Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites.

An imbalance in the estrogen metabolism has been associated with an increased risk of breast cancer development. Evaluation of the estrogen biotransformation capacity requires monitoring of various estrogen metabolites. Up to now, only some estrogen metabolites could be measured in urine. However, in order to offer tailor made nutritional support or therapies, a complete estrogen metabolite profile is required in order to identify specific deficiencies in this pathway for each patient individually. Here, we focused on this need to quantify as many as possible of the estrogen-related metabolites excreted in urine. The method was developed to quantify 27 estrogen-related metabolites in small urine quantities. This entailed sample clean-up with a multi-step solid phase extraction procedure, derivatisation of the metabolites in the less water-soluble fraction through dansylation, and analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The metabolites accurately quantified by the method devised included parent estrogens, hydroxylated and methylated forms, metabolites of the 16α-hydroxyestrogen pathway, sulphate and glucuronide conjugated forms, precursors and a related steroid hormone. This method was validated and enabled quantification in the high picograms and low nanograms per millilitre range. Finally, analyses of urine samples confirmed detection and quantification of each of the metabolites.

Comprehensive profiling of free and conjugated estrogens by capillary electrophoresis-time of flight/mass spectrometry.

The biological activity of estrogens is tightly regulated by regioselective phase I/II metabolic transformations that are critical to human health. Current methods for analysis of urinary estrogens are limited by complicated sample pretreatment and/or inadequate specificity for free estrogens and their glucuronide/sulfate conjugates that vary widely in their intrinsic polarity. In this work, direct speciation of intact estrogen conjugates and their regioisomers is demonstrated using capillary electrophoresis-time-of-flight/mass spectrometry (CE-TOF/MS) when using an alkaline buffer system with negative ion mode detection. This method allows for resolution of weakly acidic native estrogens, anionic estrogen conjugates and their positional isomers without significant matrix-induced ion suppression effects in human urine. Identification of unknown estrogen metabolites using CE-TOF/MS is supported by accurate mass together with their characteristic relative migration times, which can be predicted based on two intrinsic physicochemical properties of an ion. CE-TOF/MS offers a promising strategy for comprehensive profiling of estrogens and other classes of steroid conjugates that is needed for deeper insight into the etiology and treatment of chronic disorders associated with impaired estrogen metabolism.

The molecular etiology of breast cancer: evidence from biomarkers of risk.

Estrogens can become endogenous carcinogens via formation of catechol estrogen quinones, which react with DNA to form specific depurinating estrogen-DNA adducts. The mutations resulting from these adducts can lead to cell transformation and the initiation of breast cancer. Estrogen metabolites, conjugates and depurinating DNA adducts in urine samples from 46 healthy control women, 12 high-risk women and 17 women with breast cancer were analyzed. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of the ratios of depurinating DNA adducts to their respective estrogen metabolites and conjugates were significantly higher in high-risk women (p < 0.001) and women with breast cancer (p < 0.001) than in control subjects. The high-risk and breast cancer groups were not significantly different (p = 0.62). After adjusting for patient characteristics, these ratios were still significantly associated with health status. Thus, the depurinating estrogen-DNA adducts are possible biomarkers for early detection of breast cancer risk and response to preventive treatment.

Hydrophilic interaction liquid chromatography-tandem mass spectrometry determination of estrogen conjugates in human urine.

