Estradiol Glucuronide Research Articles

Pattern of excretion of urinary steroid metabolites during the ovarian cycle and pregnancy in the marmoset monkey

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.

In vivo estrogen synthesis by the developing chicken ( Gallus gallus) embryo

The concentrations of unconjugated and conjugated estrogens in the allantoic and amniotic fluids of chicken embryos have been followed during incubation. The estrogens estrone, estradiol-17β and estradiol-17α were present as their conjugates in the allantoic fluid of female embryos but not of male embryos. No estrogens were detectable in the amniotic fluid of embryos of either sex. Estradiol-17β glucuronide, the most abundant estrogen present in female allantoic fluid, was first detectable (159 pg/ml) at Stage 35 of development increasing in concentration to 4210 pg/ml at Stage 45. The concentration of estrogen in allantoic fluid of the partially decapitated embryos at Day 18 (Stage 41) of incubation was not significantly different from that of the intact stage 41, approximately Day 15 embryo.

A direct radioimmunoassay of estradiol 3-glucuronide using specific antiserum.

The preparation and antigenic properties of estradiol 3-glucuronide-bovine serum albumin conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-6 position on the steroid nucleus, are described. Antiserum elicited in the rabbit by the immunogen possessed high affinity and specificity to estradiol 3-glucuronide, exhibiting hardly any cross-reactivities with other estrogen conjugates except for estrone 3-glucuronide (18.66%) and no cross-reactions with free estrogens and other related steroids. This antiserum was purified by affinity chromatography on estrone 3-glucuronide-aminoethyl cellulose conjugate. The purified antiserum exhibited no cross-reaction with estrone 3-glucuronide. A direct radioimmunoassay method for urinary estradiol 3-glucuronide was developed by using the purified antiserum. The values obtained with the purified antiserum were somewhat lower than those with the native one. The within- and between-assay variations were 3.32 and 4.35%, respectively. The values obtained by the present method correlated closely with those obtained by the established indirect radioimmunoassay (RIA) method.

Preparation of specific antiserum to estradiol 3-glucuronide

The preparation and antigenic properties of estradiol 3-glucuronide-bovine serum albumin (BSA) conjugate in which the hapten is coupled to the carrier protein through the aminomethyl group at C-6 by glutaraldehyde, have been described. Antibody raised against the immunogen in the rabbit possessed high affinity ( K A = 3.7 × 10 9M −1) and excellent specificity to estradiol 3-glucuronide, exhibiting no significant cross-reactivities with estrogen glucuronides except for estriol 3-glucuronide (3.64%) and no cross-reactions with free estrogens, their sulfates and other steroids.

Prepartion and antigenic properties of estriol 16-glucuronide- and estradiol 17-glucuronide-[C-6]-bovine serum albumin conjugates

The preparation and antigenic properties of estriol 16-glucuronide-bovine serum albumin (BSA) and estradiol 17-glucuronide-BSA conjugates in which the hapten molecule is linked to the carrier protein through an (O-carboxymethyl)oxime bridge at the C-6 position on the steroid nucleus, have been described. Antibodies raised against the two immunogens in the rabbit possessed excellent specificity to estriol 16-glucuronide and estradiol 17-glucuronide, respectively, exhibiting no significant cross-reactions with estrogen glucuronides having a substituent in ring A and no cross-reactivities with free estrogens. their sulfates and related steroids.

Substrate specificity of human UDP-glucuronyltransferase in cultured lymphocytes.

1. This study establishes the presence of UDP-glucuronyltransferase activity for non-steroidal as well as steroidal substrates, in cultured human B-lymphocytes. Glucuronidation of alpha-naphthol and testosterone was demonstrated in homogenates of two cell lines, SN1006 and RPMI-1788, and that of phenolphthalein, 4-methylumbelliferone, p-nitrophenol and estradiol in the cell line with the higher glucuronyltransferase activity, SN1006. 2. Kinetic studies of testosterone glucuronidation in homogenates of both cell lines revealed a similarity in the behaviour of glucuronyltransferase of these cells. Thus, comparable apparent Km values for UDPGA (0.63 mM) and for testosterone (14 microgram) were observed, although apparent maximal velocities, Vmax, differed several-fold (3.0 versus 0.55 pmol/10(6) cells per min, in SN1006 and RPMI-1788 cells, respectively). 3. Kinetic studies of glucuronidation of testosterone, estradiol, phenolphthalein, alpha-naphthol, 4-methylumbelliferone, and p-nitrophenol yielded comparable apparent Km values for UDPGA (0.56-0.67 mM), suggesting that the same, or similar, glucuronyltransferase(s) catalyse(s) glucuronidation of this wide range of substrates in lymphocytes. This was reinforced by the observation of competitive inhibition of testosterone glucuronidation by alpha-naphthol (Ki 0.25mM), 4-methylumbelliferone (Ki 0.8mM) and p-nitrophenol (Ki 0.8 mM). Thus, lymphocyte glucuronyltransferase activity with a broad substrate specificity, for steroidal and non-steroidal aglycones, is indicated.

