estradiol-3-glucuronide and estrone-3-glucuronide

Our aim was to develop a statistical method to correct for non-parallelism in an estrone-3-glucuronide (E1G) enzyme immunoassay (EIA). Non-parallelism of serially diluted urine specimens with a calibration curve was demonstrated in an EIA for E1G. A linear mixed-effects analysis of 40 urine specimens was used to model the relationship of E1G concentration with urine volume and derive a statistical correction. The model was validated on an independent sample and applied to 30 menstrual cycles from American women. Specificity, detection limit, parallelism, recovery, correlation with serum estradiol, and imprecision of the assay were determined. Intra-and inter-assay CVs were less than 14% for high- and low-urine controls. Urinary E1G across the menstrual cycle was highly correlated with serum estradiol (r= 0.94). Non-parallelism produced decreasing E1G concentration with increase in urine volume (slope = -0.210, p < 0.0001). At 50% inhibition, the assay had 100% cross-reactivity with E1G and 83% with 17beta-estradiol 3-glucuronide. The dose-response curve of the latter did not parallel that of E1G and is a possible cause of the non-parallelism. The statistical correction adjusting E1G concentration to a standardized urine volume produced parallelism in 24 independent specimens (slope = -0.043+/-0.010), and improved the average CV of E1G concentration across dilutions from 19.5%+/-5.6% before correction to 10.3%+/-5.3% after correction. A statistical method based on linear mixed effects modeling is an expedient approach for correction of non-parallelism, particularly for hormone data that will be analyzed in aggregate.

Topics: Adult; Data Interpretation, Statistical; Enzyme-Linked Immunosorbent Assay; Estradiol; Estrone; Female; Humans; Menstrual Cycle; Middle Aged; Sensitivity and Specificity

Estrogen-induced tumors in kidneys of male Syrian hamsters have been postulated to arise from cells which are damaged by free radicals and other reactive species generated during metabolic redox cycling of catecholestrogens and which at the same time are exposed to excessive growth stimulation mediated by estrogen receptors. In this study, we have determined the rates of metabolic deconjugation of estrogen glucuronides and of catecholestrogen methyl ethers by cellular fractions from male hamster kidney and liver to evaluate the contribution of this process to renal pools of parent estrogens and of catecholestrogen metabolites. Lysosomes from male hamster kidney catalyzed the deconjugation of estradiol- and estrone-3 beta-D-glucuronides at rates of 51.7 and 64.6 pmol/mg protein/min, respectively, which were 65 and 34% higher than corresponding deconjugation rates by liver lysosomes. Treatment of hamsters with estradiol implants for 9 days increased lysosomal glucuronidase activities for these estrogen glucuronides by 15 to 25% in kidney and doubled the activities in liver, but it did not alter their corresponding Km values. Microsomal glucuronidase activities in kidney and liver were approximately 10 to 20% of lysosomal activities. Rates of demethylation of 2- and 4-methoxyestradiol by kidney microsomes were comparable (with Vmax values of 24 and 30 pmol/mg protein/min, respectively), whereas the rate of demethylation of 2-methoxyestradiol by liver microsomes was approximately fivefold higher than that of 4-methoxyestradiol. The rates of renal demethylation of methoxyestrogens were comparable with previously published rates of renal aromatic hydroxylation of estradiol, whereas rates of hepatic demethylation were about one-fifth of the corresponding hydroxylation rates. It is concluded that metabolic deconjugation is an important source of primary estrogens and of catecholestrogen metabolites in hamster kidney, a target of estrogen-induced tumorigenesis. The increased renal estrogen glucuronidase activity during prolonged estradiol treatment may also facilitate the development of estrogen-induced tumors in this target organ.

Topics: 2-Methoxyestradiol; Animals; Cell Fractionation; Cricetinae; Estradiol; Estrogens, Conjugated (USP); Estrone; Glucuronidase; Kidney; Kidney Neoplasms; Liver; Lysosomes; Male; Mesocricetus; Methylation; Oxidoreductases, N-Demethylating

