Time-resolved fluoroimmunoassay of plasma daidzein and genistein

We present a method for the determination of the phytoestrogens daidzein an d genistein in plasma (serum). These weakly estrogenic isoflavones occur in soybeans and in smaller amounts in some other beans and plants. It has bee n suggested that they may afford protection against prostate and breast can cer. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After synthesis of 4'-O-carboxymethyl-daidze in and 4'-O-carboxymethyl-genistein the compounds are coupled to bovine ser um albumin (BSA), then used as antigens to immunize rabbits. The tracers wi th the europium chelate are synthesized using the same 4'-O-derivative of t he isoflavones. After enzymatic hydrolysis and ether extraction the immunoa ssay is carried out using the VICTOR 1420 multilabel counter (Wallac Oy, Tu rku, Finland). The antisera cross-reacted to some extent with some isoflavo noids but not with flavonoids. The cross-reactivity seems not to influence the results, which were highly specific for both compounds. The correlation coefficients between the TR-FIA methods and the reference method based on isotope dilution gas chromatography-mass spectrometry were high; r-values w ere about 0.95-0.99 depending on concentration. The intra-assay coefficient s of variation (CV%) for daidzein and genistein at three different concentr ations vary 3.2-4.5 and 3.2-4.1, respectively. The inter-assay CVs vary 5.0 -6.3 and 4.5-5.3, respectively. The working ranges of the daidzein and geni stein assays are 1.0-216 and 1.7-370 nmol/l, respectively. The plasma value s (n = 80) of daidzein and genistein are very low in Finnish subjects (mean for daidzein, 3.8 +/- 6.8 and for genistein, 3.2 +/- 7.6 nmol/l; median va lue for daidzein 1.5 and for genistein 1.4 nmol/l). (C) 2000 Elsevier Scien ce Inc. All rights reserved.