Conjugated and unconjugated plasma oestrogens—oestrone, oestradiol and oestriol—in normal human males

The possible influence of ageing on the plasma concentrations of unconjugated and conjugated oestrogens was studied in 53 healthy males, 20–87 years old Unconjugated oestrogens were separated on LH 20 micro columns after extraction with diethyl ether. Oestrogen sulphates were extracted and separated after enzymatic hydrolysis with Mylase P and glucuronides were extracted and separated after acid hydrolysis of the residue. A wide scattering of the results was observed for all oestrogens within all age groups. Unconjugated oestrogens showed a significant age related increase in plasma values for both oestrone and oestradiol, whereas a significant decrease was found for oestriol. For oestrogen sulphates a significant age related decrease was observed for oestrone sulphate, whereas a significant increase with ageing was found for both oestradiol—and oestriol sulphates. Only few males had measureable plasma values for oestrone glucuronide and the mean plasma value was below detection limit. Small amounts of oestradiol glucuronide were measured and no correlation with ageing was observed. For oestriol glucuronide a significant age related increase was observed.

The direct radioimmunoassay of oestrogen glucuronides in human female urine

Radioimmunoassays for five oestrogen metabolites in urine are described; they are oestrone 3-glucuronide, oestradiol 3-glucuronide, oestradiol 17 beta-glucuronide, oestriol 3-glucuronide and oestriol 16 alpha-glucuronide. These assays have proved accurate and reliable and can be performed rapidly; they have been carried out directly in diluted menstrual cycle urine and pregnancy urine. No sample pretreatment was required. Preliminary results suggest that clinically useful information can be obtained by performing these assays on random urine specimens.

Effect of contraceptive steroids on mammary gland of beagle dog and its relevance to human carcinogenicity

Differences in the inhibitory potentials against UDP-glucuronosyltransferase (UGT) between species have been reported only rarely, even though the information would be useful for the precise characterization of drug candidates. In this study, the inhibition potentials of nonsteroidal anti-inflammatory drugs (NSAIDs) against UGT-catalyzed estradiol 3β-glucuronidation (E3G) in the liver microsomes of rats, dogs, and humans were compared. Rat liver microsomes (RLMs) and human liver microsomes (HLMs) exhibited homotropic activation kinetics with S50 values of 22 and 12 µM, respectively. However, dog liver microsomes (DLMs), exhibited Michaelis–Menten kinetics with no activation. Among the NSAIDs investigated (diclofenac, diflunisal, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, mefenamic acid, niflumic acid, and sulindac), only niflumic acid and mefenamic acid inhibited E3G potently in all three species. The IC50 values of NSAIDs against E3G in RLMs and HLMs were within a threefold difference of each other, while those in DLMs was more than three times higher than the other two. In conclusion, RLMs showed an inhibitory pattern similar to that of HLMs, whereas DLMs presented a distinct pattern. These results indicate that a rat animal model would be useful for evaluating the inhibitory potentials of drugs against estradiol glucuronidation, but a dog model would not.

Determination of estrogen ring D glucuronides in pregnancy plasma by direct radioimmunoassay without hydrolysis

A direct radioimmunoassay method using highly specific antisera without prior deconjugation has been developed for determination of estradiol 17-glucuronide and estriol 16-glucoronide in human plasma. Antisera were elicited in the rabbit by immunization with antigens in which the steroid haptens are linked to a carrier protein through the C-2 or C-4 position. After treatment with Rivanol the protein of albumin-free antiserum was covalently bound to a p-arylamine glass bead support through the cross-linkage with glutaraldehyde. A simple and reliable assay method employing the antibody-glass preparation was established and applied to measurement of estrogen ring D glucuronide concentration in peripheral plasma throughout normal pregnancy.

16-Hydroxylation of Estrone-3-Sulfate and Estrone in the Guinea Pig in Vivo*

[6,7-3H]Estrone-3-sulfate or [6,7-3H]-estrone of high specific activity was injected into adult female English Shorthair guinea pigs. Blood, liver, kidney, gall bladder bile, and urine were obtained and investigated for metabolites. Chromatographic procedures followed by enzymatic or solvolytic cleavage of conjugates and subsequent crystallization with appropriate carrier steroids revealed the pattern of metabolites formed. Injected estrone sulfate was partially hydrolyzed and reconjugated, resulting in the production of estrone and estradiol glucuronides. The main metabolites, however, were monosulfates of 16alpha-hydroxyestrone, 16-keto-17beta-estradiol, and estriol as well as disulfates of 16alpha-hydroxyestrone, estriol, and 16beta-hydroxyestrone. Particularly high amounts of these were found in urine. By far the main metabolites of injected estrone were glucuronides of estrone and estradiol, although the pattern of mono- and disulfated steroids was qualitatively similar to that found after estrone sulfate injection. It is concluded that the guinea pigs employed in the study hydroxylated estrogen in the 16alpha- and 16beta-configurations and that this activity was much more pronounced after injection of estrone sulfate than after estrone.

