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.

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.

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.

Urinary estrone conjugate and pregnanediol 3-glucuronide enzyme immunoassays for population research.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: These enzyme immunoassays can be used for the field conditions and population variation in hormone metabolite concentrations encountered in cross-cultural research.

Hormonal indicators of pregnancy in llamas and alpacas.

OBJECTIVE: To determine concentrations of estrone sulfate in serum, estrone sulfate in urine, relaxin in serum, and progesterone in serum in pregnant llamas and alpacas and to assess the potential of these hormones as pregnancy indicators. DESIGN: Prospective study. ANIMALS: 19 parous pregnant camelids (8 llamas and 11 alpacas). PROCEDURE: Estrone sulfate concentrations (in serum and in urine) and progesterone concentrations (in serum) were determined by enzyme immunoassay. Relaxin concentrations (in serum) were measured by radioimmunoassay. Serum and urine samples were collected daily for the first 30 days after breeding and, thereafter, once weekly until parturition. RESULTS: Estrone sulfate concentrations (in serum and in urine) peaked twice during pregnancy. The first took place 21 days after breeding and the second during the last month of pregnancy. Relaxin concentrations increased at 3 months of gestation to > 20 ng/mL, decreased at 5 months to 5 ng/mL, then increased from 8 months of pregnancy until parturition. Progesterone concentrations were detectable 4 days after breeding and were maintained > 2 ng/mL throughout pregnancy. CLINICAL IMPLICATIONS: The first increase in estrone sulfate concentration over basal values may indicate early interaction between mother and embryo, whereas the second increase may reflect fetal viability. Use of estrone sulfate concentration to diagnose pregnancy in llamas and alpacas is highly dependent on time of sampling. Relaxin concentration in serum is a superior indicator of pregnancy after the second month in the Ilama and alpaca because its existence is suggestive of interaction between mother and fetus, and concentrations are greater than basal values for a long period of pregnancy. Progesterone is not a direct product of the embryo or fetus and only indirectly confirms a diagnosis of pregnancy.

Serotonin metabolite excretion after postmenopausal estradiol therapy.

Serotonin, known for its beneficial action on mood and well-being, is also involved in cardiovascular functions. Thus the current work was undertaken to study the effect of hormone replacement therapy on serotonin turnover in postmenopausal women. Eighteen women received estradiol transdermally and 17 women estradiol valerate orally for 4 weeks. The serotonin metabolite 5-hydroxyindole acetic acid (5-HIAA) was determined in the urine before, and after 2 and 4 weeks' estradiol treatment. With both administration routes estradiol produced a significant increase in urinary 5-HIAA excretion, greatest with transdermal estradiol after 28 days of treatment. The enhancement of serotonin turnover may contribute not only to an improvement of mood and well-being but also to a cardioprotective effect of estradiol observed after hormone substitution in postmenopausal women.

Usefulness of early morning urine estrone-3-glucuronide assay in the monitoring ovarian secretory function in precocious puberty.

To evaluate the usefulness of the urinary estrone-3-glucuronide (EI-3-G) in the monitoring of the ovarian function in girls, we studied 11 girls with idiopathic central precocious puberty (ICPP) treated with LHRH analogs (LHRHa) for 2-5 years. Plasma LH, FSH, 17-beta-Estradiol (E2) levels, early morning urine (EMU) E1-3-G concentrations, were assessed before and 3, 6, 12 months after the onset of treatment. As expected, mean basal plasma LH, FSH and E2 concentrations, as well as mean basal EMU E1-3-G levels were significantly (p < 0.01) higher in patients studied than in normal, age matched, prepubertal controls. Three out of the 11 sexually advanced girls showed undetectable (< 15 pg/ml) basal plasma E2 values. On the contrary, in each patient studied, individual basal E1-3-G levels were higher than in normal age-matched prepubertal girls. LHRHa treatment significantly suppressed both basal and peak stimulated plasma gonadotropins, plasma E2 and EMU E1-3-G. However, while serum E2 levels were below the assay detection limit, not allowing to assess the degree of gonadal suppression, E1-3-G urinary concentrations were detectable in each subject treated, in the range of the normal prepubertal values. EMU E1-3-G determination seems to be a very sensitive and reliable approach to the monitoring of the effectiveness of LHRHa treatment in sexually advanced girls, allowing to detect very low estrogen concentrations and to achieve the desired ovarian suppression.

