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 possible. The conjugated forms of isoflavones present in a soy nutritional supplement (predominately acetyl glucosides) and in blood from two human volunteers after consuming the supplement (7- and 4'-glucuronides and sulfates) were identified using liquid chromatography coupled with electrospray/tandem mass spectrometry. Circulating conjugates of genistein and daidzein were quantified using selective enzymatic hydrolysis and deuterated internal standards for liquid chromatography-electrospray/mass spectrometry. The levels of isoflavone glucuronides were much greater than the corresponding sulfates or aglycones. The substrate activities of genistein and daidzein were evaluated with recombinant human UDP glucuronosyl transferase (UGT) and sulfotransferase (SULT) by using enzyme kinetics. The SULTs 1A1*2, 1E, and 2A1 catalyzed formation of a single genistein sulfate; however, SULTs 1A2*1 and 1A3 had no observed activity. None of the SULTs showed activity with daidzein. Although several UGTs (1A1, 1A4, 1A6, 1A7, 1A9, and 1A10) catalyzed 7- and 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 genistein. Glucuronidation of only genistein was observed in human colon microsomes, although nearly equal activity was observed for daidzein in human liver and kidney microsomes. These findings suggest a prominent role for glucuronidation of genistein in the intestine concomitant with absorption, although hepatic glucuronidation of absorbed genistein and daidzein aglycones is also likely.
Doerge
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National Center for Toxicological Research, Jefferson, Arkansas 72079-9502, USA. ddoerge@nctr.fda.gov
Chang
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Churchwell
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Holder
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D000818:Animals; D000970:Antineoplastic Agents / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics; D002417:Cattle; D002853:Chromatography, Liquid; D005260:Female; D019833:Genistein / Q000097:blood / Q000378:metabolism / Q000493:pharmacokinetics; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000378:metabolism; D006027:Glycosides / Q000097:blood / Q000737:chemistry / Q000493:pharmacokinetics; D006801:Humans; D007529:Isoflavones / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics; D007700:Kinetics; D008297:Male; D013058:Mass Spectrometry; D008861:Microsomes / Q000378:metabolism; D013025:Glycine max / Q000737:chemistry; D013379:Substrate Specificity; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases / Q000378:metabolism
D000970:Antineoplastic Agents; D020719:Glucuronides; D006027:Glycosides; D007529:Isoflavones; D013431:Sulfates; C004742:daidzein; C086566:glycitein; D019833:Genistein; D014453:Glucuronosyltransferase; D015238:Sulfotransferases