allopurinol and 2-5-dihydroxybenzoic-acid

Reactive oxygen species are implicated in the aetiology of a range of human diseases and there is increasing interest in their role in the development of cancer.. To develop a suitable method for the detection of reactive oxygen species produced by the faecal matrix.. A refined high performance liquid chromatography system for the detection of reactive oxygen species is described.. The method allows baseline separation of the products of hydroxyl radical attack on salicylic acid in the hypoxanthine/xanthine oxidase system, namely 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, and catechol. The increased efficiency and precision of the method has allowed a detailed evaluation of the dynamics of reactive oxygen species generation in the faecal matrix. The data show that the faecal matrix is capable of generating reactive oxygen species in abundance. This ability cannot be attributed to the bacteria present, but rather to a soluble component within the matrix. As yet, the nature of this soluble factor is not entirely clear but is likely to be a reducing agent.. The soluble nature of the promoting factor renders it amenable to absorption, and circumstances may exist in which either it comes into contact with either free or chelated iron in the colonocyte, leading to direct attack on cellular DNA, or else it initiates lipid peroxidation processes whereby membrane polyunsaturated fatty acids are attacked by reactive oxygen species propagating chain reactions leading to the generation of promutagenic lesions such as etheno based DNA adducts.

Topics: Ascorbic Acid; Bacteria; Catechols; Chromatography, High Pressure Liquid; Edetic Acid; Feces; Free Radical Scavengers; Gentisates; Humans; Hydroxybenzoates; Hydroxyl Radical; Hypoxanthine; Phytic Acid; Reactive Oxygen Species; Salicylic Acid; Xanthine Oxidase

The kinetics of xanthine oxidase has been investigated with the aim of addressing several outstanding questions concerning the reaction mechanism of the enzyme. Steady-state and rapid kinetic studies with the substrate 2,5-dihydroxybenzaldehyde demonstrated that (kcat/Km)app and kred/Kd exhibit comparable bell-shaped pH dependence with pKa values of 6.4 +/- 0.2 and 8.4 +/- 0.2, with the lower pKa assigned to an active-site residue of xanthine oxidase (possibly Glu-1261, by analogy to Glu-869 in the crystallographically known aldehyde oxidase from Desulfovibrio gigas) and the higher pKa to substrate. Early steps in the catalytic sequence have been investigated by following the reaction of the oxidized enzyme with a second aldehyde substrate, 2-aminopteridine-6-aldehyde. The absence of a well defined acid limb in this pH profile and other data indicate that this complex represents an Eox.S rather than Ered.P complex (i.e. no chemistry requiring the active-site base has taken place in forming the long wavelength-absorbing complex seen with this substrate). It appears that xanthine oxidase (and by inference, the closely related aldehyde oxidases) hydroxylates both aromatic heterocycles and aldehydes by a mechanism involving base-assisted catalysis. Single-turnover experiments following incorporation of 17O into the molybdenum center of the enzyme demonstrated that a single oxygen atom is incorporated at a site that gives rise to strong hyperfine coupling to the unpaired electron spin of the metal in the MoV oxidation state. By analogy to the hyperfine interactions seen in a homologous series of molybdenum model compounds, we conclude that this strongly coupled, catalytically labile site represents a metal-coordinated hydroxide rather than the Mo=O group and that this Mo-OH represents the oxygen that is incorporated into product in the course of catalysis.

Topics: Aminopterin; Animals; Benzaldehydes; Binding Sites; Catalysis; Cattle; Gentisates; Hydroxybenzoates; Kinetics; Milk; Oxidation-Reduction; Oxygen; Substrate Specificity; Xanthine Oxidase

The present study examined the effect of glibenclamide, an ATP-sensitive K+ (K(ATP)) channels antagonist, on the potassium chloride (KCl)-induced hydroxyl free radical (.OH) generation. Sodium salicylate in Ringer's solution (0.5 nmol/microl per min) was infused directly through a microdialysis probe to detect the generation of .OH as reflected by the formation of dihydroxybenzoic acid (DHBA) in the myocardium of anesthetized rat. The high concentration of KCl (70 mM) significantly increased the level of 2,3- and 2,5-DHBA by the action of depolarization by KCl. However, in the presence of glibenclamide (10 microM), KCl failed to increase the 2,3- and 2,5-DHBA formation. Moreover, when allopurinol (10 mg/kg), a xanthine oxidase inhibitor, was administered by i.v. injection, the elevation of DHBA was not observed. These results suggest that openings of cardiac K(ATP) channel by depolarization evokes .OH generation via xanthine oxidase reaction.

Topics: Allopurinol; Animals; Enzyme Inhibitors; Free Radicals; Gentisates; Glyburide; Heart; Hydroxybenzoates; Male; Microdialysis; Myocardium; Perfusion; Potassium Channel Blockers; Potassium Channels; Potassium Chloride; Rats; Rats, Wistar; Sodium Salicylate; Sucrose