Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Estrogen metabolism in nonhuman primates I. In vitro biosynthesis of estrogen glucosiduronates in rhesus monkey liver

Estrone glucosiduronate, 17β-estradiol-3-glucoslduronate, 17β-estradiol-17-glucosiduronate and estriol-16α-glucoslduronate have been biosynthesized in substantial yield by incubation of radioactive estrone, 17β-estradiol, estriol and uridlne diphosphoglucosiduronic acid with rhesus monkey liver homogenates. The metabolites were characterized by chromatography on Celite and DEAE-Sephadex, enzyme hydrolysis, derivative formation and crystallization to constant specific activity. The percent conversion to 17β-estradiol-17-glucosiduronate from 17β-estradlol ranged between 56–71%; from estrone, the conversion was 49–54%. Other metabolites formed from estradiol were estrone glucosiduronate(12–21%) and 17β-estradiol-3-glucosiduronate(5–12%). The same metabolites derived from estrone accounted for 18–28% and 10–14% respectively. After estriol incubation, more than 90% of the steroid was converted to estriol-16α-glucosiduronate with no detectable estriol-3-glucosiduronate. This report represents the first time that 17β-estradiol-17-glucosiduronate has been reported as a metabolite in the rhesus monkey and this is the only known species which forms 17β-estradiol-17-glucosiduronate as the predominant metabolite of either estrone or estradiol $\text{in vitro}$.Rhesus monkey liver is similar to the human and baboon in that it metabolizes estriol exclusively to estriol-16-glucosiduronate.     

Splanchnic and intestinal uptake and formation of estriol and estriol conjugates in the dog in vivo.

A loading dose of 3H-estriol was given to male dogs followed by a constant infusion. The concentrations of total radioactivity, conjugated estriol metabolites, estriol, estriol-o-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-3-sulfate and estriol-3-sulfate, 16alpha-glucosiduronate were determined in plasma from the femoral artery(A), hepatic vein(HV) and superior mesenteric vein (SMV). From these values the splanchnic (100[1-HV/A]) and intestinal (100[1-SMV/A]) extractions were calculated. The mean splanchnic extraction of total radioactivity was positive (23, SE 3, P less than .01), indicating net uptake by the splanchnic area, possibly due to biliary excretion. The mean splanchnic extraction of estriol was 77, SE 1, P less than .01, also indicating net uptake. The splachnic extractions of estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate and estriol-3-sulfate were negative (-15, SE 3, P less than .01; -23, SE 6, P less than .01; -31, SE 8, P less than .01 respectively) indicating net formation of these conjugates for release into the systemic circulation. The mean intestinal extraction of estriol was 12, SE 4, P less than .01, indicating net uptake by the intestine. This net uptake was associated with mean negative intestinal extractions of estriol-3-glucosiduronate (-15, SE 7, P approximately .05), estriol-3-sulfate (-33, SE 10, P less than .01) and estriol-3-sulfate, 16alpha-glucosiduronate (-53, SE 13, P less than .01), indicating net formation of these conjugates by the intestine.     

Dynamics of estrone glucosiduronate metabolism in dogs.

Interest in the metabolism of estrogen conjugates has been increased by the recent demonstration that some of these conjugates can be hydrolysed in vivo, and are thus a source of physiologically-active circulating estrogens. In this report the metabolism of the quantitatively important conjugate estrone glucosiduronate has been studied in dogs, following the intravenous infusion of 3H-estrone glucosiduronate. Arterial plasma levels of radioactive estrone glucosiduronate and of the radioactive metabolites estrone, estradiol-17beta-3-glucosiduronate and estradiol-17alpha-3-glucosiduronate were measured. The metabolic clearance rate of estrone glucosiduronate (MCREG) was determined, as well as conversion ratios for estrone glucosiduronate to estrone (CREG.E), estradiol-17beta-3-glucosiduronate (CREG.E2betaG) and estradiol-17alpha-3-glucosiduronate (CREG.E2alphaG). Sequential values for the above mentioned conversion ratios were obtained and the relationship to time was analysed. Transfer constants for estrone glucosiduronate to estrone (rhoEG.E) were measured. The mean MCREG was 329 L/day/m2, SE 35. The values for CREG.E2betaG and CREG.E2alphaG did not become constant until 80 minutes following the start of infusion. The mean values at a steady state were: CREG.E.021, SE .002, CREG.E2betaG .068, SE .005, CREG.E2alphaG .022, SE .002. The mean value for rhoEG.E was .057, SE .006.     

