment. Patients completed a 24-h urine collection for measurement of urinary tetrahydroaldosterone (THALDO) and tetrahydrocorticosterone (THDOC) excretion rates (measured using gas chromatography-mass spectrometry) and urinary electrolytes. The factors which correlated significantly with THALDO and THDOC excretion were entered into linear regression models. RESULTS: Patients with EH and CKD were well matched with no significant differences in gender, age or weight. The mean estimated glomerular filtration rate (eGFR) in CKD patients was 38.6/min/1.73 m(2). The mean urinary excretion rates of THALDO, THDOC and 24-h urinary sodium (24-h USod) were not significantly different between CKD and EH patients. The level of renal function did not correlate with THALDO or THDOC excretion. In patients with CKD, 24-h USodium (r = 0.614, P < 0.001) and 24-h UPotassium (r = 0.538, P < 0.001) were positively correlated with THALDO excretion. On multivariate linear regression analysis, 24-h USod was the strongest independent predictor (P = 0.004) of THALDO and THDOC excretion in CKD. In patients with EH, no relationship was seen between mineralocorticoid excretion and 24-h urinary sodium excretion. CONCLUSIONS: In patients with CKD, 24-h urinary sodium excretion is the strongest positive predictor of urinary mineralocorticoid excretion. The nature of this relationship is unexpected, novel, not seen in patients with EH and may explain the association seen between high urinary sodium excretion, mineralocorticoids and poor outcomes in patients with CKD.

Urinary corticosteroid excretion predicts left ventricular mass and proteinuria in chronic kidney disease.

Blockade of the MR (mineralocorticoid receptor) in CKD (chronic kidney disease) reduces LVMI [LV (left ventricular) mass index] and proteinuria. The MR can be activated by aldosterone, cortisol and DOC (deoxycorticosterone). The aim of the present study was to explore the influence of mineralocorticoids on LVMI and proteinuria in patients with CKD. A total of 70 patients with CKD and 30 patients with EH (essential hypertension) were recruited. Patients underwent clinical phenotyping; biochemical assessment and 24 h urinary collection for THAldo (tetrahydroaldosterone), THDOC (tetrahydrodeoxycorticosterone), cortisol metabolites (measured using GC-MS), and urinary electrolytes and protein [QP (proteinuira quantification)]. LVMI was measured using CMRI (cardiac magnetic resonance imaging). Factors that correlated significantly with LVMI and proteinuria were entered into linear regression models. In patients with CKD, significant predictors of LVMI were male gender, SBP (systolic blood pressure), QP, and THAldo and THDOC excretion. Significant independent predictors on multivariate analysis were THDOC excretion, SBP and male gender. In EH, no association was seen between THAldo or THDOC and LVMI; plasma aldosterone concentration was the only significant independent predictor. Significant univariate determinants of proteinuria in patients with CKD were THAldo, THDOC, USod (urinary sodium) and SBP. Only THAldo excretion and SBP were significant multivariate determinants. Using CMRI to determine LVMI we have demonstrated that THDOC is a novel independent predictor of LVMI in patients with CKD, differing from patients with EH. Twenty-four hour THAldo excretion is an independent determinant of proteinuria in patients with CKD. These findings emphasize the importance of MR activation in the pathogenesis of the adverse clinical phenotype in CKD.

Urinary tetrahydroaldosterone is associated with circulating FGF23 in kidney stone formers.

The spectrum of diseases with overactive renin-angiotensin-aldosterone system (RAS) or elevated circulating FGF23 overlaps, but the relationship between aldosterone and FGF23 remains unclarified. Here, we report that systemic RAS activation sensitively assessed by urinary tetrahydroaldosterone excretion is associated with circulating C-terminal FGF23. We performed a retrospective analysis in the Bern Kidney Stone Registry, a single-center observational cohort of kidney stone formers. Urinary excretion of the main aldosterone metabolite tetrahydroaldosterone was measured by gas chromatography-mass spectrometry. Plasma FGF23 concentrations were measured using a C-terminal assay. Regression models were calculated to assess the association of plasma FGF23 with 24 h urinary tetrahydroaldosterone excretion. We included 625 participants in the analysis. Mean age was 47 ± 14 years and 71% were male. Mean estimated GFR was 94 ml/min per 1.73 m2. In unadjusted analyses, we found a positive association between plasma FGF23 and 24 h urinary tetrahydroaldosterone excretion (ß: 0.0027; p = 4.2 × 10-7). In multivariable regression models adjusting for age, sex, body mass index and GFR, this association remained robust (ß: 0.0022; p = 2.1 × 10-5). Mineralotropic hormones, 24 h urinary sodium and potassium excretion as surrogates for sodium and potassium intake or antihypertensive drugs did not affect this association. Our data reveal a robust association of RAS activity with circulating FGF23 levels in kidney stone formers. These findings are in line with previous studies in rodents and suggest a physiological link between RAS system activation and FGF23 secretion.

