Regioselectivity of Phase II Metabolism of Luteolin and Quercetin by UDP-Glucuronosyl Transferases

Abstract

The regioselectivity of phase II conjugation of flavonoids is expected to be of importance for their biological activity. In the present study, the regioselectivity of phase II biotransformation of the model flavonoids luteolin and quercetin by UDP-glucuronosyltransferases was investigated. Identification of the metabolites formed in microsomal incubations with luteolin or quercetin was done using HPLC, LC-MS, and (1)H NMR. The results obtained demonstrate the major sites for glucuronidation to be the 7-, 3-, 3'-, or 4'-hydroxyl moiety. Using these unequivocal identifications, the regioselectivity of the glucuronidation of luteolin and quercetin by microsomal samples from different origin, i.e., rat and human intestine and liver, as well as by various individual human UDP-glucuronosyltransferase isoenzymes was characterized. The results obtained reveal that regioselectivity is dependent on the model flavonoid of interest, glucuronidation of luteolin and quercetin not following the same pattern, depending on the isoenzyme of UDP-glucuronosyltransferases (UGT) involved. Human UGT1A1, UGT1A8, and UGT1A9 were shown to be especially active in conjugation of both flavonoids, whereas UGT1A4 and UGT1A10 and the isoenzymes from the UGTB family, UGT2B7 and UGT2B15, were less efficient. Due to the different regioselectivity and activity displayed by the various UDP-glucuronosyltransferases, regioselectivity and rate of flavonoid conjugation varies with species and organ. Qualitative comparison of the regioselectivities of glucuronidation obtained with human intestine and liver microsomes to those obtained with human UGT isoenzymes indicates that, in human liver, especially UGT1A9 and, in intestine, UGT1A1 and UGT1A8 are involved in glucuronidation of quercetin and luteolin. Taking into account the fact that the anti-oxidant action as well as the pro-oxidant toxicity of these catechol-type flavonoids is especially related to their 3',4'-dihydroxyl moiety, it is of interest to note that the human intestine UGT's appear to be especially effective in conjugating this 3',4' catechol unit. This would imply that upon glucuronidation along the transport across the intestinal border, the flavonoids loose a significant part of these biological activities.

Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans

“…We found no evidence of kaempferol-7-glucuronide in any samples (lack of UV absorbance at a retention time equivalent to that of the authentic standard). Although there is some evidence to suggest that glucuroni-dation of flavonols at the 7-position does not occur in vivo (no quercetin-7-O-glucuronide was present in plasma of volunteers fed onions; Day et al, 2001), human liver cell-free extracts and microsomal preparations generate the 7-glucuronide of quercetin as the major product (Day et al, 2000a;Boersma et al, 2002), and human hepatocytes produce kaempferol-7-glucuronide when treated with kaempferol . The cause of these differences (in vivo compared with in vitro/ex vivo) are not clear, but may in part be due to differences in accessibility between cell-free systems and intact cells.…”

Effect of edible oils on quercetin, kaempferol and galangin transport and conjugation in the intestinal Caco-2/HT29-MTX co-culture model

“…The human sulphotransferases SULT1A1 and 1A3 are active on phenolics 23 and are both well expressed in HT29 cells 24 . On the other hand, Caco-2 cells highly express UDP-glucuronosyl transferases UGT1A1, 1A3, 1A6 and 2B7 25 and also UGT1A8 26 ; UGT1A1, 1A8 and 1A9 were particularly active on quercetin 18 . Both flavonol aglycone and substantial amounts of conjugates were transported across Caco-2/HT29-MTX co-cultures monolayers.…”

“…The statistical significance was set at the 5% level (SPSS version 18). 2 The mass spectrometric behaviour of all authentic standards and enzymatically synthesised metabolites of flavonols using negative-ion ESI are shown in Table 1 Since conjugation at the 5-position is reported to be very unlikely unless it is the only hydroxyl available 18 , Q3 is tentatively assigned as quercetin-4'-O-glucuronide. The availability of authentic standards confirmed peak K2 as kaempferol-3-O-glucuronide (20.9 min) and Q6 as quercetin-3'-O-sulphate.…”

“…Prior to the experiments, the culture medium was replaced by fresh medium for at least [12][13][14][15][16][17][18][19][20][21][22][23][24] hours. Transport experiments were carried out with transport buffer solution consisting of HBSS supplemented with 1.8 mM CaCl 2 (to maintain integrity of the tight junction) and 100 µM ascorbic acid (to minimise degradation of flavonols).…”

Flavonoid conjugates interact with organic anion transporters (OATs) and attenuate cytotoxicity of adefovir mediated by organic anion transporter 1 (OAT1/SLC22A6)

“…For the analysis of quercetin and their conjugates, elution was performed at 0.75 mL/min and gradient started at 30 % (B) and increased linearly to 50 % in 12 min, and then equilibrated at 30 % for 3 min. Quantification of quercetin metabolites was based on peak area of quercetin at 370 nm, assuming similar response factor for quercetin conjugates [30,31]. For the analysis of isoflavones and their conjugates, elution started at 20 % (B), increasing to 30 % in 5 min and to 70 % in 8 min, after which it returned to 20 % for 3 min at a flow rate of 0.75 mL/min.…”