HPLC-fluorescence determination of equilin and equilenin in postmenopausal women's urine.

A high-performance liquid chromatographic (HPLC) method with fluorescence detection (lambda(ex) = 280 nm; lambda(em) = 410 and 312 nm) in combination with a post-column on-line photochemical derivatization is described for the determination of equilin and equilenin in urine from normal postmenopausal women after therapy with conjugated oestrogens. The column effluents were subjected on-line to UV irradiation (254 nm) and the photo-induced modifications were useful for the identification of the analytes. The conjugated (sulphate and glucuronide) forms were analysed after enzymatic or chemical hydrolysis and extracted with chloroform. Solid-phase extraction using strong anion-exchange sorbent was applied to the analysis of unconjugated oestrogen fraction to obtain a practical and reliable sample clean-up. The HPLC separations were achieved using ODS columns with a mobile phase consisting of 0.05 M triethylamine phosphate buffer (pH 4.0)-acetonitrile (64:36, v/v) at a flow rate of 1.0 mL/min. The method was accurate and reproducible; for the equilin and equilenin separation isocratic conditions were satisfactory, allowing a sensitive detection in urine samples with a detection limit of about 50 fmol for equilin (lambda(ex) = 280 nm; lambda(em) = 312 nm, after photoderivatization) and 10 fmol for equilenin (lambda(ex) = 280 nm; lambda(em) = 410 nm).

Metabolism of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate in normal postmenopausal women.

The metabolism of 17 beta-dihydroequilin and 17 beta-dihydroequilin sulfate was investigated after intravenous administration of [3H] 17 beta-dihydroequilin and [3H] 17 beta-dihydroequilin sulfate to postmenopausal women. Urine was collected for 3 days and 46.2 +/- 10.5% and 54.5 +/- 8.7% of the injected dose of [3H] 17 beta-dihydroequilin and [17 beta-3H]dihydroequilin sulfate was excreted in the urine respectively. The estrogens present in urine were extracted and fractionated into unconjugated, sulfate, and glucuronide conjugated forms. With both precursors, the major amount (63-64%) of metabolites were excreted in the urine conjugated with glucuronic acid. From the unconjugated, sulfate, and glucuronide fraction, 17 beta-dihydroequilenin, 17 beta-dihydroequilin, equilenin, and equilin were isolated. The conversions with both precursors were similar and 17 beta-dihydroequilenin was the major metabolite isolated from all three fractions; however, the highest levels of all four metabolites were present in the glucuronide fraction. Along with these identifiable metabolites, a large amount (51-81%) of radioactivity was present in the form of metabolites which are more polar than any of the known ring-B unsaturated estrogens. These appear to be polyhydroxy 17 beta-reduced ring-B unsaturated estrogens which remain to be identified. The in-vivo formation of equilenin and 17 beta-dihydroequilenin indicates the presence of the enzyme 6.8(9) steroid dehydrogenase in humans.

Metabolism of [3H]equilin-[35S]sulfate and [3H]equilin sulfate after oral and intravenous administration in normal postmenopausal women and men.

The absorption of equilin sulfate and equilin from the gastrointestinal tract was determined in normal men after the ingestion of [3H]equilin-[35S]sulfate or a mixture of [3H]equilin and equilin-[35S]sulfate, while the metabolism of equilin sulfate was investigated after iv administration of [3H]equilin sulfate to postmenopausal women. After the oral administration of [3H]equilin-[35S]sulfate, equilin sulfate containing both 3H and 35S was isolated from plasma; however, only in the first sample taken at 10 min was the 3H/35S ratio the same as that of the [3H]equilin-[35S]sulfate ingested. The 3H/35S ratio then increased, and by 12 h only traces of equilin-[35S]sulfate were detectable. Similarly, after the ingestion of [3H]equilin and equilin-[35S]sulfate, [3H]equilin-[35S]sulfate was isolated from plasma. The 3H/35S ratio was at all time points greater than the 3H/35S ratio of the ingested mixture. Analysis of urine indicated that over 98% of 35S was not associated with any steroid and was most likely inorganic sulfate. After iv administration of [3H] equilin sulfate to postmenopausal women, equilin, equilenin, 17 beta-dihydroequilin, and 17 beta-dihydroequilenin were isolated from the urine. These results indicate that 1) some of the orally administered equilin sulfate was absorbed from the gut without prior hydrolysis, 2) some equilin sulfate was hydrolyzed in the gut before absorption; 3) equilin was absorbed more efficiently than equilin sulfate; 4) equilin absorbed was readily sulfated and circulated in this form; and 5) equilin sulfate was extensively metabolized, and the metabolites were excreted in the urine mainly conjugated with glucuronic acid.

The metabolism of equilin in normal men.

Healthy adult males received either [3H]-equilin intravenously (one subject) or a larger mass of unlabelled equilin orally (three subjects). Blood samples were taken at 20, 40, 60, 90 and 120 min and every h thereafter until eight h after injection. Urine was collected in 24 h aliquots for five days from all subjects. The half-life of the disappearance of the unconjugated radioactivity from blood was 30 min and that in the conjugated sulfate fraction was 5 1/2 h. Approximately 50% of the injected radioactivity was recovered in the urine over 5 days. After extraction, hydrolysis and fractionation, most (83%) of the radioactive material found in the urine was present in the glucuronide fraction while only 2 and 6% were present in the unconjugated and sulfate fractions, respectively. The three fractions were combined for further isolation and identification of the metabolites. Radiochemically pure equilin, equilenin, 17 beta-dihydro-equilin and 17 beta-dihydroequilenin were isolated and identified but the largest fraction of radioactivity (70.5%) was present in the form of metabolites which are more polar than any of the known ring B unsaturated estrogens. These appear to be polyhydroxy 17-reduced ring B unsaturated estrogens. These results indicate that the ring B unsaturated estrogen equilin is being metabolized in man in a somewhat similar manner to that of the classical estrogen estrone. Knowledge of the formation of 17 beta-dihydroequilin from equilin in man is of importance because this estrogen is approximately 8 times more potent as a uterotrophic agent than the commonly used estrogen, equilin.

Patterns of urinary oestrogen excretion in individual pregnant mares.

Urine collections (24 hr) were made at weekly intervals from four Pony mares from the 3rd or 4th month of pregnancy until parturition. Separation of oestrogens on Celite columns was followed by Kober measurements of oestrone and equilin. Individual differences were noted in peak amounts of total oestrogens excreted (200 to 800 mg/day), when oestrone constituted 80 to 95% at the 6th to 7th months. Although equilin increased in later gestation, oestrone remained the major product. Total oestrogen values decreased rapidly from the peak and then more gradually towards the end of pregnancy. During the last 3 weeks the decline in the ratio of oestrone to equilin was reversed in all mares.