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.

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.

Determination of ovarian cycle in Goeldi's monkey (Callimico goeldii) via the measurement of steroids and peptides in plasma and urine.

Goeldi's monkey (Callimico goeldii) is an endangered species of New World primate. The present study provides the first description of the non-conception ovarian cycle in this species based on circulating reproductive steroid and peptide hormones. The data obtained were used to validate a non-invasive system for monitoring cyclicity based on urinary reproductive steroid metabolites. Nine sexually mature females were studied. In three females, matched blood and urine samples were collected once every 2-3 days for 90-120 days; in three other females, matched blood and urine samples were collected daily for 14-20 days for one peri-ovulatory period; and in the remaining three females, urine samples only were collected once every 1-3 days for 40-60 days. Plasma progesterone, oestrone-3-conjugates and bioactive LH were measured, in addition to urinary pregnanediol-3 alpha-glucuronide and oestrone-3-conjugates. The mean maximum concentration of plasma LH occurred 1-2 days before a significant rise in plasma progesterone, which was considered to occur 1 day after ovulation. On the basis of plasma progesterone titres, the duration of the ovarian cycle was estimated as 23.9 +/- 0.4 days (n = 9), and constituted a follicular phase of 10.7 +/- 0.3 days and a luteal phase of 13.5 +/- 0.3 days. Urinary pregnanediol-3 alpha-glucuronide demonstrated a high correlation with plasma progesterone (r = 0.8), and demonstrated a significant rise at the same time as plasma progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian and endocrine patterns associated with reproductive abnormalities in llamas and alpacas.

Ovarian activity was characterized in llamas and alpacas with hypoplastic ovaries, cystic follicles, or ovulatory failure. Ovarian follicular activity was determined by transrectal ultrasonography and urinary estrone sulfate analysis; pituitary response was determined by measurement of plasma concentrations of luteinizing hormone. Llamas and alpacas with hypoplastic ovaries had follicles < or = 6 mm (minimal ovulatory size, 7 mm). Cystic follicles, defined as > 12 mm, were maintained for a mean of 9 days. Follicular activity in the ovary contralateral to the cystic follicle tended to be suppressed while the cystic follicle was present. Ovarian response to copulation in females with cystic follicles varied according to the stage of the cystic follicle. Animals with ovulatory failure did not release adequate luteinizing hormone after copulation, even though they had mature and normal follicles (8 to 12 mm). The cystic follicle syndrome appears to be temporary, whereas the syndromes involving hypoplastic ovaries and ovulatory failure may permanently affect fertility.

Estrone 3-glucuronide chemiluminescence immunoassay (LIA) and 17beta estradiol radioimmunoassay (RIA) in the monitoring of superovulation for in vitro fertilization (IVF): correlation with follicular parameters and oocyte maturity.

Many works in the literature of the last years had reported that urinary approach to superovulation study is a suitable method to evaluate ovarian response to pharmacological stimulation. Before applying urinary determination of hormonal levels with a chemiluminescence immuno assay (LIA) method in early morning urine (EMU) samples, we had studied the correlation of RIA-LIA procedures with reference to follicular volumes at hCG day and to recovered oocyte maturity; in fact follicular growth and oocyte morphological features are the main parameters to evaluate a successful induced cycle. In our department the IVF cycles are daily monitored with RIA seric E2 and LIA E1-3G determination, besides ultrasound examination of follicular growth. We have studied E2 and E1-3G levels on the hCG administration day and their correlation with follicular areas and volumes; moreover, we have evaluated hormonal values on oocyte pick-up day with reference to recovered oocyte number and maturity. We have assumed as good timing for oocyte pick-up when more than 50% of recovered oocytes were of good quality (maturity score 4). We have observed that the highest pre ovulatory E1-3G value is consistent with the best timing for oocyte pick-up; it's possible to obtain a conversion coefficient follicular volumes and urinary E1-3G excretion. We have not found significant differences between plasmatic and urinary estrogenic parameters. It is important to remember the advantages connected by a not isotopic and not invasive method. The absence of discomfort for the patients may be a decisive factor to choose the monitoring method and LIA procedure may represent a valid alternative to RIA.

