Monoclonal Antibody-Based Time-Resolved ... - Semantic Scholar

Clinical Chemistry 53:4 748 –756 (2007)

Automation and Analytical Techniques

Monoclonal Antibody-Based Time-Resolved Fluorescence Immunoassays for Daidzein, Genistein, and Equol in Blood and Urine: Application to the Isoheart Intervention Study Duncan C.S. Talbot,1* Richard M. Ogborne,1 Tony Dadd,1 Herman Adlercreutz,4 Geoff Barnard,3 Susanne Bugel,5 Fortune Kohen,2 Sandra Marlin,1 Jerry Piron,1 Aedin Cassidy,6 and Jonathan Powell1

Background: Time-resolved fluorescence immunoassays (TR-FIAs) for phytoestrogens in biological samples are an alternative to mass spectrometric methods. These immunoassays were used to test urine and plasma samples from individuals in a dietary intervention trial aimed at determining the efficacy of dietary isoflavones in reducing the risk of coronary heart disease in postmenopausal women. Methods: We established murine monoclonal TR-FIA methods for daidzein, genistein, and equol. These assays could be performed manually or adapted to an automated analyzer for high throughput and increased accuracy. Analysis of urine was conducted on nonextracted samples. Blood analysis was performed on nonextracted samples for daidzein, whereas genistein and equol required diethyl-ether extraction. Results: Comparison of monoclonal TR-FIA, commercial polyclonal antibody– based TR-FIA, and gas chromatography–mass spectrometry showed correlations (r,

0.911– 0.994) across the concentration range observed in the Isoheart study (50 mg/day isoflavones). The concentrations of urinary daidzein and genistein observed during intervention demonstrated good compliance, and a corresponding increase in serum daidzein and genistein confirmed bioavailability of the isoflavonerich foods; 33 of the 117 volunteers (28.2%) were classified as equol producers on the basis of their urinary equol concentration (>936 nmol/L), and significant differences in the numbers of equol producers were observed between Berlin and the 3 other European cohorts studied. Conclusions: The validated monoclonal TR-FIA methods are applicable for use in large-scale human phytoestrogen intervention studies and can be used to monitor compliance, demonstrate bioavailability, and assess equol producer status. © 2007 American Association for Clinical Chemistry

Interest in the role of phytoestrogens in human health has increased during the past decade, with numerous dietary interventions and epidemiological studies investigating the potential health benefits of these compounds, particularly in relation to cardiovascular and bone health and cancer prevention (1–3 ). A recent consensus based on the available data concluded that although the use of soy products and soy isoflavones may be beneficial for bone, cardiovascular risk, and hot flushes, the benefits demonstrated in available studies are subtle. Adequately powered human intervention studies are needed to demonstrate the benefits of encapsulated isoflavones or isoflavone-fortified foods on clinical outcomes, such as incidence of heart disease and bone fractures (2 ). Largescale studies are hampered by the lack of availability of rapid, validated assays for assessment of these com-

1

Unilever Corporate Research, Sharnbrook, United Kingdom. Department of Biological Regulation, The Weizmann Institute of Science, Rehovot, Israel. 3 Centre for Veterinary Science, Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom. 4 Folkhalsan Research Center, Institute for Preventive Medicine, Nutrition and Cancer and Division of Clinical Chemistry, University of Helsinki, Helsinki, Finland. 5 Department of Human Nutrition, The Royal Veterinary and Agricultural University, Frederiksberg, Denmark. 6 School of Medicine, Health Policy & Practice, University of East Anglia, Norwich, United Kingdom. * Address correspondence to this author at: Unilever Corporate Research, Colworth Park, Sharnbrook, Bedfordshire MK44 1LQ, United Kingdom. Fax 44-0-1234-248010; e-mail [email protected]. Received June 20, 2006; accepted January 23, 2007. Previously published online at DOI: 10.1373/clinchem.2006.075077 2

