Enzymatic synthesis of 3H-labeled Ring-A reduced metabolites of aldosterone and their separation by high pressure liquid chromatography.
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.
Latif
S A
SA
Department of Pathology, Miriam Hospital, Brown University, Providence, RI 02912.
Camara
P
P
Rosen
M P
MP
Morris
D J
DJ
D000450:Aldosterone / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000302:isolation & purification; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008297:Male; D008722:Methods; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains
C013282:tetrahydroaldosterone; D000450:Aldosterone; C018920:5-dihydroaldosterone