Regioselectivity of human UDP-glucuronosyltransferase isozymes in flavonoid biotransformation by metal complexation and tandem mass spectrometry

Abstract

Based on reactions with five flavonoids, the regioselectivities of twelve human UDP-glucuronosyltransferase (UGT) isozymes were elucidated. The various flavonoid glucuronides were differentiated based on LC–MS/MS fragmentation patterns of [Co(II)(flavonoid-H)(4,7-diphenyl-1,10-phenanthroline)₂]⁺ complexes generated upon post-column complexation. Glucuronide distributions were evaluated to allow a systematic assessment of the regioselectivity of each isozyme. The various UGT enzymes, including eight UGT1A and four UGT2B, displayed a remarkable range of selectivities, both in terms of the positions of glucuronidation and relative reactivity with flavanones versus flavonols.

Introduction

Flavonoids, a class of polyphenols found in fruits and vegetables, have been shown not only to have anti-inflammatory properties but also to exhibit promising chemopreventive properties against cancer and cardiovascular disease [1], [2]. The positive bioactivities of flavonoids have been demonstrated in a variety of in vitro, in vivo, and case control studies [2], [3], [4]. In recent years the presumed chemopreventive properties have been under closer scrutiny due to the poor bioavailability of most unmodified flavonoid aglycones in the body [5], coupled with the growing documentation that flavonoids undergo extensive biotransformation [6], [7], [8]. The metabolism of flavonoids has a great impact on their absorption and distribution, and importantly biotransformation can substantially alter the chemical properties of the flavonoids, such as altering the bioactivities [9].

Most flavonoids are found naturally as glycosylated forms in fruits and vegetables. When ingested the flavonoid glycosides undergo deglycosylation by β-glucosidase or lactose phloridzin hydrolase enzymes primarily found in the small intestine [10]. After loss of their sugar side-chains, flavonoids are rapidly metabolized by mainly Phase II enzymes found in small intestine, kidneys, and most importantly the liver [10]. This process results in glucuronidation, sulfation, methylation, or hydroxylation depending on the nature of the interacting enzyme [11]. Any flavonoid compound metabolized or unmodified that is not absorbed prior to reaching the large intestine may be absorbed by microflora, a process leading to decomposition of the flavonoid by ring fission and causing the release of small phenolic acids that are excreted in the urine [10]. Since most of the flavonoids ingested are conjugated and consequently absorbed as conjugates, there has been increasing interest in understanding the formation, uptake, distribution, and chemopreventive properties of the conjugates. To facilitate such investigations, the development of sensitive analytical methods to characterize, identify and track the flavonoid conjugates is paramount.

Glucuronidation of flavonoids is carried out in the body by the UDP-glucuronosyltransferase (UGT) family of enzymes. These enzymes have been found in every major organ involved in digestion, as well as the kidneys and liver [11]. To date, nineteen different isomers of the UGT enzyme have been identified [12], which are categorized into three different subgroups (UGT1As, UGT2As and UG2Bs). There are nine isoforms of the UGT1A group and seven in the UGT2B group, and together they play a major role in Phase II metabolism. The role of UGT2A isoforms remains unknown [12]. UGT enzymes catalyze the addition of glucuronic acid at a hydroxyl group, carboxylic acid, sulfide group, amine, or in rare cases a methyl group [11]. Flavonoids possessing one to multiple hydroxyl groups may undergo O-glucuronidation at various positions when metabolized by UGT enzymes. However, the specific positions which are glucuronidated by each enzyme are still not fully established.

The structural characterization of flavonoids and their metabolites has proven to be a challenging task. All flavonoids share the same basic three-ring structure and may differ by the position of a single functional group, making their positive identification difficult by many analytical methods. Mass spectrometry has proven to be one of the most effective tools for identification of flavonoids, in large part due to the informative fragmentation patterns generated by collision induced dissociation (CID) upon application of MS/MS strategies [13], especially when coupled with HPLC to allow separation of complex mixtures of flavonoids [14], [15], [16], [17], [18], [19], [20]. We have extended the capabilities of MS/MS methods for differentiation of flavonoids by formation of complexes containing a flavonoid, a metal, and an auxiliary organic ligand [21]. These complexes, upon CID, give unique fragmentation patterns that allow confident identification and differentiation of flavonoids, even for isomers. We have evaluated a number of metal complexation approaches and shown their versatility [22], [23], [24], [25], [26], [27], [28], [29], including the adaptation of the methods for identification of metabolites in urine and plasma [30], [31], [32], [33]. More recently, we applied the metal complexation/MS/MS methodology to gain insight into the regioselectivity of the UGT1A1 enzyme with various flavonoids [34]. In this prior study, the products were confidently identified and the distributions of various glucuronidated products were quantified.

In this present study, we have expanded our investigation of the selectivity of glucuronidation of the twelve most common UGT enzymes (1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, 2B17) for five of the most commonly consumed flavonoids (hesperetin, isorhamnetin, kaempferol, naringenin, quercetin). (Fig. 1) While biotransformation of flavonoids has been an area of much interest [12], [35], [36], [37], [38], [39], [40], this is the first time the isomeric flavonoid glucuronide products of such a large array of enzymatic syntheses have been determined, thus providing detailed insight into the selectivities of the UGT isoenzymes. This systematic study provides benchmark data for assessment of UGT enzymatic regioselectivity and establishes predictive correlations of biotransformation upon consumption of flavonoids.