To determine the absorption and metabolism of 17 beta-estradiol (E2) by the stomach and liver of the pig, crystalline E2 was placed in the stomach of prepubertal gilts. Blood samples were subsequently obtained from the hepatic portal and jugular veins and plasma was assayed for E2, estrone (E1), 17 beta-estradiol-glucuronide (E2G), estrone-glucuronide (E1G) and estrone-sulfate (E1S). Concentrations of E2, E1, E2G and E1S rose in the hepatic portal vein within five min and remained elevated for several hr. Concentration of E2 represented only 6% of the total estrogen detected in the hepatic portal vein during the sampling period, indicating that most of the E2 was converted or conjugated prior to entering the hepatic portal vein. The metabolism of E2 presumably occurred in the stomach mucosa because food had been withheld for 26 hr before infusion of E2. Concentrations of E2G, E1G and E1S, but not E2 and E1, rose in the jugular vein and remained elevated for several hr. The lack of a rise in E2 and E1 in the jugular vein indicates that the E2 and E1 from the hepatic portal vein were completely converted and/or removed by the liver. Most of E2 was converted to E1 and then to E1G. The infusion of bile containing normal estrogens from pregnant gilts into the duodenum of prepubertal gilts resulted in a peak of E1G and E2G in the hepatic portal and jugular veins within a few minutes. This was followed in about 180 min by a second sustained rise. The first peak was essentially abolished by extracting E1 and E2 from the bile before infusion. The second peak failed to occur in gilts given antibiotics orally to reduce gut bacteria before infusion of bile.

Topics: Absorption; Animals; Duodenum; Estradiol; Estrone; Female; Gastric Mucosa; Intestinal Absorption; Jugular Veins; Kinetics; Liver; Portal Vein; Swine

Urinary oestrone-3-glucuronide, oestradiol-3-glucuronide, oestrone and oestradiol were measured by radioimmunoassay methods adapted for the very low levels found in postmenopausal women. Oestrogen concentrations related to creatinine in morning urine samples from ten postmenopausal women were found to correlate well with total oestrogen excreted in 24 h (r = 0.934, 0.867, 0.947, 0.909, respectively; p less than 0.002). Day to day variation in five individuals, measured over 5 weeks, showed random fluctuations, with no obvious cyclical variation. Reference ranges, based on two consecutive morning urine samples from 131 postmenopausal women, were 0.73-4.57 mumol/mol creatinine for oestrone-3-glucuronide, 0.66-3.00 mumol/mol creatinine for oestradiol-3-glucuronide, 4.8-30.9 nmol/mol creatinine for oestrone and 3.8-16.8 nmol/mol creatinine for oestradiol. We suggest that such assays may have a part to play in the screening for women at greatest risk of developing osteoporotic fractures.

Topics: Estradiol; Estrogens; Estrone; Female; Humans; Menopause; Microchemistry; Middle Aged; Radioimmunoassay

Non-invasive methods for monitoring reproductive status based on the measurement of urinary steroid conjugates were examined. Levels of urinary oestrone-3-glucuronide, oestrone-3-sulphate, oestradiol glucuronide, oestradiol sulphate and pregnanediol-3 alpha-glucuronide were determined during the ovarian cycle and pregnancy. Sequential hydrolysis showed oestradiol conjugates to be more abundant than oestrone conjugates. The levels of sulphates and glucuronides were similar in the follicular phase whereas sulphates predominated during the luteal phase and pregnancy. Although levels of oestrone-3-sulphate were two- to fourfold lower than those of oestradiol sulphate, measured after hydrolysis, the profiles throughout the cycle and pregnancy were similar. Levels of oestrone-3-sulphate, measured by direct assay, were below 1 mumol/mmol creatinine during the follicular phase, rising 3-4 days after ovulation to reach maximum values (2-8 mumol/mmol creatinine) in the mid-luteal phase. There was no consistent increase before ovulation. Levels during pregnancy rose gradually until days 70-90, after which there was no further increase (gestation length = 144 days). The pattern of pregnanediol-3 alpha-glucuronide was similar to that of oestrone-3-sulphate during the ovarian cycle but levels did not increase during pregnancy. The patterns of excretion of oestrogen and progesterone metabolites were similar to the pattern of the circulating hormones during the ovarian cycle. Circulating and urinary hormone patterns were similar for oestrogens throughout pregnancy but pregnanediol-3 alpha-glucuronide did not reflect progesterone secretion beyond day 70 of gestation.

