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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.

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The extent of bioactivation of the herbal constituent estragole to its ultimate carcinogenic metabolite 1′-sulfooxyestragole depends on the relative levels of bioactivation and detoxification pathways. The present study investigated the kinetics of the metabolic reactions of both estragole and its proximate carcinogenic metabolite 1′-hydroxyestragole in humans in incubations with relevant tissue fractions. Based on the kinetic data obtained a physiologically based biokinetic (PBBK) model for estragole in human was defined to predict the relative extent of bioactivation and detoxification at different dose levels of estragole. The outcomes of the model were subsequently compared with those previously predicted by a PBBK model for estragole in male rat to evaluate the occurrence of species differences in metabolic activation. The results obtained reveal that formation of 1′-oxoestragole, which represents a minor metabolic route for 1′-hydroxyestragole in rat, is the main detoxification pathway of 1′-hydroxyestragole in humans. Due to a high level of this 1′-hydroxyestragole oxidation pathway in human liver, the predicted species differences in formation of 1′-sulfooxyestragole remain relatively low, with the predicted formation of 1′-sulfooxyestragole being twofold higher in human compared with male rat, even though the formation of its precursor 1′-hydroxyestragole was predicted to be fourfold higher in human. Overall, it is concluded that in spite of significant differences in the relative extent of different metabolic pathways between human and male rat there is a minor influence of species differences on the ultimate overall bioactivation of estragole to 1′-sulfooxyestragole.

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A structure-activity study on the quinone/quinone methide chemistry of a series of 3',4'-dihydroxyflavonoids was performed. Using the glutathione trapping method followed by HPLC, (1)H NMR, MALDI-TOF, and LC/MS analysis to identify the glutathionyl adducts, the chemical behavior of the quinones/quinone methides of the different flavonoids could be deduced. The nature and type of mono- and diglutathionyl adducts formed from quercetin, taxifolin, luteolin, fisetin, and 3,3',4'-trihydroxyflavone show how several structural elements influence the quinone/quinone methide chemistry of flavonoids. In line with previous findings, glutathionyl adduct formation for quercetin occurs at positions C6 and C8 of the A ring, due to the involvement of quinone methide-type intermediates. Elimination of the possibilities for efficient quinone methide formation by (i) the absence of the C3-OH group (luteolin), (ii) the absence of the C2=C3 double bond (taxifolin), or (iii) the absence of the C5-OH group (3,3',4'-trihydroxyflavone) results in glutathionyl adduct formation at the B ring due to involvement of the o-quinone isomer of the oxidized flavonoid. The extent of di- versus monoglutathionyl adduct formation was shown to depend on the ease of oxidation of the monoadduct as compared to the parent flavonoid. Finally, unexpected results obtained with fisetin provide new insight into the quinone/quinone methide chemistry of flavonoids. The regioselectivity and nature of the quinone adducts that formed appear to be dependent on pH. At pH values above the pK(a) for quinone protonation, glutathionyl adduct formation proceeds at the A or B ring following expected quinone/quinone methide isomerization patterns. However, decreasing the pH below this pK(a) results in a competing pathway in which glutathionyl adduct formation occurs in the C ring of the flavonoid, which is preceded by protonation of the quinone and accompanied by H(2)O adduct formation, also in the C ring of the flavonoid. All together, the data presented in this study confirm that quinone/quinone methide chemistry can be far from straightforward, but the study provides significant new data revealing an important pH dependence for the chemical behavior of this important class of electrophiles.

The periplasmic Fe-hydrogenase from Desulfovibrio vulguris (Hildenborough) contains three ironsulfur prosthetic groups : two putative electron transferring [4Fe-4S] ferredoxin-like cubanes (two Fclusters), and one putative Fe/S supercluster redox catalyst (one H-cluster). Combined elemental analysis by proton-induced X-ray emission, inductively coupled plasma mass spectrometry, instrumental neutron activation analysis, atomic absorption spectroscopy and colorimetry establishes that elements with Z > 21 (except for 12-15 Fe) are present in 0.001-0.1 mol/mol quantities, not correlating with activity. Isoelectric focussing revmIs the existence of multiple charge conformers with PI in the range 5 7-6.4. Repeated re-chromatography results in small amounts of enzyme of very high H,-production activity determined under standardized conditions (z 7000 Ujmg). The enzyme exists in two different catalytic forms: as isdated the protein is 'resting' and 02-insensitive; upon reduction the protein becomes active and 02-sensitive. EPR-monitored redox titrations have been carried out of both the resting and the activated enzyme. In the course of a reductive titration, the resting protein becomes activated and begins to produce molecular hydrogen at the expense of reduced titrant. Therefore, equiiibrium potentials are undefined, and previously reported apparent The iron-sulfur protein hydrogenase catalyzes the reversible activation of molecular hydrogen, a process involving the transfer of two electrons. Most presently known hydrogenases are also nickel proteins. The nickel ion is generally assumed to be the redox-active catalytic center [l, 21. A small subclass is formed by the Fe-hydrogenases; these enzymes presumably contain no other potentially redox active transition metals than iron [3]. By exclusion, this implies that the H2 activation is located on an iron-sulfur cluster. Redox catalysis is not Correspondence to W.