Study of the fate of phyto-oestrogens in the sheep by determination of isoflavones and coumestrol in the plasma and adipose tissue

Abstract

A method is presented for the specific determination in plasma or adipose tissue of sheep of the phyto-oestrogens genistein (G), biochanin A (BA), formononetin (F), daidzein (D), pratensein, and coumestrol. The method is based on selective extraction, paper chromatography, spectrophotometry and fluorometry, and use of radioactive recovery standards. A modification including gas chromatography is also described. Intraruminal administration to sheep of the synthetic isoflavones G, BA, or F gave rise within 2½ hr to detectable blood levels of the compound infused. In addition, intraruminal administration of BA and F was followed by the appearance of G and D, respectively, in blood and fat depots. Such O-demethylation at the C4 position was also observed after intramuscular injection of BA or F, and in sheep grazing subterranean clover (Trifolium subterraneum L.) and red clover (T. pratense). Plasma levels of free G above 5 µg/100 ml (excluding circulating isoflavone conjugated as glucuronide) were associated with maximal uterine growth response in ovariectomized ewes, while levels of 1–5 µg/100 ml appeared to elicit a graded uterine response; free plasma F and D levels above 0.5 �g/100 ml seem to be required for detectable uterotrophic action in the 5-day assay used. G, BA, F, and D were present in the plasma of sheep grazing subterranean clover var. Yarloop in concentrations adequate to explain the observed uterine growth response, but in sheep grazing red clover, the plasma levels seemed too low to account fully for the oestrogenic action of this pasture. No free isoflavones were detected in plasma from sheep grazing subterranean clover var. Marrar, despite the high isoflavone content of the pasture.