We report a hydrophilic interaction liquid chromatography (HILIC) separation with tandem mass spectrometry (MS) detection method for analysis of seven urinary estrogen conjugates. HILIC separation employing a mobile phase with high organic solvent content resulted in enhanced electrospray ionization efficiency and MS sensitivity compared with reversed-phase (RP) LC-MS methods. Solid-phase extraction (SPE) was used to further improve the limit of detection and to eliminate interferences for the analysis of urine samples. No hydrolysis or derivatization was required in the sample pretreatment. This SPE/HILIC-MS/MS method provided limits of quantification (LOQs at S/N = 10) for the seven conjugates ranging from 2 to 1000 pg/mL with only 1 mL of urine sample, representing an improvement of 1 order of magnitude over the RPLC tandem MS methods previously reported. This method provided a linear dynamic range of 3 orders of magnitude, recovery of 92-109%, intraday accuracy of 84-109%, intraday precision of 1-14%, interday accuracy of 80-111%, and interday precision of 1-22%. We have successfully applied this technique to determine the seven estrogen conjugates in urine samples of a pregnant woman and found unique concentration changes of six estrogen conjugates at different stages of pregnancy while the concentration of estriol-3-glucuronide (E3-3G) remained constant. We further studied the profiles of individual estrogen conjugates in breast cancer patients before and after treatment and found patient-dependent effects of aromatase inhibitor treatment on estrogen phase-II metabolism, which have not been reported previously. This study demonstrates the potential clinical application of the HILIC-MS/MS technique for sensitive monitoring of the changes of urinary estrogen conjugates in a clinical setting.

Urinary ovarian and gonadotropin hormone levels in premenopausal women with low bone mass.

We hypothesized that lower ovarian and gonadotropin hormone concentrations would be associated with lower levels of peak bone mineral density (BMD) in apparently normally menstruating women who did not exercise intensively and did not report anorexia or bulimia. This hypothesis was evaluated using a case-with-control study design (n = 65) which was nested within a population-based longitudinal study of peak bone mass (Michigan Bone Health Study) with annual assessment in women aged 25-45 years (n = 582). Cases were 31 premenopausal women with BMD of the lumbar spine, femoral neck, and total body less than the 10th percentile of the distribution, where controls were 34 premenopausal women with BMD between the 50th and 75th percentile. BMD was measured by dual-energy X-ray absorptiometry. In addition to their annual measurement, these 65 participants collected first-voided morning urine specimens daily through two consecutive menstrual cycles. The urine from alternating days of this collection was analyzed for estrone-3-glucuronide (E1G), pregnanediol glucuronide (PdG), testosterone, and follicle-stimulating hormone by radioimmunoassay and these values adjusted for daily creatinine excretion levels. Additionally, analyses of daily urine specimens for luteinizing hormone (uLH) was undertaken to better characterize the possible uLH surge. Cases had significantly lower amounts of E1G (p = 0.009) and PdG (p = 0.002) than did controls, whether amounts were characterized by a mean value, the highest value, or the area under the curve, and after statistically controlling for body size. Further, when B-splines were used to fit lines to the E1G and PdG data across the menstrual cycle, the 95% confidence intervals (CIs) about the line for the controls consistently excluded and excluded and exceeded the 95% confidence bands for the cases in the time frame associated with the luteal phase in ovulatory cycles. Likewise, 95% CIs for the LH surge in controls exceeded the fitted line for cases around the time associates with the LH surge. The cases and controls were not different according to dietary intake (energy, protein, calcium), family history of osteoporosis, reproductive characteristics (parity, age at menarche, age of first pregnancy), follicular phase serum hormone levels, calciotropic hormone levels, or by evidence of perimenopause. We conclude that these healthy, menstruating women with BMD at the lowest 10th percentile from a population-based study had significantly lower urinary sex steroid hormone levels during the luteal phase of menstrual cycles as compared with hormone levels in premenopausal women with BMD between the 50th and 75th percentile of the same population-based study, even after considering the role of body size. These data suggest that subclinical decreases in circulating gonadal steroids may impair the attainment and/or maintenance of bone mass in otherwise reproductively normal women.

Identification and measurement of urinary estrone, estradiol-17 beta, estriol, pregnanediol and androsterone during the menstrual cycle of the orangutan.