High aldosterone-to-renin variants of CYP11B2 and pregnancy outcome.

BACKGROUND: Increased aldosterone concentrations and volume expansion of normal pregnancies are hallmarks of normal pregnancies and blunted in pre-eclampsia. Accordingly, we hypothesized an active mineralocorticoid system to protect from pre-eclampsia. METHODS: In pregnant women (normotensive n = 44; pre-eclamptic n = 48), blood pressure, urinary tetrahydro-aldosterone excretion and activating polymorphisms (SF-1 site and intron 2) of the aldosterone synthase gene (CYP11B2) were determined; 185 non-pregnant normotensive individuals served as control. Amino acid-changing polymorphisms of the DNA- and agonist-binding regions of the mineralocorticoid receptor were evaluated by RT-PCR, SSCP and sequencing. RESULTS: Urinary tetrahydro-aldosterone excretion was reduced in pre-eclampsia as compared to normal pregnancy (P < 0.05). It inversely correlated with blood pressure (r = 0.99, P < 0.04). Homozygosity for activating CYP11B2 polymorphisms was preferably present in normotensive as compared to pre-eclamptic pregnancies, identified (intron 2, P = 0.005; SF-1 site, P = 0.016). Two mutant haplotypes decreased the risk of developing pre-eclampsia (RR 0.16; CI 0.05-0.54; P < 0.001). In contrast, intron 2 wild type predisposed to pre-eclampsia (P < 0.0015). No functional mineralocorticoid receptor mutant has been observed. CONCLUSIONS: High aldosterone availability is associated with lower maternal blood pressure. In line with this observation, gain-of-function variants of the CYP11B2 reduce the risk of developing pre-eclampsia. Mutants of the mineralocorticoid receptor cannot explain the frequent syndrome of pre-eclampsia.

Enabling insights into the maturation of the renin-angiotensin-aldosterone system in children-Development of a low-volume LC-MS assay for the simultaneous determination of aldosterone, its precursor, and main metabolite.

INTRODUCTION: As part of the renin-angiotensin-aldosterone system (RAAS), aldosterone is key to the pathology of cardiovascular and renal diseases, leading to end-organ damage and cardiovascular death. Because of different aetiology and metabolism, pharmacotherapy in adults shows only limited transferability to children. Comprehensive investigations of humoral parameters, their precursors, and metabolites are necessary to establish a more rational and safe therapy in children. The LENA (Labeling of Enalapril from Neonates up to Adolescents) project aims to generate these missing data in neonates up to adolescents and provide insight into the maturing RAAS. METHODS: A HRMS (high-resolution mass spectrometry) assay was developed, utilizing blank serum depleted of the endogenous aldosterone, its precursor, 18-hydroxycorticosterone, and its main metabolite, tetrahydroaldosterone. A TOF-MS (time-of-flight-mass spectrometry) scan run in parallel with the simultaneous determination of all three analytes enriches the acquired data. Validation of aldosterone was conducted according to EMA and FDA bioanalytical guidelines. RESULTS: Using the Sciex TripleTOF 6600, a reliable determination in 50 µL serum was successfully shown. Appropriate calibration ranges from 19.53 pg/mL for aldosterone, 39.06 pg/mL for 18-hydroxycorticosterone, and 78.13 pg/mL for tetrahydroaldosterone to 2500 pg/mL were established to ensure the applicability in diseased paediatric patients. Between-run accuracy and precision for aldosterone ranged between -1.21 and -6.99 % and 2.07 and -10.22 %, respectively, confirming compliance with international guidelines. CONCLUSION: A simultaneous bioanalytical LC-HRMS assay for the determination of the biomarker aldosterone, its precursor, and main metabolite, utilizing 50 µL serum, was successfully established. This assay facilitates insight into the maturing RAAS from neonates up to adolescents.

Urinary Cadmium Excretion Is Associated With Increased Synthesis of Cortico- and Sex Steroids in a Population Study.

