Analysis of soy isoflavone conjugation in vitro and in human blood using liquid chromatography-mass spectrometry

Soybean products containing isoflavones are widely consumed in Western and Asian diets for putative health benefits, but adverse effects are also poss ible. The conjugated forms of isoflavones present in a soy nutritional supp lement (predominately acetyl glucosides) and in blood from two human volunt eers after consuming the supplement (7- and 4'-glucuronides and sulfates) w ere identified using liquid chromatography coupled with electrospray/tandem mass spectrometry. Circulating conjugates of genistein and daidzein were q uantified using selective enzymatic hydrolysis and deuterated internal stan dards for liquid chromatography-electrospray/mass spectrometry. The levels of isoflavone glucuronides were much greater than the corresponding sulfate s or aglycones. The substrate activities of genistein and daidzein were eva luated with recombinant human UDP glucuronosyl transferase (UGT) and sulfot ransferase (SULT) by using enzyme kinetics. The SULTs 1A1*2, 1E, and 2A1 ca talyzed formation of a single genistein sulfate; however, SULTs 1A2*1 and 1 A3 had no observed activity. None of the SULTs showed activity with daidzei n. Although several UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) catalyzed 7- a nd 4'-glucuronidation of genistein or daidzein, the UGT 1A10 isoform, which is found in human colon but not liver, was found to be specific for genist ein. Glucuronidation of only genistein was observed in human colon microsom es, although nearly equal activity was observed for daidzein in human liver and kidney microsomes. These findings suggest a prominent role for glucuro nidation of genistein in the intestine concomitant with absorption, althoug h hepatic glucuronidation of absorbed genistein and daidzein aglycones is a lso likely.