Features of female reproductive senescence in tamarins (Saguinus spp.), a New World primate.

Cyclical changes in concentration of plasma progesterone, urinary oestrone-conjugates and urinary luteinizing hormone (LH) were compared in young and older cotton-top tamarins (Saguinus oedipus) and saddle-backed tamarins (S. fuscicollis). A group of six young adult tamarin females (4-5 years of age) was sampled over eight periods of 6-8 weeks and six older (14-20 years of age) females were sampled over thirteen periods. There was hormonal evidence of ovulation in all of the sampling periods for young females; in five of thirteen periods, older females displayed no evidence of ovulation. Of the six older females, two were anovulatory in one sampling period, while one female displayed no evidence of ovulation in any of three sampling periods. Generally, females over 17 years of age either did not ovulate or displayed abnormally long periods of moderate concentrations of progesterone and oestrone conjugates. Basal concentrations of LH differed in individuals, but were not always higher in older females. In contrast to patterns of reproductive senescence in other primates, older, anovulatory tamarins displayed moderate concentrations of urinary oestrone conjugates (5-50 micrograms/mg creatinine) and plasma progesterone (8-19 ng/ml), both of which are hormones of probable luteal origin in these species. This result suggests continued production of steroids by the luteal cells of the prominent interstitial gland in reproductively senescent tamarins. This suggestion was reinforced by histological examination of the ovaries of four older, anovulatory females; few primary follicles were found. Three females had no normal antral follicles, but all females had large luteal masses. The presence of functional luteal cells in the older ovaries, which do not experience regular follicular development, may distinguish ovarian ageing in New World primates from that of Old World primates.

Long-term effects of porcine zonae pellucidae immunocontraception on ovarian function in feral horses (Equus caballus).

Ten feral mares free-roaming in Maryland, USA, were inoculated with porcine zonae pellucidae (PZP) protein before the breeding season for three consecutive years (1988-90). Ovarian function was monitored for 51 days during the peak of the breeding season after the third annual PZP inoculation, in seven of these mares and in four untreated control mares, by means of urinary oestrone conjugates and nonspecific progesterone metabolites. None of the ten inoculated mares became pregnant in 1990, compared with 55% of 20 control mares, which included two of the four monitored for ovarian function. Three of the untreated mares demonstrated apparent normal ovarian activity, characterized by preovulatory oestrogen peaks, concurrent progesterone nadirs at ovulation, breeding activity, and luteal-phase progesterone increases after ovulation. Two of the seven monitored PZP-treated mares demonstrated ovulatory cycles that did not result in conception. One was pregnant as a result of conception in 1989 and demonstrated a normal, late-gestation, endocrine profile. The remaining four PZP-treated mares revealed no evidence of ovulation, and urinary oestrogen concentrations were significantly depressed. The experiments indicated that (i) a third consecutive annual PZP booster inoculation is greater than 90% effective in preventing pregnancies in mares and (ii) three consecutive years of PZP treatment may interfere with normal ovarian function as shown by markedly depressed oestrogen secretion.

Antigen recognition in feral mares previously immunized with porcine zonae pellucidae.

Twenty-six free-roaming feral mares were immunized against porcine zonae pellucidae (PZP) between February and May, 1988. Eight sexually mature mares received 2 inoculations 2 weeks apart, and 18 mares received 3 inoculations at intervals of 2 and 4 weeks. Analysis of urinary oestrone conjugates (E1C) and non-specific progesterone metabolites (iPdG) in samples collected in October, 1988, revealed that none of the 18 mares that received 3 and only 1 of the 6 mares that received two inoculations were pregnant, whereas 3 of 6 sham-injected control mares and 5 of 11 untreated mares were pregnant. In February and March, 1989, 14 of the immunized mares that were not pregnant were given a single booster inoculation of PZP. Urinary E1C and iPdG analysis, from samples collected in August and October, 1989, revealed only a single pregnancy among the 14 boosted mares, whereas 33% of mares treated in 1988, but not given a booster inoculation in 1989, and 7 of 16 untreated controls, were pregnant. Foaling was reflected accurately by the urinary steroid metabolite analyses. These results suggest that once recognition of PZP antigen occurs in mares, subsequent annual inoculations will raise antibody titres sufficiently to inhibit fertility.

