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

Validations of time-resolved fluoroimmunoassays for urinary estrone 3-glucuronide and pregnanediol 3-glucuronide James S. Kesner,* Edwin A. Knecht,* Edward F. Krieg, Jr,* Geoff Barnard, t Heikki J. Mikola,:~ Fortfine Kohen,§ Mohamed M. Gani[[ and John Coleyll *Division of Biomedical and Behavioral Science, National Institute for Occupational Safety and Health, Cincinnati, Ohio, USA; ~Diagnostics Research Unit, Ebenezer House, Bournemouth, United Kingdom; ~ Wallac Oy and Department of Chemistry, University of Turku, Turku, Finland; §Department of Hormone Research, The Weizmann Institute of Science, Rehovot, Israel; IIUnilever Research Colworth Laboratory, Colworth House, Sharnbrook, Bedford, United Kingdom Competitive time-resoivedfluoroimmunoassays (FIAs) were developedfor measuring 1,3,5( lO)-estratrien-3ol-17-one glucosiduronate ( estrone 3-glucuronMe, E I3G) and 5[3-Pregnane-3~,20~-diol 3-glucasiduronate (pregnanediol 3-glucuronide, Pd3G) in unextracted urine. The assays are specific, detect 0.98 ng Ej3G/mL and 0.035 #g Pd3G/mL, measure 102.8 + 2.0°A of EI3G 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 El 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 FI As 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 menstrualfunction in epidemiological, medical, and athletic populations. (Steroids 59:205-211, 1994). Keywm-ds:competitivefluorescenceimmunoassay;estrogen; progestin; women; menstrual cycle;europium

Introduction Measuring the principal metabolites of estradiol and progesterone in urine, 1,3,5(10)-estratrien-3-ol-17-one glucosiduronate (estrone 3-glucuronide, E13G ) and 5~.Pregnane-3~,20~-diol 3-glucosiduronate (pregnanediol 3-giucuronide, Pd3G), respectively, has proven to be useful in evaluating menstrual function in epidemiological, 1-6 medical, 7-11 and athletic populations. 12 Daily collection of urine is more convenient and non-invasive than serial sampling of blood, 4 and urine integrates the secretory episodes which complicate interpretation of blood sample measurements. 3'j°

Procedures have been described for measuring Ex3G and P d 3 G by RIA 1'2'7'9'12 and enzyme immunoassay (EIA). 6'1°'11 The present report describes competitive time-resolved fluoroimmunoassays (FIAs) for these steroid giucuronides. This procedure offers a precise and sensitive alternative to RIA and EIA, while avoiding the toxicological and logistical constraints inherent to radioisotopic reagents.

Experimental Subjects and samples

This work originated at the National Institute for Occupational Safety and Health (NIOSH). Mention of a trade name, proprietary product, or specificequipmentin this manuscriptdoes not constitutean endorsement,guarantee,or warranty by the NIOSH. Address reprint requeststo Dr James S. Kesner,Divisionof Biomedical and Behavioral Science, National Institute for Occupational Safety and Health, 4676 Columbia Parkway, Mall Stop C-23, Cincinnati, OH USA 45226-1998. ReceivedAugust 8, 1993; accepted September 13, 1993.

(~) 1994 Butterworth-Heinemann

Urine samples were collected from healthy men and non-pregnant women for me in validation studies. Firstmorning urine was collected daily by 10 women for a total of 19 menstrual cycles3.4 for determining urinary endocrine profiles. All aspects of the study protocol were reviewed and approved by the National Institute for Occupational Safety and Health Human Subject Review Board. All donating subjects provided signed consent.