Context: Urinary cadmium (Cd) excretion is associated with cancer and cardiovascular morbidity. A potential mechanism could be disturbance of steroidogenesis in gonads and adrenal glands. Objective: We tested whether urinary excretion of Cd is correlated with that of cortico- and sex steroid metabolites in the general adult population. Setting: The Swiss Kidney Project on Genes in Hypertension is a multicentric, family-based population study. Measures: Urinary excretions of steroid hormone metabolites and Cd were measured with separate day and night collections. Associations were analyzed by mixed linear models. Results: Urinary Cd and testosterone excretions in men were significantly correlated (respective day and night ß values [standard error (SE)], 1.378 [0.242], P < 0.0005; and 1.440 [0.333], P < 0.0005), but not in women [0.333(0.257), P = 0.2; and 0.674 (0.361), P = 0.06]. Urinary Cd and cortisol excretions were positively associated in both sexes [day: ß = 0.475 (SE, 0.157), P = 0.0025, and 0.877 (SE, 0.194), P < 0.0005, respectively; night: ß = 0.875 (SE, 0.253), P < 0.0005 and 1.183 (SE, 0.277), P = 0.00002, respectively]. Cd excretion was correlated with mineralocorticoid metabolites excretion, except tetrahydroaldosterone, in both sexes (P < 0.01). There was an independent effect of Cd on sex hormone and corticosteroid synthesis and an interdependent effect on gluco- and mineralcorticoid production. Conclusion: Our findings provide evidence for a global stimulating effect on steroid synthesis already at low-dose Cd exposure. These findings might explain the association of Cd with diseases such as steroid-sensitive cancers or metabolic disorders.

The renal tubular handling of aldosterone and its acid-labile conjugate.

Stop-flow studies using infusions of aldosterone-(3)H or its (3)H acid-labile conjugate were done on five rhesus monkeys. The aldosterone-(3)H urine-to-plasma (U/P) ratio decreased in the same distal urine samples as sodium. The (3)H acid-labile conjugate U/P-to-inulin U/P ratio increased in the more proximal samples either with conjugate formed endogenously during aldosterone-(3)H infusions or with labeled conjugate infused alone. Aldosterone reabsorption occurred at a distal site in the renal tubule, and secretion of its acid-labile conjugate occurred at a proximal site.

[Diagnosis of primary hyperaldosteronism]. / Diagnostik des primären Hyperaldosteronismus.

Primary hyperaldosteronism is the most common secondary form of hypertension. Diagnosis of this entity is recommended in hypokalemic hypertension, in therapy-resistant hypertension (at least three 3 drugs and RR > 140/90 mmHg), and in adrenal incidentalomas (= incidentally discovered adrenal tumors). For screening, the ratio between plasma aldosterone (PAC) and plasma renin concentration (PRC) should be measured. In the assessment of PAC/PRC ratio, the discontinuation of some antihypertensive medication and assay-specific cutoff values must be noticed. After a positive screening test, saline infusion test should be done as confirmatory test. In contraindications/impracticability of this test, 24-h urine collection for aldosterone-18-glucuronide under high-sodium diet can be used as alternative confirmatory test. After confirmation of primary hyperaldosteronism, differential diagnosis between aldosterone-producing adenoma and idiopathic hyperaldosteronism has to be done. For this approach, adrenal CT or MRT, posture test and adrenal vein catheterization as gold standard test are available. Whereas therapy of aldosterone-producing adenoma is surgery, idiopathic hyperaldosteronism is to be treated medically by spironolactone.

Association between aldosterone production and variation in the 11beta-hydroxylase (CYP11B1) gene.

CONTEXT: Variation in the region of chromosome 8 including the genes steroid 11beta-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) influences mineralocorticoid and glucocorticoid metabolism. However, the relative importance of polymorphisms in CYP11B1 and CYP11B2 in determining these phenotypes is unknown. OBJECTIVE: Our objective was to investigate genetic influences of the CYP11B1 and CYP11B2 genes on mineralocorticoid metabolism. DESIGN: We measured 24-h urinary excretion of the key metabolites of the principal mineralocorticoids, glucocorticoids and androgens secreted by the adrenal cortex. We genotyped polymorphisms spanning the CYP11B1 and CYP11B2 genes, which together capture all common variations at the locus. PARTICIPANTS: Participants included 573 members of 105 British Caucasian families ascertained on a hypertensive proband. MAIN OUTCOME MEASURES: We assessed heritability of urinary tetrahydroaldosterone (THAldo) excretion and association of THAldo excretion with genotype. RESULTS: The heritability of THAldo excretion was 52% (P < 10(-6)). There was significant association between THAldo and genotype at several of the CYP11B1/B2 polymorphisms. The strongest association was observed at the rs6387 (2803A/G) polymorphism in intron 3 of CYP11B1 (P = 0.0004). Association followed a codominant model with a 21% higher THAldo excretion per G allele. Genotype at rs6387 accounted for 2.1% of the total population variability of THAldo. We found significant association between THAldo excretion and urinary total androgen excretion, urinary tetrahydrodeoxycortisol level, and urinary cortisol metabolites (all P < 0.001). CONCLUSIONS: Aldosterone synthesis is highly heritable and is affected by genotype at CYP11B1. Our findings support the hypothesis that genetically determined differences in 11-hydroxylation efficiency can have downstream effects on mineralocorticoid synthesis. Such effects may be of relevance to the development of low-renin essential hypertension.

