Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestr ogens as tracers, was developed for the quantitative determination of enter olactone, genistein and daidzein in human urine. The aim was to create a me thod for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases. After the synthesis of the 5'-carboxymethoxy derivative of enterolactone an d 4'-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen wer e used in preparing the europium tracers. After the enzymatic hydrolysis, t he TR-FIA was carried out using the Victor 1420 multilabel counter. The met hod has sufficient sensitivity to measure the phytoestrogens at concentrati ons even below 5 nmol/l. The intra- and inter-assay coefficients of variati on, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively. We measured urinary enterolactone, genistein and daidzein in 215 samples fr om Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, < 0.1 and 0.6 and below 0.6 mu mol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than tho se measured by gas chromatography-mass spectrometry (GC-MS). However, the a ssay results by the present method showed strong correlation with those obt ained by GC-MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens. (C) 2000 Elsevier Science Ltd. All rig hts reserved.