Analysis of [3H]estradiol-17beta metabolites in calf perirenal fat

Residues of estradiol-17 beta (E2 beta) in the kidney fat of one milk-fed calf were studies using radiometric methodologies. A three-month old Friesian male veal calf was injected intramuscularly daily for 3 d with 333 mg of [6,7(n)-3H]-E2 beta (specific activity: 7.55 MBq mmol-1) dissolved in 2 ml of propylene glycol and slaughtered 3 h after the last administration. Total estrogens were about 280 ng g-1 in perirenal fat. After a de-lipidation step, the relatively polar metabolites that were extractable with dichloromethane represented the main fraction of the metabolites, which accounts for almost 50% of the total radioactivity of the tissue, of which E2 beta was the major metabolite (19.7%) and E1 and E2 alpha represented only 7.7 and 3.2%, respectively. Conjugated estrogens accounted for only 15.2% of the total estrogen content. Non-polar estrogens (about 25% of total estrogens) were removed specifically with isooctane during the de-lipidation step and were further purified on silica and alumina columns before being chromatographed by normal-phase HPLC. The radioactive metabolites were eluted as estrogen-17 esters. The HPLC analysis of the estrogens released following hydrolysis of the esters indicated that E2 beta was the main estrogen acylated by long-chain fatty acids in the fraction of lipoidal estrogens. The presence of such a class of estrogens in fat could be of interest for the detection of estrogens a considerable time after estradiol administration.