Determination of Ethinyl Estradiol in Human Urine by Radiochemical GLC

A radiochemical GLC analysis was developed for 3H-labeled ethinyl estradiol in human urine. The technique was applied to the unconjugated and aglycone fractions of urine collected from women who were dosed orally with: (a) single capsules containing 2.0mg of 3H-quinestrol (900μCi) and 2.5mg of unlabeled quingestanol acetate dissolved in sesame oil and (6) single tablets containing 100μg of 3H-quinestrol (86μCi). Unconjugated ethinyl estradiol in Day 1 urine collections represented means of 0.02% of the high quinestrol dose and 0.12% of the low dose. Ethinyl estradiol glucuronide in the same collections represented means of 0.55% of the high drug dose and 1.35% of the low dose. The method could detect 1-ng quantities of 3H-ethinyl estradiol and 3H-quinestrol.

Sex differences in estrogen excretion in early infancy

Urinary estrone (E1) and estradiol (E2) glucuronide and sulfate were determined separately by RIA in 44 newborn and young infants (26 boys, 18 girls). In 7 cases the estrogen excretion was followed up over one month. The conjugates were hydrolyzed enzymatically; the estrogens were purified and separated by Sephadex LH 20 chromatography. During the first month, children excreted more sulphated than glucuronized estrogens with a ratio of 8:2, but within the first year, this ratio was reversed to 3:7. In both sexes E1 + E2 excretion was 10 times higher during the first 2 months than at the end of the first year. Boys showed a sharp decline during the third month of life; girls, however, did not reach low values until the sixth month. While the E2/E1 ratio in boys was one, girls showed predominance of E2-excretion with a mean ratio of 2.5:1 during the first 6 months of life.

Effect of hypophysectomy on estrogen conjugation and on plasma and tissue concentrations of tritium after administration of 3H-estradiol-17β

Estradiol-17β-6,7- 3H was injected into ovariectomized (control) and ovariectomized, hypophysectomized (hypox) rats in order to study the binding of estradiol in the brain. Hypophysectomy resulted in a significant increase in the concentration of tritium in the hypothalamus and preoptic area, as well as cortex, muscle, plasma and liver. However, since the liver was lighter in hypox rats, the total tritium content in the liver was unchanged from controls. Part of the weight reduction in the liver was due to a loss of stainable glycogen, which took place within 24 hours of hypox. The increase in circulating tritiated estradiol after hypox led us to investigate hepatic metabolism of estradiol. In vitro studies on liver slices from control and hypox rats demonstrated a significant reduction in the formation of conjugates of estrogen. Specifically, estradiol glucuronide and estrone sulfate formation were reduced in hypox rats and conversely the unmetabolized estradiol concentration was higher. Hypophysectomy for 24 hours results in a significant decrease in hepatic metabolism of estradiol-17β.

The radioimmunoassay of steroid glucuronides. The oestrogen C-3 glucuronides as haptens.

Antisera were prepared against three related oestrogen ring-A glucuronides, oestrone 3-glucuronide, oestradiol 3-glucuronide and oestriol 3-glucuronide. The corresponding 6,7-3H-labelled conjugates were synthesized as radioligands and the cross-reactions of the antisera against ring-A oestrogen glucuronides and other steroid conjugates were examined. The specificity of the antiserum against oestriol 3-glucuronide was compared with that raised against oestriol 16alpha-glucuronide, and the measurement of the former conjugate in late-pregnancy urine is discussed.

Studies on bilirubin and steroid glucuronidation by rat liver microsomes

The addition of digitonin or other detergents to liver microsomes from control and phenobarbital-treated rats increased the rate of glucuronidation of bilirubin, testosterone, estradiol, estrone and estriol. The extent of activation by digitonin as well as the concentration of detergent causing maximal activation was substrate dependent. Treatment of rats with sodium phenobarbital (75 mg/kg/day, i.p.) for 3 days significantly increased the glucuronidation of testosterone and estradiol per mg of protein but had no effect on estrone glucuronidation. Administration of 0.1% sodium phenobarbital in the drinking water for 20 days increased bilirubin, testosterone and estrone glucuronidation per mg of protein, but had little or no effect on estradiol and estriol glucuronidation. When UDP-glucuronyl transferase activity was calculated on a per g liver (wet wt) basis, however, an increase in the glucuronidation of estradiol and estriol was seen; this increase could be accounted for by a large stimulatory effect of phenobarbital on the synthesis of total microsomal protein. Liver microsomes from homozygous Gunn rats, which are deficient in the ability to conjugate bilirubin, did conjugate testosterone, estradiol. estrone and estriol, but some substrates were conjugated more rapidly than others. The addition ofdigitonin to liver microsomes from the homozygous Gunn rat increased the glucuronidation of testosterone but not that of estrone. Microsomal steroid glucuronidation in this animal was stimulated to varying degrees by the administration of sodium phenobarbital. The data suggest the presence of several glucuronyl transferases in liver microsomes.