Identification of some plasma metabolites of 6, 7-3-H-estrone glucosiduronate in male dogs.

Following the constant infusion of 6, 7-3-H-estrone glucosiduronate in male dogs for a period of 120 minutes, the radioactive metabolites present in the plasma were separated by solvent partition, DEAE-Sephapadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and steroids and their derivatives. Most of the radioactivity in the plasma was identified as estrone glucosiduronate. The major metabolite present was estradiol-17-beta-3-glucosiduronate. Small amounts of estradiol-17-alpha-3-glucosiduronate and free estrone were also identified. Three other minor conjugates were separated, but positive identification could not be made.     

Serum and urinary estrone sulfate during the menstrual cycle, measured by a direct radioimmunoassay, and fate of exogenously injected estrone sulfate

Serum and early-morning urinary levels of estrone sulfate during the menstrual cycle were measured by a direct radioimmunoassay without hydrolysis. These levels were high and showed prominent peaks [serum, 2.67 +/- 0.37 ng/ml (mean +/- SE); urine, 5.82 +/- 2.3 micrograms/l] around the day of the preovulatory estradiol-17 beta peak, and increased again during the luteal phase. Following intravenous injection of estrone sulfate, serum estrone sulfate, estrone and estradiol-17 beta were measured. The conversion of estrone sulfate to estrone and/or estradiol-17 beta was very small during their transit in the general circulation.     

Biliary metabolites of estriol in the rat

Following the subcutaneous administration of estriol-6,7-³H to rats, biliary metabolites were identified and quantitated. Approximately 70% of the metabolites were excreted in the form of “glucosiduronate” conjugates. 3, 17β-Dihydroxy-2-methoxy-1,3,5(10)-estratrien-16-one was the major metabolite in this conjugate fraction. Significant amounts of 3,17β-dihydroxy-1,3,5(10)-estratrien-16-one and 2,3,17β-trihydroxy-1,3,5(10)-estratrien-16-one, as well as smaller quantities of 1,3,5(10)-estratriene-2,3,16α,17β-tetrol and 2-methoxy-1,3,5(10)-estratriene-3,16α, 17β-triol, were also found. In 17α-ethinylestradiol - treated animals, the rate of excretion of radioactivity and the proportion of 16-oxo-17β-ol metabolites found in the “glucosiduronate” fraction were reduced.     

Radioimmunoassay of 17-hydroxyprogesterone in serum with or without thin-layer chromatographic purification.

A radioimmunoassay for the measurement of 17-hydroxyprogesterone (17-OHP) is described. The antigen 11-desoxycortisol-21-hemisuccinate-bovine serum albumin has been used to produce two antisera of different antibody populations in the same animal. The thin-layer chromatographic system described can be used to separate all the cross-reacting steroids investigated from 17-OHP. A simplified method is also presented using preliminary solvent extraction only. The mean 17-OHP levels measured, using this method, were, for normal men, 0.86 +/- 0.19 ng/ml (SD), for normal females 0.30 +/- 0.19 ng/ml in the follicular phase and 1.72 +/- 0.18 ng/ml in the luteal phase of the menstrual cycle, and 0.45 +/- 0.17 ng/ml in normal postmenopausal women.     

Identification of radioactive 17beta-estradiol-17-beta-D-glucuronide and 17alpha-estradiol-17-beta-D-glucuronide in the urine of the domestic fowl after intramuscular injection of [4-14C]estrone.

Two previously uncharacterized radioactive estrogen conjugates, 17beta-estradiol-17-beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17beta-D-glucopyranosiduronate) and 17alpha-estradiol-17beta-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17alpha-yl-beta-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.     