Urinary estrone, estradiol-17beta, estriol, pregnanediol and androsterone were identified and measured during 3 menstrual cycles in 2 female orangutans. In 2 of the cycles, the animals excreted 1-8 mug/day estrone, 0.5-6 mug/day estradiol-17beta, 1-8 mug/day estriol, 20-206 mug/day pregnanediol and 120-522 mug/day androsterone during the first half of the menstrual cycle. In the second half of the cycle, corresponding values were 3-21 mug/day estrone, 2-10 mug/day estradiol-17beta, 1-9 mug/day estriol, 54-800 mug/day pregnanediol and 90-1158 mug/day androsterone. In 1 cycle, the estrogen values for the second half were considerably higher, possibly due to the animal becoming pregnant just before this study commenced. The values for estrone and estradiol-17beta are similar to those found in the human and chimpanzee menstrual cycle. The values for estriol were lower than in the human but higher than in the chimpanzee. Levels for urinary pregnanediol and androsterone were significantly lower than in the human. Variations during the menstrual cycle for estrone were characterized by a midcycle peak followed by a second peak in the luteal phase. No definite pattern was apparent for estradiol-17beta or estriol. Both urinary pregnanediol and androsterone levels were low during the first half of the cycle, started to rise just after midcycle, and showed a peak during the second half of the menstrual cycle.

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.

Characterization of reproductive hormonal dynamics in the perimenopause.

Medical therapy for women in the perimenopausal period is controversial, in part due to varying degrees of ovarian hormone secretion characteristic of this time of life. To extend our understanding of the reproductive endocrine milieu of perimenopausal women, we studied 6 cycling women, aged 47 yr and older, for 6 months with daily collections of first morning voided urine. Five additional older reproductive aged (43-47 yr old) women were studied with daily urine and serum sampling for a single menstrual cycle; their urinary hormone data were combined with the former group for menstrual cycle comparisons. Urine was assayed for LH, FSH, estrone conjugates, and pregnanediol glucuronide and normalized for creatinine (Cr). Eleven midreproductive aged (19-38 yr old) normally cycling women, 5 women with well defined premature ovarian failure, and 5 women aged 54 yr and older who were at least 1 yr postmenopausal were used for comparison. Perimenopausal women had shorter follicular phases (11 +/- 2 days vs. 14 +/- 1 days; P = 0.031) and, hence, shorter menstrual cycles than midreproductive aged controls. FSH excretion in perimenopausal women was greater than that in younger women (range of means, 4-32 vs 3-7 IU/g Cr; P = 0.0005). LH secretion was overall greater than that in younger normal subjects (range of means, 1.4-6.8 vs. 1.1-4.2 IU/g Cr; P < 0.026). Overall mean estrone conjugate excretion was greater in the perimenopausal women compared to that in the younger women [76.9 ng/mg Cr (range, 13.1-135) vs. 40.7 ng/mg Cr (range, 22.8-60.3); P = 0.023] and was similarly elevated in both follicular and luteal phases. Luteal phase pregnanediol excretion was diminished in the perimenopausal women compared to that in younger normal subjects (range for integrated pregnanediol, 1.0-8.4 vs. 1.6-12.7 microg/mg Cr/luteal phase; P = 0.015). Compared to postmenopausal women, perimenopausal women had more overall estrone excretion (2.5-6.2 ng/mg Cr in postmenopausal women; P = 0.02) and lower mean FSH (range of means for postmenopause, 24-85 IU/g Cr; P = 0.017) and LH (range for postmenopause, 4.3-14.8 IU/g Cr; P = 0.041). Compared to women with premature menopause, perimenopausal women again had lower FSH (range of means for premature menopause, 36-82 IU/g Cr; P = 0.0022), lower LH (range of means for premature menopause, 5.5-23.8 IU/g Cr; P = 0.0092), borderline higher mean estrone conjugates (range of means for premature menopause, 4-44 ng/mg Cr; P = 0.064), and far longer periods of ovarian activity (one to two cycles in prematurely menopausal women vs. three to six cycles in perimenopausal women). We conclude that altered ovarian function in the perimenopause can be observed as early as age 43 yr and include hyperestrogenism, hypergonadotropism, and decreased luteal phase progesterone excretion. These hormonal alterations may well be responsible for the increased gynecological morbidity that characterizes this period of life.

Studies on phenolic steriods in human subjects. XXI. renal metabolism, conjugation and excretion of four androgens: a comparison with estriol.

