Regioselective Monosulfation and Disulfation of the Phytoestrogens Daidzein and Genistein by Human Liver Sulfotransferases

Abstract

Regioselective sulfation of the phytoestrogens daidzein (DZ, 7,4'-dihydroxyisoflavone) and genistein (GS, 5,7,4'-trihydroxyisoflavone) was investigated using human liver cytosol and purified recombinant human sulfotransferase (SULT) isoforms, SULT1A1, SULT1A3, SULT2A1, and SULT1E1. 7-Position-preferential sulfation of DZ and GS was observed in human hepatic cytosols from 3 male and 3 female subjects. Average ratios for 7- to 4'-sulfate formation were 4.5:1 from DZ and 8.4:1 from GS in these human liver cytosols. Apparent K(m) values for the 7- and 4'-sulfation of DZ and GS by these cytosols were similar and in a range from 0.46 to 0.66 microM. All recombinant human SULTs had activity for 7- and 4'-sulfation of these phytoestrogens except for 7-sulfating activity of SULT1A3. SULT1A1 and SULT1E1 exhibited much higher catalytic efficiency, k(cat)/K(m), for 7- and 4'-sulfation of these substrates than did the other two, SULT1A3 and SULT2A1. SULT1A1 showed K(m) values of 0.47 and 0.52 microM for the mono-sulfation of DZ and GS, respectively, which were very similar to those of human cytosol. The observed k(cat)/K(m) indicated that SULT1A1 catalyzed 7-sulfation of DZ and GS at rates 4.4- and 8.8-fold higher, respectively, than such 4'-sulfation. However, with SULT1E1, catalytic efficiency was very similar for the sulfation of both positions. These data strongly suggest that SULT1A1 plays a major role in monosulfation of the phytoestrogens and determines the regioselectivity of sulfation in human hepatic cytosol. A kinetic study for 7,4'-disulfate formation of DZ and GS from their 7- and 4'-monosulfates indicated that SULT1E1 most efficiently catalyzed both reactions among human SULTs.

Plasma Profiling of Intact Isoflavone Metabolites by High-Performance Liquid Chromatography and Mass Spectrometric Identification of Flavone Glycosides Daidzin and Genistin in Human Plasma after Administration of Kinako

“…The purities of D-7-G and G-7-G (chemically synthesized) and of D-4Ј-G and G-4Ј-G purified by preparative HPLC were determined to be more than 98% by HPLC. D-7-S, D-4Ј-S, G-7-S, and G-4Ј-S were previously synthesized in our laboratory (Nakano et al, 2004). A mixture of the 7-and 4Ј-glucuronide conjugates of Dein and Gein was also prepared on a micro scale using the microsomal recombinant human UGT1A1 according to the method reported by Doerge et al (2000).…”

“…Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, 4Ј, 5, dmd.aspetjournals.org 7-trihydroxyisoflavone) was purchased from Extrasynthese (Genay, France). Daidzein-7-sulfate (D-7-S), daidzein-4Ј-sulfate (D-4Ј-S), genistein-7-sulfate (G-7-S), and genistein-4Ј-sulfate (G-4Ј-S) were synthesized in our laboratory (Nakano et al, 2004). Phosphoric acid (99.999%) was purchased from Aldrich Chemical Co. (Milwaukee, WI).…”

Metabolism and Disposition of Isoflavone Conjugated Metabolites in Humans after Ingestion of Kinako

“…Daidzein-4Ј-glucuronide (D-4Ј-G) and genistein-4Ј-glucuronide (G-4Ј-G) were isolated from human urine (Hosoda et al, 2008). Daidzein-7-sulfate (D-7-S), daidzein-4Ј-sulfate (D-4Ј-S), D-4Ј,7-diS, genistein-7-sulfate (G-7-S), genistein-4Ј-sulfate (G-4Ј-S), and genistein-4Ј,7-disulfate (G-4Ј,7-diS) were synthesized in our laboratory (Nakano et al, 2004). Phosphoric acid (99.999%) was purchased from SigmaAldrich (St. Louis, MO).…”

Inhibitory Effects of Herbal Extracts on the Activity of Human Sulfotransferase Isoform Sulfotransferase 1A3 (SULT1A3)

“…13) Bacterial expression and purification of His-tagged recombinant human SULTs by one-step column chromatography were performed as described previously. 14) Fractions containing the homogenous enzyme protein, appearing as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were pooled as described previously.…”