Isotope tracer measures of meal fatty acid metabolism: reproducibility and effects of the menstrual cycle

Oxidation and adipose tissue uptake of dietary fat can be measured by adding fatty acid tracers to meals. These studies were conducted to measure between-study variability of these types of experiments and assess whether dietary fatty acids are handled differently in the follicular vs. luteal phase of the menstrual cycle. Healthy normal-weight men (n = 12) and women (n = 12) participated in these studies, which were block randomized to control for study order, isotope ([3H]triolein vs. [14C]triolein), and menstrual cycle. Energy expenditure (indirect calorimetry), meal fatty acid oxidation, and meal fatty acid uptake into upper body and lower body subcutaneous fat (biopsies) 24 h after the experimental meal were measured. A greater portion of meal fatty acids was stored in upper body subcutaneous adipose tissue (24 +/- 2 vs. 16 +/- 2%, P < 0.005) and lower body fat (12 +/- 1 vs. 7 +/- 1%, P < 0.005) in women than in men. Meal fatty acid oxidation (3H2O generation) was greater in men than in women (52 +/- 3 vs. 45 +/- 2%, P = 0.04). Leg adipose tissue uptake of meal fatty acids was 15 +/- 2% in the follicular phase of the menstrual cycle and 10 +/- 1% in the luteal phase (P = NS). Variance in meal fatty acid uptake was somewhat (P = NS) greater in women than in men, although menstrual cycle factors did not contribute significantly. We conclude that leg uptake of dietary fat is slightly more variable in women than in men, but that there are no major effects of menstrual cycle on meal fatty acid disposal.     

Long-term data on basic reproductive parameters and evaluation of endocrine,morphological, and behavioral measures for monitoring reproductive status in a group of semifree-ranging Tonkean macaques (Macaca tonkeana)

Cycle and gestation lengths, menstruation patterns, female genital swelling characteristics, and male-female consortship durations are reported in a semifree-ranging group of Tonkean macaques (Macaca tonkeana) studied over a 12 year period. In addition, profiles of urinary estrone conjugates (E1C) and immunoreactive pregnanediol glucuronide (PdG) throughout four complete menstrual cycles in two females and three full-term pregnancies are presented. Based on intermenstrual intervals, a mean cycle length of 37–41 days (n = 55 cycles in 10 females) was found. Gestation length averaged 173 days (n = 27 pregnancies in eight females). Measurement of PdG immunoreactivity in urine revealed a cyclic pattern with a 5–15-fold increase between follicular and luteal phase concentrations, suggesting that PdG is a reliable indicator of ovarian cyclicity and luteal function. In contrast to PdG, E1C excretion showed no clear pattern throughout the cycle; however, highest values of E1C were usually found shortly before the onset of the luteal phase PdG rise at the presumed time of ovulation. Levels of both hormones were elevated during the first half of gestation and showed a marked increase throughout the second half, with maximum E1C concentrations being up to 100-fold higher than nonpregnant levels. Consortships by the male and occurrence of female genital swelling were long lasting (on average 5–10 days and 13 days, respectively) and were restricted to the follicular phase of the cycle. The day of maximal swelling and day of detumescence as well as the end of male consortship were closely associated with the periovulatory period. Swellings and consortships were longer following lactational ammenorhea than for subsequent cycles. The evolutionary significance of the cyclical changes undergone by females upon their relations with males is discussed. © 1996 Wiley-Liss, Inc.     

Preparation of specific antisera to estrone-3-glucuronide and estriol-3-glucuronide

The preparation and antigenic properties of estrone-3-glucuronide- and estriol-3-glucuronide-bovine serum albumin conjugates 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, have been described. Antibodies raised against the two immunogens in the rabbit possessed high specificity to estrone-3-glucuronide and estriol-3-glucuronide, respectively, exhibiting little cross-reactivities with other estrogen conjugates and no cross-reactions with related steroids except for free estrogens, their 3-methyl ethers and 3-sulfates. The cross-reactive antibodies were eliminated by partial immunoadsorption on affinity chromatographic media using the estrone-3-methyl ether 17-(O-carboxymethyl)oxime- and estriol-3-methyl ether 16 (or 17)-hemisuccinate-aminohexyl Sepharose conjugates, respectively. The purified antisera exhibited no cross-reactivities with free estrogens and ring A conjugates of estrone and estriol.     