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Reagents Competitive double antibody FIAs were developed for Ea3G and Pd3G. For both assays, 4#g/well rabbit anti-mouse immunoglobulin (Dako; Glostrup, Denmark; code no. Z 259) in 200 mmol/L sodium phosphate buffer (pH = 3.5) was coated onto polystyrene 96-well microtiter plates (Nunc; Roskilde, Denmark; MaxiSorb C12 Framed Plate; cat. no. 473709). Coated plates were sealed with tape and stored at 4 C for up to 10 days before use. The primary monoclonal antibody for EI3G was the 3 F I I clone; ~3 the ascitic fluid was added at a dilution of 1:10,000. The primary monoclonal antibody for Pd3G was the 2555.6 clone ~4 and was added at a dilution of 1.81/~g protein/mE Europium-labeled Ex3G and Pd3G were synthesized as described below and were diluted 1:6,000 and 1:1,000, respectively, in assay buffer. E~3G purified by recrystallization (Dr. Delwood Collins, Lexington, Kentucky, USA) and commercial Pd3G (Sigma Chemical Co., St. Louis, MO, USA; cat. no. P-3635) served as standards. Standard curves, plotted as percent tracer bound in the presence of unlabeled analyte divided by tracer bound in the absence of competitor (B/Bo) versus log standard concentrations, extend from 1.56 to 200 ng E13G/mL and 0.078 to 20.0/~g Pd3G/mL. Potency of the commercial Pd3G was 118.8% relative to the synthetic Pd3G (Dr. James Brown, Carlton, Victoria, Australia). The E13G standard stock was 1 mg/mL in 60% ethanol: 40% water; the working solution contained 0.012% ethanol. Pd3G standard stock was 1 mg/mL in 100% ethanol; the working solution contained 2% ethanol. Double demineralized water (DDM H 2 0 ) was used for all aspects of the assays. A wash solution ~5 and assay buffer described previously ~5 were used with slight modifications. Wash solution at pH 7.75 contained 5 mmol TRIS, 0.154 mol NaC1, 7.7 mmol sodium azide, and 50/~L Tween 20 per liter DDM H20. Assay buffer at pH 7.75 contained 250 mmol TRIS-HCI, 5g bovine serum albumin, 500rag bovine globulin, 20/~mol diethylenetriaminepentaacetic acid, 0.154 mol NaC1, 7.7 mmol sodium azide, and 100/~L Tween 40 per liter DDM HzO. Enhancement solution (DELFIA, Wallac Oy; Turku, Finland; cat. no. 1244-104) dissociated the europium from the labeled steroids, enabling the free europium to rapidly form a highly fluorescent chelate with the components of the enhancement solution, a5 The enhancement solution contained 1 g Triton X-100 surfactant, 50/~mol 2-naphthoyltrifluoroacetone, and 100 mmol acetic acid, and was adjusted to pH 3.2 with 6.8 mmol potassium hydrogen phthalate per liter DDM H20. is

Synthesis of Europium-labeled Ex3G and Pd3G E13G and Pd3G were synthesized using a modification of a previously described procedure? 5 Materials for organic syntheses were obtained from Aldrich Chemical Co. (Milwaukee, WI, USA) or E. Merck (Darmstadt, Germany); solvents, thin-layer chromatography (TLC) plates, silica for shortcolumn chromatography, and ion-exchange resin XAD-2 were obtained from E. Merck. Solvents were used as received. Nuclear magnetic resonance spectra of synthesized derivatives were recorded using a Jeol JNM-GX400 FT-NMR spectrometer (Tokyo, Japan). Purification of europium-labeled steroid derivatives was performed using a Trisacryl GF05 M column (IBF Biotechnics; Villeneuve-la-Garenne, France) with detection at 254 nm using a Uvicord ultraviolet monitor (Pharmacia LKB Biotechnology AB; Uppsala, Sweden). Methyl [2,3,4-tri-O-acetyl- 1-bromo- 1-deoxy-ct-D-glucopyran] uronate (Bromosugar) was synthesized from D-glucurono6,3-1actone, using base-catalyzed esterification, acid-catalyzed

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acetylation, and HBr in acetic acid for bromination, and purified by recrystallization. ~6 Pregnane-3,20-diol 20-acetate was synthesized from pregnane-3,20-diol using acetic anhydride for acetylation and KHCO 3 solution for partial deacetylation. The monoacetate was purified by short-column chromatography. The glucuronyl residue was coupled to pregnane-3,20-diol 20-acetate and estrone by the Koenigs-Knorr reaction in anhydrous toluene using finely powdered cadmium carbonate (Aldrich Cat. No. 22,950-4) as a catalyst? 5'~7 All the protecting groups were removed simultaneously using methanolic NaOH. The synthesized steroid glucuronides were purified by ion-exchange on XAD-2 resin ta and then further by TLC. The Pd3G and Ea3G were labeled with europium chelate of 4-amino-benzyldiethylenetriaminetetraacetic acid x9 using 1-[3-dimethyl amino propyl] ethylcarbodiimide as a condensation agent as described previously, z° The europiumlabeled steroid glucuronides were purified by TLC followed by a Trisacryl GF05 M column, z~