The renal physiology of pendrin (SLC26A4) and its role in hypertension.

SLC26A4 (pendrin, PDS) is a Na+-independent, Cl-/HCO3-/OH- exchanger that is expressed in the apical regions of type B and non-A, non-B intercalated cells within the cortical collecting duct (CCD), the connecting tubule and the distal convoluted tubule where it mediates HCO3- secretion and Cl- absorption. SLC26A4 is upregulated with aldosterone analogues and with Cl- restriction. While under basal conditions no renal abnormalities are observed in mice and humans with genetic disruption of SLC26A4 (Pendred syndrome), differences become apparent under conditions wherein the transporter is stimulated. Following treatment with aldosterone analogues, e.g. deoxycorticosterone pivalate (DOCP), weight gain and hypertension are observed in Slc26a4+/+ but not in Slc26a4-/- mice. During dietary NaCl restriction, a model in which serum aldosterone is appropriately increased, urinary volume and urinary excretion of Cl- are greater in Slc26a4-/- than in wild-type mice which results in apparent vascular volume contraction in Slc26a4-/- mice. Moreover, during NaCl restriction or following DOCP treatment, Slc26a4-/- mice have a higher serum HCO3- than wild type mice from an impaired ability to excrete OH- equivalents. In conclusion, SLC26A4 regulates blood pressure and arterial pH, likely by participating in the renal regulation of net acid and Cl- excretion.

New mineralocorticoids: 5alpha-dihydroaldosterone and 5alpha-dihydro-11-deoxycorticosterone.

Mineralocorticoid activity of several delta4-3-ketosteroids and their 5alpha-dihydro analogs were evaluated by bioassay using urinary Na:K ratio of adrenalectomized rats as an index of mineralocorticoid activity. Among delta4-3-ketosteroids, aldosterone, 11-deoxycorticosterone, corticosterone, cortisol, 11-dehydrocorticosterone, and cortisone showed mineralcorticoid activity with aldosterone, the most potent of the series, showing virtually maximum activity at a dose of 0.25 microgram/rat. 5alpha-Dihydroaldosterone and 5alpha-dihydro-11-deoxycorticosterone possessed distinct mineralcorticoid activity, albeit less than aldosterone and 11-deoxycorticosterone. 5alpha-Dihydrocorticosterone, 5alpha-dihydrocortisol, 5alpha-dihydro-11-dehydrocorticosterone, and 5alpha-dihydrocortisone did not show mineralocorticoid activity in doses up to 100 microgram/rat. It is concluded that reduction of the 4.5 double bond diminishes mineralocorticoid activity of delta4-3-ketosteroids. Nevertheless, 5alpha-dihydroaldosterone has distinct mineralocorticoid activity with potency of 1.8% of that of aldosterone and approximately the same as that of 11-deoxycorticosterone.

Antinatriuretic and kaliuretic activities of the reduced derivatives of aldosterone.

The mineralocorticoid activities of the two dihydro- and the four tetrahydroisomers of the ring A-reduced derivatives of aldosterone were tested in adrenalectomized male rats. Potency was assessed by three criteria. Overall mineralocorticoid activity is expressed as the ability to reduce the urinary Na+/K+ ratio; antinatriuretic activity is represented by decreases in urinary Na+/creatinine; kaliuretic activity is shown by increases in K+/creatinine. All measurements were made on urine collected in the period 1-3 h postinjection. Measurements of overall activity indicate that the potency of aldosterone is greater than 5 alpha-dihydroaldosterone (DHA) greater than 3 alpha, 5 alpha-tetrahydroaldosterone (THA) greater than 3 alpha, 5 beta-THA greater than 3 beta, 5 alpha-THA greater than 5 beta-DHA greater than 3 beta, 5 beta-THA. Measurements of individual cation effects indicated that reduced derivatives generally, and the 5 alpha-reduced derivatives in particular, have greater antinatriuretic than kaliuretic activity. For example 5 alpha-DHA possesses between 7% and 17% of the antinatriuretic activity of aldosterone but only 0.7-2.7% of the kaliuretic activity. 5 alpha-DHA and 3 alpha, 5 beta-THA at concentrations of 10(-7)M were also shown to have mineralocorticoid activity in the isolated toad bladder; both caused an increase in the short circuit current across this epithelium although not to the level shown by a similar concentration of aldosterone. 5 beta-DHA appeared to be inactive at this dose.

