Regioselectivity of human UDP-glucuronosyl-transferase 1A1 in the synthesis of flavonoid glucuronides determined by metal complexation and tandem mass spectrometry

Abstract

A three-part tandem mass spectrometric strategy that entails MS n analysis and a post-column LC-MS cobalt complexation method is developed to identify flavonoid monoglucuronide metabolites synthesized using the 1A1 isozyme of human UDP-glucuronosyltransferase (UGT). Ten flavonoid aglycons were used as substrates, spanning the subclasses of flavones, flavonols, and flavanones. The products were characterized by LC-MS and LC-MS n , with post-column cobalt complexation employed to pinpoint the specific sites of conjugation. The dissociation of complexes of the form [Co(II) (flavonoid glucuronide Ϫ H) (4,7-diphenyl-1,10-phenanthroline) 2 ] ϩ allowed identification of the products and differentiation of isomers. The correlation between glycosylation site and elution order is used to provide additional structural confirmation. Flavonoids lacking a 3= hydroxyl group were glucuronidated only at position 7, while those containing this functionality also formed 3=-O-glucuronides and sometimes 4=-O-glucuronides, thus supporting the conclusion that the presence or absence of the 3=-OH group is the major determinant of the regioselectivity of glucuronidation. Moreover, the specific distribution of multiple glucuronide products ( The currently accepted paradigm involves the consumption of flavonoid glycosides in plant-based food products, deglucosylation in the small intestine by ␤-glucosidase or lactose phloridzin hydrolase, and rapid metabolism by Phase I and (especially) Phase II enzymes [1][2][3]. Glucuronidation and sulfation are important metabolic routes for most flavonoids, while methylation or hydroxylation may also occur depending on the structure of the molecule in question [2,3]. There has also been a report of glutathionerelated metabolites in human urine [4]. As a result of these rapid conjugation reactions, neither the original flavonoid glycosides (except anthocyanins) nor the aglycon forms (except catechins) are found in plasma [5][6][7][8]. Early reports of unmodified flavonoid glycosides circulating in the bloodstream [9 -11] were likely mistaken identifications of flavonoid glucuronides, which have similar chromatographic and ultraviolet (UV) spectroscopic characteristics [7,12]. Flavonoids that fail to be absorbed in the small intestine may be broken down by microflora in the large intestine [1][2][3]. This process may release the free aglycons, which can then be absorbed and metabolized, but mostly results in the release of small phenolic acids, which are expelled in the urine [1][2][3]. Quantitative in vivo studies generally show that only a small percentage of consumed flavonoid glycosides is recovered in urine as conjugated Phase II metabolites [13][14][15]. Walle et al. used 14 Clabeled quercetin to show that up to 81% of the administered dose ultimately is exhaled in the form of carbon dioxide [16]. There remains considerable interest in the conjugated metabolites as they may retain some of the bioactivity of the original molecules [5,17].In spite of breakthroughs in the field of flavonoid metabol...

Metabolites of puerarin identified by liquid chromatography tandem mass spectrometry: Similar metabolic profiles in liver and intestine of rats

“…It has been reported that 7-O-glucuronide was eluted prior to the elution of 4 -O-glucuronide during analysis by reversed-phase high performance liquid chromatography (HPLC) on a C18 or C8 column [15][16][17]. Our experiments demonstrated that the retention times for M1 and M2 were 3.9 and 5.0 min, respectively.…”

“…It has been reported that different conjugation sites of the glucuronide group results in different retention behaviors in HPLC analysis [15][16][17]. An elution rule was reported when separation of flavonoid monoglucuronide isomers was performed by reversed-phase HPLC, that is, the 4 -glucuronide isoflavones were retained for longer than their 7-isomers [15][16][17], regardless of the exact chromatographic method employed. In our experiments, the retention times for M1 and M2 were 3.9 and 5.0 min, respectively, so that presumably, M1 is puerarin-7-O-glucuronide and M2 is puerarin-4 -O-glucuronide.…”

Iron Chelating Activity of Gossypitrin Isolated from the Petals of Talipariti elatum Sw. (Fryxell) Malvaceae

“…This ability to chelate metals has been used to enhance the capabilities of MS; it has been used to assist the elucidation of flavonoid glucuronides (Davis et al, 2006;Davis et al, 2007) and various diglycosides (Pikulski et al, 2007). This is a result of the spectral changes observed when flavonoids are complexed with metals, giving rise to simpler yet more intense spectra (Satterfield & Brodbelt, 2000).…”

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

“…Previous studies reported that the Caco-2 cell model has the ability to digest Tween 80 in the absence of pancreatin to liberate oleic acid, which was then used by the cells to enhance the basolateral secretion of chylomicrons 31 . Further, co-administration of olive oil and Tween 80 increased the formation of chylomicrons in the rat 32 and quercetin was transported into lymph after being metabolised in the gastrointestinal mucosa of rats 33,34 . In humans, a suspension in pure soybean oil increased the bioavailability of coenzyme Q10 compared to lipid formulations that included Tween 80 and/or phosphatidylcholine as surfactant.…”