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pounds; current validated assays rely on expensive and laborious methods such as gas chromatography–mass spectrometry (GC-MS),7 which is considered the reference method (4, 5 ) for assessment of these compounds and offers good sensitivity and excellent specificity, although HPLC (generally with coulometric detection) has gained acceptance because of reduction in the number of required purification steps (6, 7 ). Liquid chromatography– mass spectrometry is an excellent method, but the high costs of instrumentation limit its use in laboratories. These methods require sample extraction and have relatively low throughput. Immunoassays offer the potential advantages of small sample volume, direct sample testing, speed, simplicity, low cost, throughput, and availability of automated equipment. The development of rapid immunoassays for assessment of both parent compounds and intestinal metabolites has allowed biological effects in relation to isoflavone metabolism to be addressed in intervention trials, an area of particular interest because of increasing evidence that the clinical efficacy of isoflavones in humans depends on the production of a gut metabolite, equol. Equol is a microbial metabolite of daidzein (8 ), and its production in humans is highly variable; only 30%– 50% of any given population group can produce equol after ingestion of soy foods (9 –11 ). The capacity to produce equol is associated with circulating reproductive hormones (9, 12 ), inversely associated with mammographic density (13, 14 ), and positively associated with bone mineral density in response to isoflavone intervention (15, 16 ). Equol production may enhance the action of isoflavones because it has a lower affinity for serum proteins and a greater affinity for estrogen receptors and exhibits antioxidant activity greater than that of genistein and daidzein (17–19 ) As part of the European Union Isoheart project to investigate the potential cardioprotective effects of isolated isoflavones in healthy postmenopausal women, we developed and validated methods for the assessment of the isoflavone metabolites daidzein, genistein, and equol. When the Isoheart project commenced, commercial phytoestrogen assays were not available, but an appropriate monoclonal antibody against daidzein was available (20 ). We therefore prepared monoclonal antibodies to 2 other key isoflavone metabolites, genistein and equol. Our aim was to use validated phytoestrogen immunoassays to demonstrate bioavailability and monitor compliance within the Isoheart trial.

7 Nonstandard abbreviations: GC-MS, gas chromatography–mass spectrometry; BSA, bovine serum albumin; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay; TR-FIA, time-resolved fluorescence immunoassay; CI, confidence interval.

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Materials and Methods preparation of monoclonal antibodies Preparation of the daidzein monoclonal antibody has previously been described (20 ). Genistein– bovine serum albumin (BSA) immunoconjugates were synthesized by addition of 2.2 g/L genistein to 0.1 mol/L carbonate buffer (pH 9.6) and solubilized with 0.2 mol/L NaOH. Genistein was mixed with 10 g/L BSA in carbonate buffer before addition of formaldehyde (66 ␮mol/L in MES/ NaCl) and the reaction stirred at 38.2 °C for 24 h. Reaction products were purified through a PD10 column. The conjugate was eluted in phosphate buffered saline (7 mmol/L Na2HPO4, 3.7 mmol/L H2NaPO4, and 0.15 mol/L NaCl, pH 7.4), supplemented with 0.05% sodium azide, and analyzed by size-exclusion chromatography at the carrier protein maximum wavelength (280 nm) and the hapten maximum wavelength (262 nm). The ratio of incorporation was 18.7:1 (genistein:BSA). A genisteinovalbumin screening conjugate was prepared in the same manner as the genistein-BSA immunoconjugate, yielding an incorporation ratio of 1:8 to 1:12. Carboxymethyl equol immunoconjugate was synthesized by addition of 23.4 mg equol to a round bottomed flask containing 14 mL HPLCgrade dry n-propanol, followed by addition of 100 mg dry sodium that was cut into small pieces. After dissolution of sodium, 150 mg dry bromoacetic acid was added. The reaction was refluxed for 8.5 h and cooled, and then 1 mL water was added and the precipitate was collected. The precipitate was extracted into methanol (0.6 mL), insoluble products were discarded, and the filtrate was evaporated. The residue was chromatographed on silica gel 60. Elution with methanol:CHCl3:HOAc (89.7:10:0.3) yielded the desired monoaddition product of equol (1.5 mg), with an RF of 0.2 in the solvent system. In the same system equol showed an RF of 0.9. The electron mass spectrum of the carboxy derivative of equol showed a peak at 298.9 (M-1), corresponding to a compound with a formula of C17O5H15. Carboxymethyl equol was conjugated to KLH via a 2-step reaction. The reactive equol derivative (2 mg) was dissolved in 200 ␮L dry dioxane and 1 mg N-hydroxysuccinimide, and then 2 mg carbodiimide (2 mg) was added to the reaction mixture. After an overnight reaction at room temperature, urea was formed, indicating that an active ester of equol was formed. The active ester of equol (0.6 mg) was then used in the next step without further purification. KLH (5 mg) was dialyzed twice against phosphate-buffered saline (7mmol/L Na2HPO4, 3.7 mmol/L H2NaPO4, 0.15 mol/L NaCl, pH 7.4, pH 7.4). To this solution (0.9 mL) we added 50 ␮L of 1 mol/L sodium phosphate, followed by drop-wise addition of 100 ␮L of the active ester. The reaction mixture was stirred for 2 h at room temperature and then dialyzed against phosphatebuffered saline and stored at ⫺20 °C until use. We immunized 6- to 8-week-old female Balb/c mice subcutaneously and intraperitoneally with ⬃50 ␮g of each immunogen. Booster injections were given on day 14, and