The present study was conducted in order to ascertain whether the human kidney can conjugate androgens to an extent similar to that of estriol (E3). Differently labeled androgens (testosterone, DHT and androstenedione) were injected simultaneously into a peripheral vein and the renal artery. The excretion of the radioactivity in the early urine collections served as an index of the ability of the kidney to conjugate and/or metabolize the various steroids administered. It was shown that the human kidney can conjugate testosterone to some extent as the 17-glucuronide of DHT, but to a much lesser degree that E3. Androstenedione was not conjugated by the kidney and the excretion DHT was paradoxically lower following its renal artery administration than following its peripheral injection. We interpret the latter to indicate that some kidney cells may contain receptors with very high affinity for DHT, thus leading to the lower excretion observed. The administration of androstenediol (into the renal artery) and E3 (peripherally) indicated that the diol was not conjugated as readily as E3. The results point to the ability of the kidney to conjugate testosterone to some extent; however, in no case was it able to conjugate an androgen with the same facility as it does E3.

Studies of phenolic steroids in human subjects. XIX. Renal conjugation of 15alpha-hydroxyestradiol-3-sulfate.

In order to elucidate further the role of the kidney in the conjugation and excretion of 15alpha-hydroxy-estrogens, we synthesized doubly and singly labeled 15alpha-hydroxyestradiol-3-sulfate (15alpha-OHE-S) and determined its urinary excretory pattern after administering differently labeled 15alpha-OHE2-S into the renal artery and concomitantly into a peripheral vein and by the infusion of doubly labeled 3H-15alpha-OHE2-S-35S into the renal artery. The excretion of radioactivity in the early urinary samples and the conjugate pattern in these urines were determined. About twice as much radioactivity was excreted in the early urine samples originating from the renal artery injection than from samples obtained following peripheral administration, indicating renal conjugation and excretion of the 15alpha-OHE2-S. The latter was preponderantly excreted as the S-NAG with only a small part (about 10%) of the administered sulfate being hydrolyzed to the aglycone. The latter was then either conjugated with N-acetylglucosamine (NAG) or glucuronide, probably also in the kidney. Most of the 15-alpha-OHE2-S could not be accounted for, possibly because of biliary excretion and enterohepatic circulation. The results obtained and those already published indicate that the human kidney is capable of conjugating 15alpha-OHE2, its NAG, and its sulfate conjugates.

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.

Lignan and isoflavonoid conjugates in human urine.

Lignans and isoflavonoids are two groups of diphenolic phytoestrogens of plant origin which have gained increasing interest because of their possible cancer protective properties. High excretion of these compounds occur in populations at low risk of breast, prostate and colon cancer consuming either high amounts of whole-grain (lignans and some isoflavonoids) or soy products (isoflavonoids and some lignans). We determined the pattern of conjugation of the phytoestrogens in four urine samples from vegetarian or semivegetarian women and in two samples from men. Seven compounds were investigated: enterodiol, enterolactone, matairesinol, diadzein, equol, genistein and O-desmethylangolensin. The fractions quantified are the free fraction, mono- and disulfate, as well as the mono-, di- and sulfoglucuronide fractions. For the fractionation and purification we used ion-exchange chromatography and the determination of the concentrations of each compound in all fractions was done by isotope dilution gas chromatography-mass spectrometry (GLC-MS) using deuterated internal standards of all diphenols. More than 60% of all compounds determined, occurred in the monoglucuronide fraction. Daidzein, enterodiol and equol are excreted to a relatively high extent as sulfoglucuronides and genistein as diglucuronide. We conclude that the general pattern of lignan and isoflavonoid conjugates in urine is similar to that of endogenous estrogens.

Use of Sep-pak cartridges for urinary steroid extraction: evaluation of the method for use prior to gas chromatographic analysis.