The metabolism of 17beta-estradiol-3-glucosiduronate and estrone-3-glucosiduronate in the rabbit.

[6, 7-3H]-17beta-Estradiol-3-glucosiduronate, [6, 7-3H]-estrone-3-glucosiduronate or [6, 7-3H]-estrone was administered intravenously into the rabbit, and analysis and identification of the urinary metabolites were carried out. In either case, the major urinary metabolite was found to be a diconjugate. The sequential enzymic hydrolysis indicated that this diconjugate was glucosiduronate-N-acetyglucosaminide of 17alpha-estradiol. From these results, the conversion of the estrogen glucosiduronate into a diconjugate was thought a rather universal phenomenon in the rabbit.     

Direct measurements of urinary estrone conjugates during the normal menstrual cycle of the gorilla (Gorilla gorilla)

A direct immunoassay for urinary estrone conjugates (estrone sulfate and estrone glucuronide) was used to assess the preovulatory estrogen rise in normal gorilla menstrual cycles. Immunoreactive estrone conjugates in samples concomitantly assessed for total estrogen immunoreactivity reflected similar profiles throughout the cycle; however, the speed and resolution of the direct assay for conjugates indicate this method to be more accurate in monitoring ovulation than the measurement of total immunoreactive estrogens. In a single conceptive ovarian cycle, urinary estrone conjugate continued to rise in the luteal phase, indicating that this test may also be useful for detecting early pregnancy. The application of this technique provides clear profile of ovarian function in gorillas as well as in other primate species.     

Sex hormones correlated with sex skin swelling and rectal temperature during the menstrual cycle of the pigtail macaque (Macaca nemestrina).

Daily measurement of serum luteinizing hormone, estradiol-17beta, and progesterone were made during the menstrual cycle in nine pigtail macaques (Macaca nemestrina). All data were normalized to the day of the luteinizing hormone peak. Serum estradiol-17beta increased from approximately 100 pg/ml during the early follicular phase to 442 +/- 156 pg/ml during the maximum midcycle concomitant with the luteinizing hormone peak, and a small increase in serum estradiol-17beta was observed during the luteal phase coincident with the progesterone peak. Serum progesterone values increased slightly at the time of the luteinizing hormone peak and increased from 0.2-0.3 ng/ml during the midfollicular phase to peak levels of 8.3 +/- 1.75 ng/ml 9 days after the luteinizing hormone surge. Serum luteinizing hormone remained low and relatively constant throughout the early and midcycle, then sharply increased approximately four-fold to peak values of 6.25 +/- 0.9 ng/ml. Sex skin swelling increased slowly during the follicular phase and declined slowly throughout the early luteal phase. Rectal temperature did not change significantly throughout the menstrual cycle. The similarity of plasma sex hormone changes during the menstrual cycle between women and the pigtail macaque suggested that this nonhuman primate should be a useful animal model for studying human reproduction.     

Sex differences in the urinary catecholamines.

Urinary excretion of dopamine, norepinephrine and epinephrine was measured in a group of adult men and women of comparable age during recumbency and then during stimulation by upright posture. Urinary norepinephrine was found to be significantly higher in women (30.3 +/- 4.4 ng/min/m2 B.S.) than in men (18.3 +/- 2.7 ng/min/m2 B.S.) during recumbency; there was no significant sex difference in dopamine and epinephrine excretion. There was no apparent trend indicating a difference in urinary catecholamine excretion during the follicular or luteal phase of the menstrual cycle. In response to upright posture, there was a significant decrease in the urinary dopamine-norepinephrine ratio in both sexes; the magnitude of the decrease was, however, significantly higher in men (-9.9 +/- 3.0) than in women (-2.05 +/- 0.72). The mechanisms of the sex differences in urinary catecholamine excretion are unknown. Clinical studies involving catecholamines have to take these sex differences into account.     