Renal receptor-binding activity of reduced metabolites of aldosterone: evidence for a mineralocorticoid effect outside of the classic aldosterone receptor system.

Reduced metabolites of aldosterone have been shown to have antinatriuretic and kaliuretic effects. We have studied the ability of four reduced metabolites of aldosterone to compete with [3H]aldosterone and [3H]dexamethasone for binding to the mineralocorticoid and glucocorticoid receptors of the kidney using adrenalectomized rat renal slices and cytosol, respectively, as sources of the binding proteins. 5 alpha-Dihydroaldosterone had 18.9% the ability to compete with [3H]aldosterone for binding to the cytoplasmic receptor of adrenalectomized rat renal slices in comparison to unlabeled aldosterone. Its antinatriuretic potency varied between 7-17%. Its ability to compete with [3H]dexamethasone for binding to the renal glucocorticoid receptor was only 1.9% in comparison to unlabeled dexamethasone. The relative competitive activities of 3 beta,5 alpha-tetrahydroaldosterone and 3 beta,5 beta-tetrahydroaldosterone with [3H]aldosterone to adrenalectomized rat renal slices cytosol were 1.26% and 0.05%, respectively, in comparison to unlabeled aldosterone. Their reported mineralocorticoid activities using the adrenalectomized rat bioassay (antinatriuresis) were 0.1-0.4% and 0.15%, respectively, in comparison to aldosterone. The most important aldosterone metabolite 3 alpha,5 beta-tetrahydroaldosterone showed negligible competitive activity with [3H]aldosterone or [3H]dexamethasone for the renal corticoid type I or type II receptors, respectively. However, this compound has been reported and confirmed to have weak but clear-cut mineralocorticoid activity (approximately 1/100th that of aldosterone). The mineralocorticoid activity of 3 alpha,5 beta-tetrahydroaldosterone cannot be explained by a mechanism involving the classic renal mineralocorticoid receptor. The mechanism could involve an alternative receptor system, a nonreceptor-mediated renal mechanism, or the conversion to a metabolite that would interact with classic receptors.

19-nor analogs of adrenal steroids: mineralocorticoid and glucocorticoid receptor activity.

The effect of absence of the C-19 methyl group from five adrenal steroids has been studied in terms of their affinity for mineralocorticoid (MR) and glucocorticoid receptors (GR). In MR assays, 19-nordeoxycorticosterone and 19-norprogesterone showed 3-fold higher affinity for MR than did their respective parent steroids; 19-norcortisol had 1.5 times the affinity of cortisol for MR. In contrast, corticosterone and 19-nororticosterone showed equal affinity, and 19-noraldosterone showed less than 1% the MR activity of aldosterone. In GR assays, the absence of the C-19 methyl group from progesterone increased GR affinity 3-fold and deoxycorticosterone affinity 1.5-fold. In contrast, the other 19-nor steroids showed decreased affinity vis à vis their parent compounds (19-norcorticosterone, 30%; 19-norcortisol, 10%; 19-noraldosterone, < 1%). These findings suggest that while the 19-nor analogs of 11-deoxy steroids are consistently more active than their parent steroid, the 19-nor 11-oxygenated adrenal steroids show no predictable pattern of binding for MR or GR.

Orally active mineralocorticoid agonists and antagonists: delta 1-derivatives of aldosterone and 18-deoxyaldosterone.

