estradiol-3-glucuronide and estriol-3-glucuronide

klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that have been shown to be involved in the catalytic activity of this family of enzymes, the function of this protein was unknown. Here, we have studied the biochemical characteristics of recombinant Klotho. The purified chimeric Klotho-human IgG1 Fc protein (KLFc) was assayed with a series of 4-methylumbelliferyl (4Mu) beta-glycosides as potential substrates. An enzymatic activity of Klotho was observed only with 4-methylumbelliferyl beta-D-glucuronide in contrast to bovine liver beta-glucuronidase, which exhibits a rather wide substrate specificity. Furthermore, the enzymatic activity of KLFc was reduced by the addition of specific inhibitors of beta-glucuronidase. A number of natural beta-glucuronides were screened by competitive inhibition for KLFc beta-glucuronidase. We found that steroid beta-glucuronides such as beta-estradiol 3-beta-D-glucuronide, estrone 3-beta-D-glucuronide, and estriol 3beta-D-glucuronide were hydrolyzed by KLFc. The artificial fluorescent substrate and the steroid conjugates share a common phenolic structure. Collectively, these data suggest that Klotho functions as a novel beta-glucuronidase and that steroid beta-glucuronides are potential candidates for the natural substrate(s) of Klotho.

Topics: Animals; Blotting, Western; Catalysis; Cattle; Cell Membrane; CHO Cells; Cricetinae; Culture Media, Conditioned; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Estradiol; Estriol; Glucuronidase; Humans; Hydrogen-Ion Concentration; Hydrolysis; Immunoglobulin G; Kinetics; Klotho Proteins; Liver; Membrane Proteins; Mice; Phenol; Phenotype; Protein Binding; Recombinant Proteins; Steroids; Substrate Specificity; Time Factors; Transfection

Monitoring of reproductive steroid hormones at the population level requires frequent measurements, hormones or metabolites that remain stable under less than ideal collection and storage conditions, a long-term supply of antibodies, and assays useful for a range of populations. We developed enzyme immunoassays for urinary pregnanediol 3-glucuronide (PDG) and estrone conjugates (E1Cs) that meet these criteria.. Enzyme immunoassays based on monoclonal antibodies were evaluated for specificity, detection limit, parallelism, recovery, and imprecision. Paired urine and serum specimens were analyzed throughout menstrual cycles of 30 US women. Assay application in different populations was examined with 23 US and 42 Bangladeshi specimens. Metabolite stability in urine was evaluated for 0-8 days at room temperature and for 0-10 freeze-thaw cycles.. Recoveries were 108% for the PDG assay and 105% for the E1C assay. Serially diluted specimens exhibited parallelism with calibration curves in both assays. Inter- and intraassay CVs were <11%. Urinary and serum concentrations were highly correlated: r = 0.93 for E1C-estradiol; r = 0.98 for PDG-progesterone. All Bangladeshi and US specimens were above detection limits (PDG, 21 nmol/L; E1C, 0.27 nmol/L). Bangladeshi women had lower follicular phase PDG and lower luteal phase PDG and E1Cs than US women. Stability experiments showed a maximum decrease in concentration for each metabolite of <4% per day at room temperature and no significant decrease associated with number of freeze-thaw cycles.. These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Topics: Adult; Bangladesh; Estradiol; Estriol; Estrogens, Conjugated (USP); Estrone; Female; Fluoroimmunoassay; Humans; Immunoenzyme Techniques; Mass Screening; Middle Aged; Pregnanediol; Specimen Handling; United States

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