Apparatus A robotic sample processor (Model RSP-8051-ID; TECAN US, Inc.; Hillsborough, NC, USA) was used to rapidly pipette standards, samples, and urine control pools. DELFIA instruments (Wallac Oy) used to perform the FIAs included a time-resolved fluorometer (Model 1232), plate washer (cat. no. 1296-024), reagent dispenser and dispensing unit (cat. no. 1296-401 and 1296-403), and plate shaker (cat. no. 1296-002). The time-resolved fluorometer excites the reaction mixture in each microtiter plate well 1000 times during one second with 340 nm pulsed light. During the period between pulses, the sample fluorescence is integrated at 615 nm after a delay of 400 microseconds.

Procedure The assay procedure for the E~3G and Pd3G is modified from one described previously~S: 1) wash antibody-coated assay plates twice; 2) add 100 #L labeled steroid glucuronide to plates; 3) wait 5 min; 4) add 20/~L standard, sample, or control urine pool then 100 #L primary antibody; 5) incubate plates on shaker for 60 min; 6) wash plates 6 times, then add 200 ttL enhancement solution; 7) incubate plates for 5 min on shaker, then 10 min off shaker; and 8) then count. All procedures were conducted at 20-25C. Plates were washed with wash buffer.

Validations Specificity for the E13G 3FI 1 monoclonal antibody has been characterized previously. 14 Specificity for the Pd3G 2555.6 monoclonal antibody was determined by competitive EIA. Assays were performed in the presence of Pd3G competing with an amount of antibody that yields 60-70% maximal binding in the absence of steroid. Cross-reaction with other steroids is defined as the amount of Pd3G relative to heterologous steroid needed to reduce B/B o to 50%. The assay standard stock solutions were used for the analytical recovery. Spikes were prepared in assay buffer and then added as 10% of the assay sample volume. Thus, 1.50, 3.19, 12.6, and 49.6ng E13G/mL and 0.077, 0.312, 1.25, and 5.01 #g Pd3G/mL were added to each of five urine samples with low or undetectable amounts of endogenous steroid glucuronide or to assay buffer. Recovery in urine was calculated as a percentage of the spiked assay buffer. Curves generated from serially-diluted assay standards and five urine samples were tested for paralldism. Steroid

F/As to urinary EI3G and Pd3G: Kesner et al. glucuronide concentrations in the undiluted urine samples used for these studies ranged from 14.3 to 69.7 ng Et3G/mL and 1.1 to 6.2/zg Pd3G/mL. Samples were progressively diluted 1 + 1 (Et3G) or 1 +3 (Pd3G) with assay buffer. The effects of urinary pH and osmolality on E~3G and Pd3G measures were examined. Urine samples were adjusted to pH 4.5 using 12 mol/L HCI and to pH 9.0 using 10 mol/L NaOH. Five of these samples were used to test the interaction of pH and TRIS-HCI concentration (50 versus 250 mmol/L) in the assay buffer. Results with 250 mmol TRIS-HCI/L assay buffer were confirmed with 5 additional urine samples. Osmolality was augmented 500 and 1000 mOsm/kg in 10 urine samples using granular NaCI. Untreated urine samples served as the baseline. Ranges of pH and osmolality for untreated samples were 5.7-6.5 and 524-1084 mOsm/kg, respectively. Precision was determined using urine pools containing low, medium, and high concentrations of analytes. Pools were measured in duplicate at the front and back of each of 2-6 microtiter plates in 5 assays. Limits of detection were calculated as the smallest concentration of analyte for which there was 95% confidence of detection by the method and were based on 6 assays. 22 EI3G and Pd3G were measured in urine samples containing 0.52 mol/L glycerol 23 collected daily throughout 19 menstrual cycles. Values were divided by creatinine (CR) concentrations for standardization. 24 These urine samples had also been assayed for luteinizing hormone (LH), E~3G, and Pd3G by RIA 3'~ and LH and follicle stimulating hormone (FSH) by time-resolved immunofluorometric assays (IFMA). 25 Presence or absence of ovulation during these cycles was also evaluated by transvaginal ultrasonography. 3 Partial correlations between FIA and RIA determinations were calculated for E13G and Pd3G.