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sera from these mice were tested on day 18. Best responders were boosted at 2-week intervals, and 3 days before splenectomy were given a final (intravenous and intraperitoneal for equol and genistein, respectively) boost with immunogen alone. Throughout the immunization schedule, the mice were treated humanely in accordance with strict U.K. Government Home Office regulations. Ethical review board meetings were held regularly, and the animal facilities were visited on a quarterly basis by U.K. Government Home Office inspectors. The mouse spleens were perfused with culture medium, and the resulting lymphocyte suspension (⬃107 cells per spleen) was combined with SP2/0 myeloma cells in a 1:1 ratio. After centrifugation at 200g for 5 min, the supernatant was discarded and the pellet was loosened. To this mixed pellet we added 1 mL 45% polyethylene glycol 3000 (Fluka AG). The cells were centrifuged gently for 3 min, and after a total contact time of 8 min, the polyethylene glycol was gradually diluted and removed by addition of culture medium and further centrifugation. The cells were finally suspended in culture medium containing hypoxanthine and azaserine and dispensed into 10 48-well plates. Hybridomas were visible at day 3, and a culture medium change was performed on day 7. On day 10 supernatants from all fusion wells were screened by enzyme immunoassay using genisteinovalbumin or equol-ovalbumin at 5 mg/L, with detection using antimouse antibody conjugated to alkaline phosphatase. Confirmation of positive clones was performed by inhibition assay using free genistein or equol, with doubling dilutions starting at 20 mg/L. Clones of interest were recloned to ensure monoclonality and then grown to a volume of 8 L in RPMI, supplemented with 5% donor horse serum using stirred fermentors or roller bottles, centrifuged and resuspended in serum-free medium, grown for 2–3 days, and allowed to die. After filtration and concentration, purification was by protein A chromatography. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm [concentration (g/L) ⫽ absorbance 280 nm/1.4]. The concentrations of the selected purified antibodies were as follows: daidzein, 0.78 g/L; equol, 6.8 g/L, serum genistein, 1.01 g/L; and urinary genistein, 2.98 g/L. Candidate antibodies were then evaluated in the dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) immunoassay format.

delfia immunoassay methods The Perkin-Elmer DELFIA time-resolved fluorescence immunoassay (TR-FIA) was selected because of the sensitivity, specificity, and wide linear range offered by this immunoassay compared with conventional enzyme immunoassays. The DELFIA reagents and instruments were obtained from Perkin-Elmer Life Sciences. These included yellow, low-fluorescence antimouse IgG– coated plates with nonremovable wells (Cat. No. AAAND-0003), assay

buffer (250 mL; Cat. No. 1244 –111), wash buffer (250 mL; Cat. No. 1244 –114), and enhancement solution (250 mL; Cat No. 1244 –105). We added 25 ␮L duplicate samples of urine (diluted in assay buffer, 1/25 daidzein, 1/5 genistein, or 1/10 equol), serum (daidzein), or extracted serum (genistein and equol), calibrator (Daidzein, Sigma D7802; Genistein, Sigma G6649; and Equol, Indofine Chemical Co. 02-1268), and QC samples to appropriate wells of an antimouse plate. QC material was prepared by diluting stock solutions of 3 known amounts of analyte in phosphatebuffered saline ⫹ 0.1% ovalbumin. These concentrations

Table 1. Assay performance. A. Intraassay CV was determined by analyzing 25 replicates of standards and determining the concentration using Multicalc. Concentration, nmol/L

Daidzein assay 31.4 157.2 786 Urinary genistein assay 231 925 3700 14 800 Serum genistein assay 92.5 462.5 2313 Equol 19.5 97.5 390 1950