Intermediary metabolism of estriol in pregnancy

Estriol (E₃), the most abundant estrogen in pregnancy is produced predominantly in the placenta from androgen precursors of fetal origin. The estriol so formed is secreted efficiently into the maternal circulation where it is converted to 4 conjugates—estriol-3-sulfate (E₃-3S), estriol-16-glucosiduronate (E₃-16G), estriol-3-glucosiduronate (E₃-3G) and estriol-3-sulfate-16-glucosiduronate (E₃-SG). The order of renal clearances is E₃-16G > E₃-3G > E₃-3S ~ E₃-SG. Unconjugated E₃ and E₃-3G differ from the other forms of estriol in that their removal from the blood compartment is essentially irreversible. E₃-3S, E₃-16G and E₃-SG undergo interconversions during enterohepatic circulation and eventual partial conversion to E₃-3G. Following delivery of the fetus and placenta, unconjugated E₃ is no longer detectable in the maternal serum within l–2h, whereas the concentrations of the conjugates decline more slowly, the rates being determined by the rates of renal clearance and enterohepatic interconversions. E₃-3G levels were dramatically elevated in a case of Group C polycystic kidney disease, providing evidence that this conjugate is indeed an end-product of estriol metabolism.     

Determination of estradiol-17-sulfate in human urine by a direct radioimmunoassay: urinary levels throughout the menstrual cycle

The measurement of urinary estradiol-17-sulfate concentration by direct radioimmunoassay was established. The urinary estradiol-17-sulfate levels measured by the radioimmunoassay correlated well with those determined by high-performance liquid chromatography equipped with electrochemical detector. Estradiol-17-sulfate concentrations in early morning urine of six healthy adult men was 8.2 +/- 2.0 ng/mL, or 5.7 +/- 1.8 ng/mg creatinine. The urinary levels in women throughout the menstrual cycle showed a characteristic three-peak excretion pattern: the first and the second peaks appeared just after and three days before the urinary LH peak, and the third peak appeared a few days before menstruation.     

Steroid metabolism in human breast cancer cell lines

The metabolism of 1,2-³H-androstenedione was studied in 2 cell lines, MCF-7 (estrogen responsive) and BT-20 (estrogen nonresponsive) over 48 hrs. Water soluble and unconjugated metabolites were separated by solvent partition and the former was submitted to chromatography on Sephadex LH-20 and enzyme hydrolysis. The resulting unconjugated steroids were separated by paper chromatography and identities were established by reverse isotope dilution. The unconjugated steroids initially obtained were separated by chromatography and identified by reverse isotope dilution. About 70% of the androstenedione was metabolized by both cell lines. However, the respective conversions to conjugates by MCF-7 and BT-20 were 31% and 0.32%. In the former, glucosiduronates predominated (94%) and consisted of androsterone (55%), etiocholanolone (9.4%) and androstanediol (5α-androstane-3α,17β-diol) (9.3%). Androsterone comprised most of the unconjugated metabolites in both cell lines. Androstanediol was found in both cell lines, 2% in MCF-7 and 12% in BT-20. Testosterone, 5α-androstane-3,17-dione and 3β-hydroxy-5α-androstan-17-one were isolated only from MCF-7. The metabolism of ³H-estriol was studied in a similar way. Both cell lines produced about equal amounts of estriol-3-sulfate (9%) and a compound with properties of estriol-3-glucosiduronate (0.15 – 0.5%). The results worthy of emphasis are: 1. The far greater conjugation of androgens exhibited by the MCF-7 cell lines as compared to the BT-20 cell lines; 2. In MCF-7, the high conversion of androstenedione to etiocholanolone (glucosiduronate form), a metabolite reported to form only in liver and sebaceous cysts; 3. The possible formation in both cell lines of estriol-3-glucosiduronate, normally a metabolite of the intestine.