The effect of delta 1 unsaturation on the oral effectiveness of a representative mineralocorticoid agonist and antagonist was investigated in an adrenalectomized rat bioassay. Dehydrogenation at the 1.2 position did not alter the qualitative nature of the mineralocorticoid activity of the parent compound. Thus delta 1-aldosterone (21,18-dihydroxy-11 beta, 18-oxido-1,4-pregnadiene-3,20-dione) retained pure mineralocorticoid agonism, and delta 1-18-deoxyaldosterone (21-hydroxy-11 beta, 18-oxido-1,4-pregnadiene-3,20-dione)demonstrated the same relative degree of predominant antagonism as 18-deoxyaldosterone (21-hydroxy-11 beta, 18-oxido-4-=pregnene-3,20-dione) itself. In each instance, receptor affinity was diminished by 1,2 unsaturation, but this effect was offset by the greater bioavailability of the delta 1 derivatives on oral administration. (Endocrinology 108: 517, 1981)

Mineralocorticoid and renal receptor binding activity of 21-deoxyaldosterone.

Since several aldosterone metabolites are known to be active, we have assessed the mineralocorticoid biological and renal receptor binding activities of the aldosterone metabolites, 21-deoxyaldosterone (21-deoxy-Aldo), 21-deoxytetrahydroaldosterone (21-deoxy-THAldo), and 3 alpha, 5 beta-tetrahydroaldosterone (THAldo). We synthesized these steroids by bioreduction of aldosterone with intestinal bacteria. Mineralocorticoid agonist activity of 21-deoxy-Aldo, 21-deoxy-THAldo and THAldo, determined by bioassay using adrenalectomized rats, was 1-5%, less than 0.01%, and 0.1-0.5% that of aldosterone, respectively. 21-Deoxy-Aldo showed no antagonist activity. The relative affinity in competing with [3H]aldosterone for binding to mineralocorticoid receptors in adrenalectomized rat kidney cytosols was 94%, less than 0.01%, and less than 0.01% that of aldosterone. The relative binding affinity for rat renal glucocorticoid receptors was 23%, less than 0.01%, and less than 0.01% that of dexamethasone, and for corticosteroid-binding globulin 17%, less than 0.01%, and less than 0.01% that of cortisol. These results show that the naturally occurring steroid, 21-deoxy-Aldo, possesses mineralocorticoid agonist activity which is equivalent to that of 11-deoxycorticosterone, and has substantial affinity for rat renal mineralocorticoid and glucocorticoid receptors. The results also implicate the pathophysiological role of 21-deoxy-Aldo as a potential mineralocorticoid in 21-hydroxylase deficiency, where urinary excretion of this steroid is invariably elevated.

Mineralocorticoid activity of 19-hydroxyaldosterone, 19-nor-aldosterone, and 3 beta-hydroxy-delta 5-aldosterone: relative potencies measured in two bioassay systems.

The mineralocorticoid (MC) activities of 19-hydroxyaldosterone (19-OH-Aldo) and 19-nor-aldosterone (19-nor-Aldo) were tested in adrenalectomized male rats. Potency was assessed by three criteria. Overall MC activity is expressed as the ability to decrease the urinary Na+ to K+ ratio; antinatriuretic activity is represented by decreases in the urinary Na+ to creatinine ratio, and kaliuretic activity by increases in the K+ to creatinine ratio. All measurements were made on urine collected 1-3 h postinjection. In this assay, 19-OH-Aldo was 1/100th to 1/140th as active as Aldo, and 19-nor-Aldo possessed MC activity similar to that of Aldo; both steroids possessed antinatriuretic and kaliuretic activities. In contrast, when assayed in vitro in the isolated toad urinary bladder, the natriferic responses of both 19-OH-Aldo and 19-nor-Aldo (10(-8), 10(-7), and 10(-6) M) were not significantly different from those caused by equivalent concentrations of Aldo. 3 beta-Hydroxy-delta 5-Aldo is active as a MC in the adrenalectomized male rat, being 1/20th to 1/35th as active as Aldo, but, in contrast to 19-OH-Aldo, was less active in the isolated toad bladder system. 19-OH-Aldo, 19-nor-Aldo, and 3 beta-hydroxy-delta 5-Aldo could represent important new classes of Aldo analogs.

The mineralocorticoid antagonist activity of an 11 beta,18-oxidopregnane.

Removal of the 18-hydroxy group of the hemiacetal form of aldosterone transforms its activity from that of pure agonist to predominant antagonist. The 18-deoxy derivative possesses one third of the binding affinity of aldosterone for the cytoplasmic mineralocorticoid receptor of rat kidney and exhibits an approximate 2:1 antagonist to agonist ratio in both toad bladder and adrenalectomized rat bioassay systems. The promising properties of the 11 beta,18-oxidopregnane tested included very low androgen receptor affinity and approximately equal effectiveness in vitro and in vivo in displacing aldosterone from mineralocorticoid-binding sites in the rat.