Statistical analyses EI3G and Pd3G concentrations were calculated using a 4-parameter logistic model 26 for each analyte. Regression analysis with a 4-parameter logistic model using ALLFIT was used to test parallelism between curves generated from preparations of standard, references, and samples) ~'27 Linear regression analysis was used to compare urinary FIA values with urinary and serum RIA values. Because time is required for circulating analytes to be excreted, linear regression analyses were also conducted between urinary FIA values and serum RIA values lagged by one day. Analysis of variance (ANOVA) for repeated measures was used to analyze recovery of mass and effects of pH and osmolality. Repeated measures were used to account for the multiple measures of mass, pH, or osmolality measured within each sample. Variance components were estimated using a mixed model ANOVA. Contrasts were performed in the case of significant main effects or interactions. Coefficients of variation were calculated using the estimated variance components and the urine pool means. Precision about the mean is expressed in the text as standard errors.

Results Cross-reactivity of the anti-Pd3G monoclonal antibody is presented in Table 1. (See Reference 14 for E13G cross-reactivity.) Mean recoveries for added urinary steroid glucuronides were 102.8 _+ 2.0% for E I 3 G and 93.6 + 2.9% for P d 3 G . Neither rate of recovery was significantly different from 100% (P = 0.23, P = 0.09, respectively). Recovery

Table 1

Percent cross-reactivity of 2555.6 anti-pregnanediol 3-glucuronide monoclonal antibody with various steroids % Cross-

Steroid

reactivity

5p- Pregnane-3a,20=-diol 3-glucosiduronate m,1 5/~- Pregnane-3=,20=-diol 2

100.0 7.3

5~- Pregnane-3p,20=-diol 20=- Hydroxy-5/]-pregnan-3-one 20=- Hydroxypregn-4-en-3-one 3/~-Hydroxypregn-5-en-20-one3 20/~- Hydroxypregn-4-en- 3-one

6.0 1.9 1.0 < 1.0 < 1.0

Pregn-4-ene-3,20-dione 4

< 1.0

5=- Pregnane-3=,20=-diol 3=- Hydroxy-5p-pregnan-20-ona 5/~-Pregnane-3=,l 7=,20=-triol 3=-Hydroxyandrost-5-en- 17-one 3-sulfate5 3=-Hydroxyandmst-5-en-17-one 3-glucosidumnatee 3#- Hydroxyandrost-5 -en- 17 - one7 3=- Hydroxy-5#-androstan- 17-one 3-glucosiduronates 5/~-Pregnane-3,20-dionee

< 1.0 < 1.0 < 1.0 < 1.0 < 1.0 < 1.0 <0.1 <0.1

aTrivial names:

lpregnanediol 3-glucuronide; 2pregnanediol;

3pregnenolone; 4progesterone; Sdehydroandrosterone sulfate; edehydroandrosterone glucuronide; 7dehydroisoandrosterone; eetiocholan-3=-ol-17-one 3-glucuronide; epregnanadione.

did not differ between concentrations of added analyte (P = 0.36, P = 0.71, respectively). Slopes for the serial-dilution curves of standards and urine samples were not different for E13G (slopes = - 1 . 0 2 9 +0.027; P=0.48) and P d 3 G (slopes = - 0 . 9 6 0 -I- 0.034; P = 0.53; Figure 1). Adjusting urine samples to p H 4.5 tended to increase values (EI3G, P = 0.09; Pd3G, P = 0.10) when assay buffer contained 50 mmol TRIS-HC1 per liter. This trend was not observed (E~3G, P = 0 . 3 6 ; Pd3G, P = 0.31) after increasing the buffering capacity to 250retool TRIS-HC1 per liter (Figure 2). Steroid glucuronide measurements in urine samples, in which osmolality was augmented 500 and 1000 mOsm/kg, were 89% and 100% for E13G (P = 0.19) and 95% and 95% for P d 3 G (P = 0.27), respectively, of unadjusted samples. The limits of detection for the E13G and P d 3 G FIAs were 0.98 ng/mL and 0.035 #g/mL, respectively. Meas-

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Steroids, 1 9 9 4 , vol. 59, M a r c h

