RADIOIMMUNOASSAY DETERMINATION OF FORMONONETIN IN MURINE PLASMA AND MAMMARY GLANDULAR TISSUE

Formononetin is an isoflavonoid phytoestrogen found in animal fodder a nd in certain human foodstuffs, Since high consumption of formononetin has been implicated in infertility among ruminants and may relate to human breast cancer, we developed a radioimmunoassay method and applie d it to quantitate formononetin in murine plasma and mammary glandular tissue for animal model studies, The radioimmunoassay utilized an ant iserum raised in rabbits following immunization with formononetin-7-O- (carboxymethyl)ether coupled to bovine serum albumin, The tracer was a n [H-3]leucine derivative of formononetin synthesized by mixed anhydri de reaction between formononetin-7-O-(carboxymethyl)ether and [H-3]leu cine, The bound and free forms of formononetin were separated by addin g dextran-coated charcoal, This radioimmunoassay procedure enabled the quantification of 4 ng/ml of plasma or 50 pg/mg of mammary tissue, an d the antiserum showed no marked crossreaction with the reactants test ed, The reliability and reproducibility of the assay were demonstrated by intra-and inter-assay variation that was 6.5% and 11.9%, respectiv ely, This radioimmunoassay was compared with a high-performance liquid chromatographic method by determining concentrations of formononetin in ethanol extracts of red clover. Good correlation existed between th e radioimmunoassay and high-performance liquid chromatographic method (r = 0.980), but the radioimmunoassay values were on the average 5% hi gher than high-performance liquid chromatographic values, Furthermore, we used the radioimmunoassay to assess the pharmacokinetics of formon onetin in castrated female BALB/c mice after a single subcutaneous inj ection at 40 mg/kg dose. The sensitivity of the radioimmunoassay permi tted the detection of a prolonged elimination phase for formononetin i n both plasma and mammary glandular tissues, with mean elimination hal f-lives of 2 and 2.5 hr, respectively. A specific, sensitive, and fast radioimmunoassay procedure has been developed for the determination o f formononetin in murine plasma and mammary glandular tissue, We concl ude that the presented radioimmunoassay is a valid alternative method to the quantification of formononetin in biological fluids and/or plan ts.