A method is described for the rapid and quantitative extraction of free and conjugated steroids from urine using Sep-pak C18 cartridges. The method was evaluated by determining the efficiency of recovery of (1) radiolabeled steroid glucuronides, (2) radiolabeled steroids freed by enzymatic hydrolysis, (3) steroid sulphates, (4) selected reference neutral free steroids of varied structure and polarity, and (5) oestrogens. In all cases the cartridges gave results equal to or better than those obtained by solvent or Amberlite XAD-2 extraction methods. Each urine extraction could be completed in 2-3 minutes and no further purification of extracts was required prior to derivatisation and gas chromatographic analysis.

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.

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.

The urinary metabolites of 17alpha-ethynylestradiol-9alpha,11xi-3H in women. Chromatographic profiling and indentification of ethynyl and non-ethynyl compounds.

Metabolites of 17alpha-ethynylestradiol (EE2) were obtained from human urine following ingestion of tritium-labeled EE2. Over 95% of the recovered activity was found as conjugated steroids and these were separated into four groups by chromatography of the urine extract on Sephadex LH-20 with chloroform-methanol (1/1) + 0.01M NaCl. The two major conjugate fractions appeared to be almost exclusively glucosiduronates. Enzymatic hydrolysis liberated at least ten different EE2 metabolites as shown by chromatography on Sephadex LH-20 with benzene-methanol (85/15). After additional separation and purification of these metabolites, positive identification was obtained for nine radioactive compounds by either gas liquid chromatography-mass spectrometry or reverse-isotope recrystallization. Five were ethynyl compounds: EE2, 2-MeO EE2, 16beta-OH EE2, 2-OH EE2 and 6alpha-OH EE2. The other four were de-ethynylated estrogens: estrone, estradiol-17beta, estriol, and 2-Me-O-estradiol-17beta.

PIP: Hysterectomized women were administered tritiated-17alpha-ethinyl estradiol either iv or orally and their urine collected and assayed for labeled-metabolites. Over 95% of the recovered activity was found as conjugated steroids. These were separated into 4 groups by Sephadex LH-20 chromatography with chloroform-methanol and .01M NaCl. 2 of the groups were almost entirely glucosiduronates. 17alpha-ethinyl estradiol itself was the major component in 3 of the fractions while 2-methoxy-17alpha-ethinyl estradiol was the major component of the 4th. Hydrolysis, chromatography with benzene-methanol, and further purification of each of the 4 fractions yielded similar metabolite patterns. Of the major compounds identified, 5 were ethinyl compounds: 17alpha-ethinyl estradiol, 2-methoxy-17alpha-ethinyl estradiol, 16 beta-hydroxy-17alpha-ethinyl estradiol, 2-hydroxy-17alpha-ethinyl estradiol, and 6 alpha-hydroxy-17alpha-eithinylestradiol; 4 were deethinylated estrogens: estrone, estreadiol-17beta, estriol, and 2-methoxy-estradiol-17 beta.

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide.

Competitive time-resolved fluoroimmunoassays (FIAs) were developed for measuring 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E(1)3G) and 5 beta-Pregnane-3 alpha,20 alpha-diol 3-glucosiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng E(1)3G/mL and 0.035 microgram Pd3G/mL, measure 102.8 +/- 2.0% of E(1)3G and 93.6 +/- 2.9% of Pd3G added, and exhibit between and within assay coefficients of variation, respectively, of 5.3% and 7.1% for E(1)3G and 6.8% and 7.8% for Pd3G. The urine matrix does not interfere with the assay. Urinary steroid glucuronide profiles measured by these FIAs conform to those of urinary steroid glucuronides and serum estradiol and progesterone measured by other established immunoassays. These FIAs afford the advantages of non-radioisotopic procedures and urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) to evaluate menstrual function in epidemiological, medical, and athletic populations.

Urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate. Significance of proportionate differences during the menstrual cycle.