Intraassay CV, %

4.9 3.0 2.2 13.4 8.8 3.9 5.3 8.3 4.0 2.6 6.7 2.9 5.0 4.6

B. Interassay CV was determined by analyzing QC samples in duplicate, 2 runs per day, for 20 days (n ⴝ 40) and determining the concentration using Multicalc. Concentration, nmol/L

Daidzein assay 15.7 94.3 2358 Urinary genistein assay 1480 7400 22 200 Serum genistein assay 55.5 1480 7400 Equol assay 9.75 58.5 975

Interassay CV, %

8.6 4.1 6.8 15.3 15.8 18.5 11.7 6.8 15.6 9.5 6.7 9.2

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were selected to appropriately represent the range of analyte likely to be encountered with each assay. These samples were termed QC low, medium, and high, respectively, and the low, medium, and high concentrations were as follows: for daidzein, 15.75, 63.6, and 2340 nmol/L (4, 24, and 600 ␮g/L); for equol, 9.25, 55.5, and 925 nmol/L (2.5, 15, and 250 ␮g/L); for serum genistein, 58.5, 1560, and 7800 nmol/L (15, 400, and 2000 ␮g/L); for urinary genistein, 1560, 7800, and 23 400 nmol/L (400, 2000, and 6000 ␮g/L). Standard dilutions were prepared from 1 g/L stocks in 96% ethanol in 0.1 mol/L phosphatebuffered saline, pH 7.2, containing 0.1% ovalbumin (Sigma A5378), and stored frozen at ⫺20 °C. Six standards were used per assay, including a zero. The highest and lowest standards were 3930 nmol/L (1000 ␮g/L) and 6.2 nmol/L (1.6 ␮g/L) for daidzein; 1950 nmol/L (500 ␮g/L) and 3.9 nmol/L (1.0 ␮g/L) for equol; 1156.25 nmol/L (312.5 ␮g/L) and 18.5 mol/L (5.0 ␮g/L) for serum genistein; and 44 400 nmol/L (12 000 ␮g/L) and 231.5 nmol/L (62.5 ␮g/L) for urinary genistein. To each well we added 100 ␮L tracer [daidzein 1/24 000 (daidzein-europium) genistein 1/4000 (genistein-ovalbumin-europium), and equol, 1/10 000 (equol-ovalbumin-europium)] diluted in assay buffer incorporating 0.4 U/L B-glucuronidase (Sigma G7396) and incubated the plates with shaking for 60 min at room temperature. The enzyme treatment is an integral part of the assay protocol. We then added 100 ␮L antibody (daidzein, 4E4 25 ␮g/L; urinary genistein, 6547:3 65 ␮g/L; serum genistein, 6066:1 200 ␮g/L; or equol, 6588:1 20 ␮g/L) diluted in assay buffer, to each well, after which the plate was incubated, with shaking, for an additional hour at room temperature. The plate was washed 6 times with 400 ␮L of wash buffer (wash concentrate diluted 1/25 in MilliQ water), 200 ␮L of enhancement solution was added, and the plate was shaken for a final 5 min before reading of the fluorescence. Assays were performed manually using a Perkin-Elmer DELFIA plate shaker (1296-003) and washer (1296-026) and were read on a Victor2 1420 multilabel counter or automated for high-throughput testing on an AutoDELFIA.

To assess the limit of detection and intraassay variability, we included a standard curve comprising 6 calibrators and 3 QC samples in each assay (in duplicate). Twentyfive replicates of each calibrator were run as samples on 3 occasions, and the mean concentration of each was determined (intraassay) using Multicalc (Perkin-Elmer). The limit of detection of the assays was defined as the concentration that was 3 SDs above the mean of the zero standards, giving a ⬎99% confidence value. The limit of quantification for each assay was determined as the concentration that was 10 SDs above the mean of the zero standards. To determine interassay CVs, low, medium, and high QC samples for each assay were analyzed in duplicate, on 2 analytical runs per day, for 20 days (n ⫽ 40), and the concentrations determined with Multicalc. Assay specificity was assessed by measuring cross-reactivity with a panel of relevant phytoestrogens dissolved in assay buffer. Cross-reactivity was defined as 100(X/Y)%, where X is the mass of homologous phytoestrogen and Y is the mass of heterologous phytoestrogen required to produce 50% inhibition of binding of the Europiumlabeled phytoestrogen.

