5-dihydroaldosterone and tetrahydroaldosterone

Specific methods are described for the enzymatic synthesis of each of the six possible 3H-labeled Ring-A reduced metabolites of aldosterone (5 alpha- and 5 beta-DHAldo; 3 alpha,5 alpha-THAldo; 3 beta,5 alpha-THAldo; 3 alpha,5 beta-THAldo; and 3 beta,5 beta-THAldo; see footnote 1 for full names). Use of heated jacketed columns (C8-reverse phase) and two HPLC solvent systems, with isocratic aqueous methanol or acetonitrile, respectively, have been developed which resolve all six Ring-A reduced metabolites of aldosterone. The relative retention times and elution order of each reduced metabolite are different with each solvent system and hence help confirm the identities of Ring-A reduced metabolites made in vivo from physiological quantities of [3H]aldosterone. The use of an on-line beta-radioactivity detector (Berthold LB-504) enhanced the sensitivity of detection and markedly improved the resolution of these metabolites, compared with that obtained by off-line scintillation counting. Thus, the use of increased temperature with these two solvent systems, together with an on-line radioactivity detector, provide a useful and efficient analytical tool for the separation and identification of each reduced metabolite of aldosterone.

Topics: Aldosterone; Animals; Chromatography, High Pressure Liquid; Female; In Vitro Techniques; Liver; Male; Methods; Oxidation-Reduction; Rats; Rats, Inbred Strains

Radioimmunoassays of two unconjugated reduced metabolites of aldosterone were developed. Excretion rates of unconjugated 5 beta-dihydroaldosterone (5 beta-DHAld) and 3 alpha,5 beta-tetrahydroaldosterone (3 alpha,5 beta-THAld) were measured in 192 hypertensive patients and 16 normal subjects and results were compared with urinary tetrahydroaldosterone-glucuronide(TH-Aldo-glu), aldosterone-18-glucuronide (Aldo-18-glu) and 'free' aldosterone. Generally, values of unconjugated reduced metabolites correlated with one another but in some cases were exceptionally elevated indicating alteration of urinary bacterial flora or impairment of the enzymatic system regulating the conjugation reaction. We suggest that in those cases of hypertension and suspected primary aldosteronism when aldosterone-18-glucuronide and tetrahydroaldosterone glucuronide values remain within normal range, determination of the free metabolite fraction may be diagnostically helpful.

Topics: Aldosterone; Chromatography, Paper; Female; Humans; Hypertension; Male; Radioimmunoassay

The experiments described in this paper demonstrate that most of the metabolic alterations of the aldosterone molecule, hitherto attributed to hepatic enzymes, equally well may be carried out by enzymes synthesized by anaerobic bacteria from the human gut. The steroid reductases synthesized by Clostridium paraputrificum, Clostridium J-1, and Clostridium innocuum convert aldosterone to the 3 alpha, 5 beta tetrahydroaldosterone (THA), 3 beta, 5 alpha-THA, and 3 alpha, 5 alpha-THA, respectively. All three enzymes metabolize 5 alpha.dihydroaldosterone to a single compound: 3 beta, 5 alpha-THA. Bifidobacterium adolescentis reduces aldosterone to 20 beta-dihydroaldosterone. In mixed cultures of B. adolescentis and clostridia, the individual enzymes operate independently of each other; however, about half of the aldosterone metabolites are in the free form and half in the acetal form. By appropriate selection of substrate and bacterial strains, therefore, it is possible to biosynthesize not only three of the THA isomers but also the hexahydroisomers in free form as well as in the acetal form.

Topics: Aldosterone; Clostridium; Intestines; Mass Spectrometry

Slices of kidney cortex and medulla from adrenalectomized male rats metabolized aldosterone to at least 4 peaks of polar metabolites as well as reduced metabolites. The majority of the products were ring A-reduced and shown chromatographically to consist of 5 alpha-reduced metabolites; significant quantities of 5 beta-reduced metabolites were also synthesized. Approximately half of the 5 alpha-reduced products cochromatographed with 3 beta,5 alpha-THA and the remainder with 5 alpha-DHA and/or 3 alpha,5 alpha-THA. The anti-mineralocorticoids, spironolactone and progesterone, inhibited renal synthesis of 5 alpha-reduced products of aldosterone by 80%. 5 beta-Reduction was also slightly inhibited. Corticosterone slightly inhibited synthesis of the 5 alpha- and 5 beta-reduced products. Corticosterone and progesterone significantly inhibited the renal synthesis of the polar metabolites of aldosterone, but the inhibition was not significant at this dosage of spironolactone. Interestingly, the nuclei and plasma membranes were shown to be the most active fractions for these aldosterone transformations. Importantly significant quantities of 5 alpha-DHA and/or 3 alpha,5 alpha-THA, which possess 1/10 and 1/30, respectively, of the antinatriuretic activity of aldosterone, were synthesized at these subcellular locations.

Topics: Adrenalectomy; Aldosterone; Animals; Cell Membrane; Cell Nucleus; Chromatography, High Pressure Liquid; Corticosterone; Kidney; Male; Progesterone; Rats; Rats, Inbred Strains; Spironolactone