The urinary excretion of estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate in men and throughout the menstrual cycle in women was measured by specific radioimmunoassay. In 9 men the mean +/- SE excretion of these conjugates was 15.9 +/- 1.4, 2.7 +/- 0.3, and 3.2 +/- 0.2 microgram/24 h respectively. In 15 women studied in the midfollicular phase (day 8) of the menstrual cycle, the excretion was 19.4 +/- 1.7, 2.9 +/- 0.2, and 5.4 +/- 1.3 micrograms/24 h. Excretion of each conjugate was significantly (P less than 0.01) elevated in the midluteal phase (day 22) to 41.9 +/- 3.9, 6.3 +/- 0.8, and 12.2 +/- 1.5 micrograms/24 h respectively (n = 14). The mean excretion of estriol-16 alpha-glucosiduronate was greater than that of 17 beta-estradiol-17-glucosiduronate in the luteal phase (P less than 0.05) but not in the follicular phase or in men (P greater than 0.05). The excretion of each of these specific conjugates measured throughout the menstrual cycle in 7 women was characterized by a sharp midcycle peak and a lower, broader luteal phase peak. The ratios of estriol-16 alpha-glucosiduronate to 17 beta-estradiol-17-glucosiduronate, estrone glucosiduronate to 17 beta-estradiol-17-glucosiduronate, and estriol-16 alpha-glucosiduronate to estrone glucosiduronate throughout the menstrual cycle were analyzed. When the mean ratio during the follicular phase was set at 1, a significant increase (P less than 0.01) occurred in the mean luteal phase ratio in each case: 1.00 +/- 0.03 to 1.66 +/- 0.09, 1.00 +/- 0.04 to 1.30 +/- 0.04, and 1.00 +/- 0.03 to 1.24 +/- 0.04 (mean +/- SE) respectively. The marked alteration in the proportions of these urinary estrogen conjugates may be due to altered metabolism of 17 beta-estradiol, but it more likely reflects a change in the pattern of estrogen secretion or production between the two phases of the menstrual cycle.

Plasma estrone-sulfate: assessment of reduced estrogen production during treatment of metastatic breast carcinoma.

Highly sensitive and specific estrogen assays are required to monitor the hormonal effects of surgical adrenalectomy or pharmacologic estrogen suppression in postmenopausal women with breast carcinoma. Because the levels of plasma estrone-sulfate are 10-fold higher than its unconjugated counterpart, we developed a radioimmunoassay for estrone-sulfate to quantitate the minimal estrogen concentrations expected under conditions of endocrine gland ablation. After establishing normal ranges, we compared plasma estrone- sulfate levels and urinary conjugated estrone basally and after surgical adrenalectomy or aminoglutethimide (estrogen suppression) therapy in 23 postmenopausal women with breast carcinoma. In response to either therapy, the plasma levels of estrone-sulfate fell by 63.5-79.2% (p less than .01) and conjugated urinary estrone by 85-94.5% (p less than .01) in all study days over a 12-week period. Correlation analyses yielded r values of 0.77-0.94 between conjugated plasma and urinary estrone concentrations in the surgical adrenalectomy and aminoglutethimide-treated groups, respectively. No significant differences in estrone-sulfate levels were observed when comparing spontaneously menopausal and surgically castrate patients.

Blood production rates of estrogens in women with differing ratios of urinary estrogen conjugates.

On the basis of the ratios of the estrogen conjugates in their urine (estriol/estrone + estradiol: E3/[E1+E2]), 19 women were divided into two groups: 9 women had ratios less than 0.6 and 10 women had ratios greater than 1.3. All women had measurements made of endogenous estrogens in their plasma by radioimmunoassay. They were then given constant infusions of 3H-estrone, 3H-estradiol and 14C-estriol during days 5-7 and days 20-22 of their cycles, and metabolic clearance rates (MCR) and blood production rates (PB) of estrone, estradiol and estriol were determined. Despite the wide disparity in their ratios of urinary estrogens, no differences could be found between the groups for the MCR's and PB's for all estrogens at either time of the cycle. The mean ratios of PB's (PB3/[PB2+PB1]) of estrone, estradiol and estriol ranged from 0.07 to 0.10 for each group during the cycle. The amounts of estriol entering the blood are small compared to the amounts of estrone and estradiol and do not correlate with the ratios of their urinary conjugates.