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Urinory pH Figure 2 Effect of adjusting pH and assay buffer on urinary E~3G and Pd3G concentrations. Each point is the mean of 5 samples expressed as the percent of values for unadjusted samples (Initial) assayed in buffer containing 250 mmol TRIS-HCI/L. Main effect of pH with 50 mmol TRIS-HCI/L: E13G, P = 0.09; Pd3G, P = 0.10; with 250mmol TRIS-HCI/L: E13G, P = 0 . 3 6 ; Pd3G, P=0.31. Results with 250 mmol TRIS-HCI/L assay buffer were confirmed with 5 additional urine samples. Initial pH levels ranged from 5.7-6.5.

ures of precision are presented in Table 2. Intra-plate drift, due to differences in incubation time, was absent for all three quality control urine pools for E13G (P = 0.80)• A small but significant front--* back drift exists for Pd3G (pool ~1, 0.38--*0.39 #g/mL; pool 4f2, 1.14-*l.15#g/mL; pool 4f3, 5.9 -, 6.2 #g/mL; P = 0.05). Figure 3 depicts mean profiles of urinary Et3G and Pd3G measured by FIA during 13 normal ovulatory menstrual cycles from seven women. Also plotted are urinary LH and FSH. Steroid glucuronide measurements obtained by FIA were regressed on those obtained by RIA and on serum steroid levels for the 13 normal cycles and 6 atypical menstrual cycles (Table 3). Table 2

Coefficients of variation (%)a

Mean

Within assay Plate

Repl.

Total

Estrone 3-glucuronide Urine Pools Low Medium High Mean

ng/mL 8.3 14.9 41.6

Urine Pools Low Medium High Mean

/,g/ml. 0.38 1.14 6.04

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a Coefficients of variation generated from duplicate determinations for urine pools positioned at the front and back of each of 2-6 microtiter plates in 5 assays. "Total" is the sum of all within assay variability; "Repl.'" = replicates.

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Steroids, 1994, vol. 59, March

6

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Figure 3 Mean urinary endocrine profiles during 13 normal ovulatory mentrual cycles (n = 12 for FSH) from 7 women. E13G, Pd3G, FSH, and LH were measured by FIA or IFMA ( O - ~ - - - Q ) ; E13G, Pd3G, and LH were also measured by RIA (O O). For Pd3G and LH, the ordinate scales are different for IFMA and RIA values. Table 3 Linear regression analyses for steroids measured in urine by FIA and urine and serum by RIA Partial R

Pregnanediol 3-glucuronide 10.1 7.5 2.7 6.8

/°

0 .............. : .............. 0 -15 -10 -5 0 5 10 5 Doyt From the Onut of the Urinary LH Surge

Precision of the E13G and Pd3G FIAs

Between assay

i

Slope

RIA urinary E13G RIA serum estradiol RIA serum estradiol/1 -day lag

FIA urinary E,3G 0.91 0.73 0.61 0.12 0.73 0.15

RIA urinary Pd3G RIA serum progesterone RIA serum progesterone/1 -day lag

FIA urinary Pd3G 0.83 2.59 0.89 0.79 0.71 0.52

P < 0.0001 for all linear regression analyses.

F/As to urinary E13G and Pd3G: Kesner et aL

Urinary FIA measurements correlated well with RIA measurements in urine and serum. However, urinary Pd3G values measured by FIA were about 2.5-fold greater than those measured by RIA. The luteal peak:follicular phase Pd3G ratio was 50% greater when measured by FIA compared to RIA. Figures 4-6 illustrate the value of these measurements to detect normal and abnormal menstrual cycles. While all three cycles exhibited normal intermenstrual intervals, one cycle conformed to normal ovulation (Figure 4), one to luteal phase deficiency (Figure 5), and one to anovulation (Figure 6).

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Days of the Menstrual Cycle Figure 4 Urinary endocrine profiles during a single ovulatory cycle. E~3G, Pd3G, FSH, and LH were measured by FIA or IFMA ((t--------O); E~3G, Pd3G, and LH were measured by RIA (O O). Note the virtual identity between the E13G values measured by FIA and RIA; and the similar Pd3G profiles measured by FIA and RIA. For Pd3G and LH, the ordinate scales are different for IFMA and RIA values.

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Figure 5 Urinary endocrine profiles during a single cycle. The short luteal phase and low progastin levels are consistent with luteal phase deficiency. E~3G, Pd3G, FSH, and LH were measured by FIA or IFMA ( @ - ~ - - O ) ; E13G, Pd3G, and LH were measured by RIA (C O ) . For Pd3G and LH, the ordinate scales are different for IFMA and RIA values.