extraction of blood samples before analysis by delfia genistein and equol immunoassay The extraction method was the diethyl ether method described previously (21 ), and was used in the commercial (Labmaster Ltd.) DELFIA immunoassay.

gc-ms and labmaster tr-fia determinations GC-MS and Labmaster TR-FIA analyses were performed at the laboratory of Herman Adlercreutz according to previously published methods (4, 5, 21–23 ).

the isoheart study and blood/urine samples for phytoestrogen analysis The Isoheart intervention study was a placebo-controlled, 2 ⫻ 8-week randomized crossover, with an 8-week washout period. Study participants consumed 2 cereal bars per day, which provided 50 mg/day isoflavones (genistein: daidzein ratio, 2:1), or the same quantity of cereal bars containing no added isoflavones (placebo). Twenty-four-

Table 2. Specificity of the monoclonal antibody based DELFIA phytoestrogen assays.a Cross-reactivity with

Apigenin Daidzein Dihydrodaidzein 17B-Estradiol Equol Genistein Glycitein Luteolin Quercitin a

Cross-reactivity, daidzein (clone 4E4), %

Cross-reactivity, urinary genistein (clone 6547:3), %

Cross-reactivity, serum genistein (clone 6066:1), %

Cross-reactivity, equol (clone 6588:1), %

⬍0.02 100 9.9 ⬍0.02 ⬍0.02 1.6 ⬍0.02 ⬍0.02 ⬍0.02

⬍2 12 ⬍2 ⬍2 ⬍2 100 ⬍2 ⬍2 ⬍2

⬍0.2 18.8 0.8 ⬍0.2 ⬍0.2 100 ⬍0.2 ⬍0.2 ⬍0.2

0.3 ⬍0.04 2 ⬍0.04 100 0.3 ⬍0.04 ⬍0.04 ⬍0.04

The listed compounds were tested in assay buffer for their cross-reactivity with the 4 selected monoclonal antibodies as described in Materials and Methods.

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Fig. 1. Correlation of Unilever TR-FIAs for the detection of urinary daidzein, genistein, and equol with GC-MS and Labmaster TR-FIA using Deming regression analysis as described in Materials and Methods. Slopes and intercepts with 95% CIs are given. Dotted line, 1:1 correlation; solid line, best fit.

hour urine samples were collected at the beginning and end of each arm of the study, and serum samples at weeks 0, 4, and 8 of each arm. The study design and protocol were approved by the University of Reading, INRAN Rome, DIFE Berlin, and the Municipal Ethical Committee of Copenhagen and Frederiksber. After receiving a detailed explanation of the study procedure, each participant gave written informed consent.

statistical analysis Method Validator (Philip Maquis, 1999; available at http://www.MultiQC.com) was used for analysis and

SAS for graphical presentation. Deming regression was used to fit straight lines to the data because, in contrast to ordinary linear regression, Deming regression realistically assumes that the measurements by both methods are subject to random errors. Linnet weighting was applied because it takes into account nonconstant analytical variances for both methods. Because of the large proportion of nonproducers in these data we did not use weighting for the urinary equol comparison with GC-MS. We used a jackknife algorithm to calculate 95% confidence intervals (CIs) for slopes and intercepts. Comparison between monoclonal TR-FIA and Labmaster TR-FIA was made


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Fig. 2. Correlation of Unilever TR-FIAs for detection of serum daidzein, genistein, and equol with GC-MS and Labmaster TR-FIA using Deming regression analysis as described in Materials and Methods. Slopes and intercepts with 95% CIs are given. Equol GC-MS values are omitted due to small numbers being available for analysis. Dotted line, 1:1 correlation; solid line, best fit.

with noncorrected Labmaster TR-FIA data (raw assay data before application of a correction factor to GC-MS units).