Metabolism of oestradiol-17 beta and progesterone in the white rhinoceros (Ceratotherium simum simum).

14C-Labelled oestradiol-17 beta and progesterone (50 mu Ci each) were injected i.v. into an adult female white rhinoceros and all urine and faeces collected separately over the next 4 days. The total recovery of injected label was 61%, 25% being present in the urine and 36% in the faeces. Of the radioactivity recovered, 69% was excreted on Day 2 of the collection period. Repeated extraction of samples obtained on Day 2 showed that, of the radioactivity in faeces, 92.4% was associated with unconjugated steroids whereas in the urine the proportion of conjugated and unconjugated steroids were similar (41.2% and 51.4% respectively). After phenolic separation of urinary steroids, HPLC followed by derivatization and recrystallization techniques identified progesterone as the major component of the unconjugated portion with 4-pregnen-20 alpha-ol-3-one as the principal metabolite in the conjugated fraction. Pregnanediol was not present. Oestrone appeared to be the most abundant oestrogen metabolite with smaller but significant amounts of oestradiol-17 beta and oestradiol-17 alpha in the unconjugated and conjugated fractions respectively. Small amounts of progesterone were found in the faecal extract in which the radioactivity consisted mainly of oestradiol-17 alpha and oestradiol-17 beta. The results have established the major excreted metabolites of oestradiol-17 beta and progesterone in the white rhinoceros and the development of more appropriate assay methods for monitoring ovarian function in African rhinoceroses should now be possible.

Changes in urinary and plasma oestrone sulphate concentrations after induction of foetal death in mares at 45 days of gestation.

Foetal death was induced in 10 Standardbred mares at day 45 of gestation by injecting 20 to 45 ml of hypertonic (24% W/V) saline into the conceptus at surgery. Ten mares underwent sham treatment and acted as controls. Blood and urine samples were collected every other day between days 30 and 45 post ovulation and at 0, 3 and 6 h relative to the infusion of saline in the treated mares, or sham treatment in control mares. Blood and urine samples were then collected daily between days 46 and 55 post ovulation. Urine oestrone sulphate (E1S) concentrations, measured by radioimmunoassay, increased between day 34 and day 36 of gestation in treated and control groups. In mares in which foetal death was induced, urine E1S concentrations declined post-operatively and were significantly (p less than .05) lower than controls by day 50. In plasma, E1S concentrations showed a major increase between days 36 and 40 in both groups. This was followed by a rapid decline after treatment in saline-injected mares, so that by day 48 plasma E1S concentrations in treated mares were significantly (P less than .05) lower than the controls. The results show that urinary and plasma E1S concentrations rise rapidly during early pregnancy, and are associated with a viable foetus after day 45 of pregnancy.

Source of oestrogen in early pregnancy in the mare.

Oestrogen secretion was determined by oestrogen conjugate (EC) analysis of urine in three groups of pregnant mares: Group I (N = 6), animals ovariectomized on Day 18-19 of gestation with pregnancy maintained by daily administration of an oral progestagen, altrenogest; Group II (N = 9), untreated, pregnant mares; Group III (N = 5) intact, pregnant mares treated daily with altrenogest. The mean EC concentrations in the ovariectomized mares in Group I increased in a constant linear manner from 17 ng/mg Cr on Day 20 to 291 ng/mg Cr on Day 70, with no apparent surge in oestrogen secretion around Day 39. Mean EC concentrations on Days 33, 39 and 44 were respectively 41, 48, and 73 ng/mg Cr. In the intact mares in Groups II and III (shown in parentheses), the mean urinary EC concentrations were 201 (171) ng/mg Cr between Days 20 and 33 of gestation, increased rapidly from 172 (77) ng/mg Cr on Day 33 to a peak of 1066 (895) ng/mg Cr on Day 39, followed by a decline to 637 (719) ng/mg Cr on Day 44. After Day 44, EC concentrations continued to increase in a linear manner to 1191 (842) ng/mg Cr on Day 70. The mean EC concentrations between Days 20 and 70 in Group I were significantly (P less than 0.05) lower than in mares in Groups II and III. EC concentrations in Group III mares were significantly lower (P less than 0.05) than in Group II mares between Days 28 and 34.(ABSTRACT TRUNCATED AT 250 WORDS)