Topics: Animals; Callitrichinae; Estradiol; Estrogens, Conjugated (USP); Estrone; Estrus; Female; Pregnancy; Pregnancy, Animal; Pregnanediol; Progesterone

Studies were conducted to determine the absorption and metabolic fate of 3H-estradiol-17 beta-glucuronide (3H-E2-G) in swine. The conjugate, 3H-E2-G (48.7 x 10(6) DPM, 45.5 Ci/mmol), was injected into ligated 15-cm sections of duodenum, proximal jejunum, distal jejunum, ileum and spiral colon of 10 kg female pigs. Blood from the jugular and portal veins and urine were collected at .5-h intervals for 5 h. Absorption from the colon was rapid and radioactivity peaked in both portal and jugular plasma by .5 h postinjection. In contrast, the highest plasma estrogen concentration from most other sections was reached at 5 h, the last sampling time. The urinary excretion patterns were nearly identical to those seen in plasma, with the radioactivity peaking early (1.5 h) after instillation of 3H-E2-G into the colon, but still rising at the end of the experiment after instillation into the duodenum, distal jejunum and ileum. The proximal jejunum, which produced low plasma estrogen concentrations, also produced low urine concentrations. The slower absorption of 3H-E2-G compared to 14C-estradiol-17 beta is consistent with the view that the limiting factor for the absorption of the conjugate is hydrolysis to a free estrogen. The predominant metabolites in portal venous plasma from all sections of the intestine at the end of the experiments were the monoglucuronides of estrone and estradiol. Because the administered 3H-E2-G was conjugated at C-17, the presence of estrone glucuronide in portal plasma indicates that, at least in the duodenum, ileum and colon, 3H-E2-G undergoes cleavage, followed by the oxidation of estradiol to estrone, which is subsequently reconjugated by the intestinal mucosa.

Topics: Animals; Chromatography, Thin Layer; Estradiol; Estrone; Female; Intestinal Absorption; Intestinal Mucosa; Swine

Assays which measure immunoreactive metabolites of the major urinary estrogens directly are described, and their applicability to ovulation detection methods is discussed. The immunoreactive materials measured were estrone glucuronide (E1G), estradiol-3-glucuronide (E2-3G), estradiol-17beta-glucuronide (E2-17G), estriol-3-glucuronide (E3-3G), estriol-16alpha-glucuronide, and pregnanediol-3alpha-glucuronide (Pd-3G); these estrogen and pregnanediol glucuronide fractions were measured in portions of 24-hour and overnight samples of urine collected daily from 7 women throughout 1 menstrual cycle. The urinary excretions were correlated with daily serum levels of estradiol, progesterone, and luteinizing hormone. The usual serum changes were noted preovulatorily in all 7 women, who were therefore considered ovulatory. 24-hour and overnight urinary excretion patterns of the estrogen glucuronides were similar to the serum estradiol pattern, and of the 5 estrogen glucuronide fractions, E2-17G showed the earliest and steepest ascending slope of preovulatory estrogen surge. Therefore, E2-17G is most suitable estrogen fraction for predicting ovulation. Pd-3G rose in parallel with progesterone serum levels and was markedly elevated 2-3 days after the midcycle luteinizing hormone peak; therefore, because milligram quantities of Pd-3G are excreted daily in urine, measurements of this glucuronide are most useful for ovulation detection by a simple immunochemical or enzymatic technique (dipstick).

Topics: Adult; Estradiol; Estriol; Estrogens; Estrone; Female; Glucuronates; Humans; Luteinizing Hormone; Menstruation; Pregnanediol; Progesterone; Radioimmunoassay; Specimen Handling

In attempt to find a more reliable and earlier predictor of ovulation, radioimmunoassay methods were investigated which could measure the levels of steroid glucuronides (follicular estrogens), varying levels of which would warn of approaching ovulation well before (up to 96 hours) its occurrence so that abstinence can be practiced during the period of sperm viability before the egg is released. To this end, daily urine collections were made on 9 subjects and the steroid glucuronides were totalled. In addition, the excretion of 5 beta-pregnanediol-3 alpha-glucuronide was measured throughout the menstrual cycle. Measurement of this parameter seemed of more practical value since the ratio of estrone-3-glucuronide/pregnanediol-3 alpha-glucuronide rises during the periovulatory period and falls sharply after the formation of the corpus lutuem. This parameter, unlike measurement of total steroid glucuronides, is not dependent on the rate of excretion or on concentration. In all 9 subjects, a significant increase in this ratio (P .05) was detected 48-120 hours before the plasma lutropin maximum. For assay, this ratio value is easier to assess because the assay kit would be applicable to early morning urine samples and would not involve any measurement of urine volume or time. A short discussion follows the experimental presentation.

Topics: Estradiol; Estriol; Estrogens; Estrone; Female; Fertility; Glucuronates; Humans; Menstruation; Ovulation

Topics: Administration, Oral; Animals; Dogs; Estradiol; Estrone; Female; Glucuronates; Injections, Intravenous