Results assay performance The limits of detection of the TR-FIA assays were determined to be 3.9 nmol/L (daidzein), 88.8 nmol/L (urinary genistein), 8.7 nmol/L (serum genistein), and 2.2 nmol/L (equol). The limits of quantification (LOQ) of the TR-FIA assays were 11.8 nmol/L (daidzein), 319.3 nmol/L (urinary genistein), 41.1 nmol/L (serum genistein), and 4.3

nmol/L (equol). The intra- and interassay CVs are summarized in Table 1. Assay specificity is summarized in Table 2, which shows the cross-reactivity of each monoclonal phytoestrogen antibody for a panel of similar compounds. Fig. 1 shows the correlation between the established urinary monoclonal TR-FIA assays, commercial (Labmaster Ltd.) polyclonal TR-FIAs, and gold-standard GC-MS determinations. Fig. 2 shows the correlation between the assay methods for comparison of nonextracted serum samples in the daidzein monoclonal TR-FIA assay and extracted serum samples in the monoclonal serum genistein and

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measured by monoclonal TR-FIA, for each volunteer in the Isoheart intervention study. Equol producers were defined as having a urinary equol concentration ⬎960 nmol/L (21 ). Volunteers with low urinary equol concentrations also showed low serum equol concentrations (⬍15 nmol/L). On the basis of their urinary equol concentration, 33 of the 117 volunteers (28.2%) were classified as equol producers. The Fisher exact test revealed a general imbalance between the proportions of equol producers in the different centers (P ⫽ 0.0057). The most obvious cause of this is the difference between Berlin (50% equol producers) and the other centers (19% equol producers, P ⫽ 0.0014). Although Rome (11% equol producers) appears to be different from Reading (26% equol producers) and Copenhagen (21% equol producers), no evidence for imbalance is observed between the 3 centers overall (P ⫽ 0.33), and there is no evidence for imbalance between Rome and Reading/Copenhagen combined (24% equol producers, P ⫽ 0.24; Table 3). Fig. 3. Equol producer status of Isoheart volunteers.

Discussion

We analyzed 8-week intervention urine and serum samples by AutoDELFIA assay as described in Materials and Methods. Producer status defined as urinary equol concentration ⬎960 nmol/L (solid vertical line) and serum equol concentration ⬎40 nmol/L (solid horizontal line).

equol assays with Labmaster and GC-MS results. Unfortunately, insufficient samples tested by GC-MS had high enough equol concentrations for a robust correlation to be established.

isoheart study: clinical analysis The validated assays were used to test urine and serum samples from the Isoheart intervention study. Mean (SE) urinary daidzein and genistein concentrations for the 117 volunteers on the study were 393 (45) nmol/L and 731 (52) nmol/L, respectively, during the placebo phase and 12038 (714) nmol/L and 13399 (631) nmol/L, respectively, during the supplemented phase of the trial. Mean (SE) serum daidzein and genistein concentrations rose from 38 (1) nmol/L and 9 (2) nmol/L during the placebo phase to 139 (5) nmol/L and 283 (21) nmol/L, respectively, during the intervention arm of the study. Fig. 3 shows plots of week 8 urinary equol concentration against week 8 extracted serum equol concentration,

Current methods for the measurement of daidzein, genistein, and equol in clinical trial samples rely predominantly on GC-MS, a labor-intensive and expensive technique. To increase throughput, we raised mouse monoclonal antibodies against these compounds, developed high-throughput AutoDELFIA assays, and validated them against GC-MS and a commercially available immunoassay reagent set (Labmaster). We subsequently used the assays to measure compliance, bioavailability, and equol production in the Isoheart trial. The monoclonal TR-FIA assays could be performed either manually or on the AutoDELFIA which, as well as offering improved precision, facilitated automated highthroughput testing of up to 12 plates (39 samples/plate) of bar-coded samples at a time. Alternatively, 4 plates (4 ⫻ 39 samples) could be tested for daidzein, genistein, and equol in 1 run. Because of the lower phytoestrogen concentrations measured in blood than in urine, serum (daidzein), or extracted serum/plasma (genistein and equol), undiluted samples were tested. The AutoDELFIA, however, enabled preparation of urinary dilutions from 1⁄2 to 1/100 depending on levels expected/observed in particular studies. The selected urinary genistein antibody

Table 3. Equol producer status by study center.a Isoheart Study Center, frequency (%) Equol producer status

Berlin, Germany

Copenhagen, Denmark

Reading, U.K.

Rome, Italy

Total number of producers or nonproducers

Nonproducer Producer Total number of study participants analyzed per study center

17 (50%) 17 (50%)b 34

22 (78.6%) 6 (21.4%) 28

20 (74.1%) 7 (25.9%) 27

25 (89.3%) 3 (10.7%) 28

84 (71.8%) 33 (28.2%) 117

a Urinary equol concentrations were analyzed by AutoDELFIA assay as described in Materials and Methods. Equol producer defined as urine equol concentration ⬎960 nmol/L. Upper number per cell represents number of study participants defined as producer or nonproducer per study center. Percentages are proportion of producers or nonproducers per study center. b Significantly different proportion of equol producers compared with other study centers (P ⫽ 0.0057).