Microsomal glucuronidation of selected steroids using a rapid radiometric assay

Glucuronidation of testosterone, estrone, estradiol and estriol was investigated in rat liver microsomes using a rapid radiometric method. The method is one-step with activity measured in the same vial in which the reaction is conducted. Unreacted substrate is partitioned from the aqueous incubation medium into the non-polar scintillation fluid. Glucuronides remain in the aqueous fraction and therefore, are not detected by liquid scintillation counting which enables one to establish the glucuronidation rate by following substrate disappearance. This rapid method is less variable than more time-consuming extraction procedures and can be used easily to obtain kinetic data. Parameters were established for reaction linearity with respect to time and the addition of microsomal protein. Male rat liver microsomal glucuronyltransferase conjugated testosterone (2.6 nmol/min/mg protein) most rapidly followed by β-estradiol, estrone, and estriol, respectively. Kidney and lung microsomes had no detectable steroid glucuronyltransferase activity, although these tissues had significant levels of p-nitrophenol glucuronyltransferase.

Liaison du sulfate d'oestrone par les proteines seriques du rat au cours de son developpement

The binding of estrone sulfate by rat serum has been studied comparatively, with an equilibrium dialysis technique, in the embryo and the pregnant rat at the 19th day of pregnancy, as well as during post-natal development. The sera of the embryo and the new-born rat almost bind no estrone sulfate; in contrast, this binding is high in the 21-days-old and in the adult animal. A significant reduction of the affinity index is shown in the pregnant rat serum. The binding of testosterone sulfate is 3–4 times lower than that of estrone sulfate. There is no fixation of estradiol glucuronide in any of the tested sera. A positive correlation has been found between the albumin level of the different sera and their affinity index for the estrone sulfate. The albumin purified from embryo sera and the adult serum albumin have similar high affinity indexes for estrone sulfate. The binding curves for the estrogen conjugate with these two purified proteins and the whole sera of normal and pregnant adult rats are not appreciably different. In all cases, two kinds of binding sites are demonstrated: relatively strong binding sites, with an association constant of about 1.5·10 5 M −1 at 25°C and numerous low affinity binding sites, with an association constant of about 3·10 3 M −1 at 25 °C. There are probably two strong binding sites per serum albumin molecule ( n 1 ∼ 2). From these results, it is concluded that albumin is the main protein that binds estrone sulfate in the rat serum. The absence of binding in the embryo is due to the low levels of this protein and not to a qualitatively different behavior of the foetal serum albumin. The lack of binding of estrone sulfate in the embryo is in contrast with the high affinity of the embryo sera for estrone and estradiol-17β.

Comparative fate of estrone and estrone sulfate in man.

Estrone-14C and estrone sulfate-3H were simultaneously injected into a 37-yr-old woman and disposition of the two isotopes was followed. The products of estrone sulfate were excreted in urine more rapidly and to a greater extent than those of the coadministered estrone. Estrone was converted to estrone and estradiol glucuronides to a much greater extent than its sulfate conjugate. Estrone sulfate on the other hand contributed more to the urinary estrone and estradiol sulfates. All of the other metabolites irrespective of their conjugation contained equal amounts of both isotopes and were therefore most probably biosynthesized after equilibration of the dose. It is likely that estrone sulfate rather than estrone is the precursor of these compounds.

Effects of ethinylestradiol-induced cholestasis on bile flow and biliary excretion of estradiol and estradiol glucuronide by the rat.

SummaryThe effects of ethinylestradiol treatment on bile flow and biliary excretion of bromsulfophthalein, estradiol, and estradiol glucuronide in the rat were studied. One day following 9 days of 0.5 mg of ethinylestradiol treatment, bile duct cannulation and femoral vein catheterization were carried out. From that time until the end of study, bile flow was strikingly reduced in the estrogen-treated animals to less than 50% of that in the control group. One day following surgery, studies of biliary excretion were performed revealing a markedly diminished biliary excretion of radioactivity from intravenously administered tracer doses of both estradiol and estradiol glucuronide in the treated animals. The effect of estrogen treatment on bile flow and estrogen excretion in the bile is similar to that previously observed for bromsulfophthalein clearance. Possible mechanisms of estrogen action and implications in human estrogen-induced cholestasis are discussed.