Discussion

The FIAs described and validated herein are specific, sensitive, precise, and accurate. The urine matrix does not affect assay values since the buffering capacity of the assay buffer was increased to negate a tendency for low pH to inflate values. These FIAs provide alternatives to RIAs and EIAs which have been described previously. 1'2'6-12 RIAs suffer from the inherent problems associated with purchasing, handling, and disposing of radioisotopes. As a general rule, EIA results may be lightly more variable due to incubation variables that affect enzyme activity (e.g., pH, temperature, ionic

Steroids, 1994, vol. 59, March 209

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Figure 6 Urinary endocrine profiles during a single cycle. The absence of preovulatory E13G, LH, and FSH peaks and luteal phase Pd3G elevation indicates that this cycle was anovulatory. E13G, Pd3G, FSH, and LH were measured by FIA or IFMA ( ( p - ~ ) ; E13G, Pd3G, and LH were measured by RIA (O O). For Pd3G and LH, the ordinate scales are different for I FMA and RIA values.

strength) and variations in optical density within and between microtiter plates. 2s The tracers used in these FIAs are stable, inexpensive, and innocuous and the europium chelate used as label gives high specific activity and stable signal. 29 Our Pd3G values measured by FIA are highly correlated with those measured by RIA, but are about 2.5-fold higher. The reason for this quantitative difference is not clear. Pd3G standards for both assays, while separate, were obtained from the same source, used unpurified, and therefore would appear similar. Furthermore, the commercial Pd3G standard used for the FIA

210

Steroids, 1994, vol. 59, March

and a pure, synthetic Pd3G standard were of similar potency. Thus the standard does not appear to be the source of difference. Rather, the difference most likely reflects a difference in assay specificity. So while extensive cross-reactivity studies demonstrated the FIA Pd3G assay to be very specific, it is quite possible that the assay recognizes other, untested conjugated progesterone metabolites. Importantly, the FIA Pd3G values reported herein correlate well with urinary Pd3G and serum progesterone measured by RIA (3), begin to increase concurrent to the onset of the LH surge as expected, and indeed present a more robust increase from follicular phase baseline to the luteal phase peak than do the RIA values. Consequently, this assay affords an excellent measure of progesterone production. The E~3G values measured by us and others 3° by FIA are very similar to those measured by RIA. Labeling steroid glucuronides with europium may be more difficult than labeling, for instance, proteins or antibodies with iodine or europium. Although the steroid glucuronides used to generate the tracers were synthesized for the present studies, sufficiently pure preparations are commercially available. Synthesis of the europium chelate used to label the steroid glucuronides, and purification of the europium-labeled steroid glucuronides may be challenging for basic endocrinology laboratories. Drawbacks of our procedure to synthesize steroid glucuronides are the limited stability of the Bromosugar and the strictly anhydrous conditions needed. We used freshly prepared or recrystallized Bromosugar, dried toluene, and flame-dried glassware during the synthesis. Lot variation of cadmium carbonate catalyst also greatly affects the reaction. 3~ Purification of the synthesized steroid tracers by TLC alone yielded unsatisfactory purity. Additional purification was accomplished using a Trisacryl GF05 M column to remove the traces of byproducts, unlabeled steroids, and free europium chelate. 2 1 On the other hand, once these tracers are produced, they are stable for months ~5 or years (unpublished observation) when stored refrigerated. The current E~3G and Pd3G europium tracers have been used for 18 and 9 months to date, respectively, without loss of activity. Queries on availability of these tracers may be directed to one of us (HJM). In summary, FIAs were developed and validated for measuring urinary E~3G and Pd3G. These assays afford the advantages of non-radioisotopic procedures along with those of urine sample collection (convenience, non-invasiveness, integration of pulsatile secretion) for application to evaluate menstrual function in epidemiological, clinical, and sports populations.

Acknowledgments The authors acknowledge the technical assistance of Ms. Catherine M. Miller, the critical review of the manuscript by Mr. Dennis W. Lynch, and the generous donations of purified E~3G by Dr. Delwood Collins, University of

F/As to urinary E13G and Pd3G: Kesner et a/.

Kentucky Medical Center, Lexington, Kentucky, USA and synthetic Pd3G by Dr. James Brown, Royal Women's Hospital, Carlton, Victoria, Australia.

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