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(6547:3), suitable for assaying intervention study samples at a single urine dilution, had a broader assay range but was less sensitive than the selected monoclonal antibody for blood genistein measurement (6066:1). The daidzein (4E4) and equol (6588:1) monoclonal antibodies were sensitive, exhibited broad assay range, and were used for both urine and blood testing. In the Isoheart intervention study, genistein analysis was performed on 1 of 5 urine samples, whereas urinary daidzein and equol analysis were performed on 1 of 25 and 1 of 10 urine samples, respectively. By incorporating B-glucuronidase in the DELFIA assay buffer (pH 7), this enzyme treatment could be performed as an integral part of the urinary daidzein, genistein, equol, and (nonextracted) serum daidzein assays. For blood genistein and equol, B-glucuronidase and sulfatase treatments were performed in pH 5 acetate buffer, an appropriate pH for both enzymes, before diethyl ether extraction (21, 22 ). Assay validation showed correlations ranging from r ⫽ 0.911 to r ⫽ 0.994 across the range of urine and serum intervention study samples with GC-MS and Labmaster TR-FIA. As previously observed (21, 23 ), higher urinary phytoestrogen concentrations were obtained by TR-FIA than GC-MS. This result could be due to cross-reactivity of the antibodies, although their specificity appeared generally good (Table 2). Losses due to the number of procedures involved in the GC-MS method (e.g., purification and derivatization, whereas TR-FIA used untreated urine) may also have contributed to losses, despite the application of a correction based on the yield of a radiolabel. Correlations between monoclonal TR-FIA and Labmaster TR-FIA were good for all 3 analytes (Figs. 1 and 2). The slope for equol was close to 1 for urine and serum. Daidzein, and to a greater extent genistein, however, were overreported in urine compared with the Labmaster TRFIA. The genistein monoclonal TR-FIA has a greater clinical range and higher limit of detection than the Labmaster TR-FIA assay and thus was selected for use in compliance monitoring. These characteristics may have led to a greater difference in the assay comparison results at higher urinary genistein concentrations. The slope of the correlation between monoclonal TR-FIA and Labmaster TR-FIA for the serum genistein assay suggests underrecovery, which may be attributable to the extraction procedure resulting in losses in the monoclonal serum genistein assay, although further work is required to confirm this. Comparison of the monoclonal and polyclonal TR-FIA results for extracted serum equol, however, showed equivalence when the same extracted samples were used as for serum genistein. The established correlation equations could be used to correct monoclonal TR-FIA values to GC-MS units, as previously described in reports on the use of the Labmaster polyclonal TR-FIA (21–23 ). Whereas the Labmaster correction factors were established with low urinary daidzein and genistein concentrations (23 ), the samples com-

pared here were across the intervention study range, facilitating robust correction across this concentration range. Urinary equol concentrations (in a 24-h collection) were considered to be a better indicator of producer status than serum equol concentrations, because the timings for cereal bar consumption and bleeding times were not standardized, and analysis required extraction, with associated losses. The plot of urinary equol concentrations clearly splits the volunteers into “producers” and “nonproducers.” If the urinary equol concentrations were converted to GC-MS units, only 6 borderline results (3 Berlin, 1 Copenhagen, and 2 Reading) would then fall below the 936 nmol/L cutoff and be considered as nonproducers. A statistically significant difference was noted in the number of equol producers in Berlin compared with the other study centers (P ⫽ 0.0057). This observation is worthy of further investigation and may reflect differences in dietary habits or antibiotic treatment (24 ). Work is ongoing to test this hypothesis by analysis of completed food questionnaires from the study volunteers.

The development of the genistein and equol DELFIA assays, assay validation, and testing of the bioavailability and intervention study samples was carried out within the EU project Isoheart no. QLK1-CT-2001-00221. We particularly acknowledge the other members of the Isoheart consortium, especially those involved in running the bioavailability and intervention studies at the 4 study centers (DIFE, Berlin, KVL, Copenhagen, Reading University, U.K., and INRAN, Rome). Reagent availability: Researchers interested in obtaining the genistein and equol antibodies should contact the corresponding author. Researchers interested in the daidzein antibody should contact F.K.

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