Xanthine oxidase/hypoxanthine

Iron-mediated generation of hydroxyl radical ( 0H) was monitored by the hypoxanthine-xanthine oxidase method as previously described (28). Formaldehyde produced by reaction of 0H with DMSO was determined spectrophotometrically by the Hantzsch reaction (29). 

Ascorbic Acid (AA). Experimental (SI, S7, S24) and clinical (B13, B17) studies have provided some evidence for the concept that oxidative stress is the common pathway for the initiation of AP (B14). The most abundant endogenous antioxidant in the aqueous phase is ascorbic acid (AA), a bioactive form of vitamin C, which scavenges oxygen-derived free radicals produced by activated neutrophils and the hypoxanthine-xanthine oxidase system (D12). Scott et al. 

D12. Dwenger, A., Funck, M., Lueken, B., Schweitzer, G., and Lehmann, U., Effect of ascorbic acid on neutrophil functions and hypoxanthine-xanthine oxidase-generated, oxygen derived radicals. Eur. J. Clin. Chem. Clin. Biochem. 30, 187-191 (1992). 

A complete hypoxanthine-xanthine oxidase system should give a final A q of about 0.65, corresponding to 150-200 nmol of hydroxyl-ated product. Formation of hydroxylated products can be inhibited almost completely by superoxide dismutase, catalase, or the iron chelator desferrioxamine (Richmond et al., 1981). 

Chemiluminescence (CL) was measured in triplicate at 23 °C under moderate agitation by use of Luminescence Reader BLR-201 (Aloka Co. Ltd Tokyo Japan). When O2/HPX/XOD was used as O2 generator, 500 pL of DIW, 100 pL of 10-fold concentrated PBS, 100 pL of 1.67 mM HPX solution, 100 pL of 1 pM MCLA solution, 100 pL of antioxidant solution were mixed in a glass test tube, and then the test tube was set in the Luminescence Reader. About 30 s after the start of CL measurement, 100 pL of 25 U/mL XOD solution was injected into the test tube through a rubber cap by a syringe. Here, HPX, XOD, DIW, and PBS denote hypoxanthine, xanthine oxidase, distilled and deionized water, and phosphate buffered saline, respectively. 

Next, the specificity of the probes for the detection of 2 was examined by reaction with H2O2, Oi (hypoxanthine-xanthine oxidase) and OH (Fenton reaction). As a result, f-MSVP provided CL signals of 0.0004, 0.1 and 10% of that for O2, respectively, which were excellent and comparable to /-MVP (0.008, 0.3 and 5%, respectively). 

To evaluate the sensitivity toward supraoxide under physiological conditions, the intensity of supraoxide-triggered chemiluminescence of la was measured in a neutral buffer with various coiKentrations of the probe and a fixed composition of hypoxanthine-xanthine oxidase systeiiL The results are shown in Fig. 2, in conqtaiison with CLA and luminol, which are comm ially available supox>xide probes. Compound la exhibited the concentration-dependent chemiluminescence intensity strongCT than luminol, although the luminescence response was lower titan that of CLA... 

Aruoma, O.I., Halliwell, B., and Dizdaroglu, M. (1989) Iron ion-dependent modification of bases in DNA by the superoxide radical-generating system hypoxanthine/xanthine oxidase. J. Biol. Chem., 264, 13024-13028. 

See also Urea Cycle Reactions, Urea, Antioxidants (from Chapter 15), Excessive Uric Acid in Purine Degradation (from Chapter 22), Purine Degradation (from Chapter 22), Pathways in Nucleotide Metabolism (from Chapter 22), HGPRT, Hypoxanthine, Xanthine Oxidase... 

Hypoxanthine/xanthine oxidase system Fenton reaction system HjOz/NaOH/DMSO system Suppressed the superoxide, hydroxyl, and methyl radicals [115, 118J... 

The antioxidant activity of alizarin was established in four different assays (1) suppression of light emission in the p-iodophenol enhanced chemiluminescent assay, (2) scavenging of superoxide anion (02 -) in a hypoxanthine-xanthine oxidase system, (3) protection of rat liver microsomes from lipid peroxidation by ADP/iron(II) ions, and (4) protection of bromobenzene-intoxicated mice from liver injury in vivo [141]. Alizarin was compared with Trolox (water soluble vitamin E), the flavonoid baicalin and green tea proanthocyanidins. In assay (1) the activity of alizarin was 76% of that of Trolox. In assay (2) the inhibition of 02 -induced chemiluminescence was 40%, 32%, 23% and 14% for Trolox, alizarin, green tea polyphenols and baicalin respectively. Alizarin was not significantly active in the lipid peroxidation assay but after baicalin the most active compound in the in vivo assay. This shows again the difficulty in the evaluation of antioxidant activity and the differences between in vitro and in vivo assays [141]. 

There has been must less work directed to exploiting endogenous or targeted oxidative reactivity in tumours, compared to work on bioreductive drugs. Glucose oxidase or hypoxanthine/xanthine oxidase has been explored as sources of hydrogen peroxide or superoxide radicals [154,155], and the effects on tumour growth of ischaemia/reperfusion (which leads to a superoxide burst ) has been studied [156,157]. 

Radical scavenging effects of tannins and related polyphenols were also evidenced by ESR studies for the effects on the superoxide anion radical generated in the hypoxanthine-xanthine oxidase system and on the 1,1-diphenyl-2-picrylhydrazyl radical which is often used as a model of free radicals in lipid peroxidation [100]. Investigation of structure-activity... 

Table 7. Scavenging Effects of Tannins and Related Polyphenols on Snperoxide Anion Radicals Generated by Hypoxanthine-xanthine Oxidase...

Significant antioxidant activity has also been observed in the hexane extracts of different cashew products (Table 10.3). The antioxidant activity was determined in the hypoxanthine/xanthine oxidase assay and expressed as inhibition of reactive oxygen species (ROS) attack on salicylic acid (%). CNSL has the highest antioxidant activity, followed by cashew fiber, cashew apple, raw cashew nut,... 

Antioxidant Activity of Hexane Extracts of Different Cashew Products as Determined in the Hypoxanthine/Xanthine Oxidase Assay... 

FIGURE 18.1 Scavenging effects of 90% chitosan( ), 75% chitosan (o), and 50% chitosan (t) on hydroxyl radical obtained in Fenton reaction system (a) and on superoxide radical obtained in hypoxanthine-xanthine oxidase system (b) at various concentrations of hetero-chitosans. Inset shows ESR spectrum of the spin adduct of DMPO-OH (a) and superoxide radical (b). (From Park, P.J. et al., Carbohydr. Polym., 55, 17, 2004c. With permission.)... 

Chrysotile, crocidolite, Ti02 Rat alveolar macrophages In vitro ROS Dose-dependent stimulation of TNF-a secretion by asbestos was inhibited by deferoxamine (iron chelator). Role of ROS confirmed by radical regenerating hypoxanthine-xanthine oxidase system SiMEONOVA and Luster (1995) ROS in sigml-ling TNF-a stimulation... 

Not only Oj " and HO, but also alkyl or alkoxyl radicals (R ) were formed when saccharides such as glucose, fructose and sucrose were added into the xanthine oxidase/hypoxanthine system containing iron (Luo et al. 2001). The generated amount of R depended on the kind and concentration of saccharides added into this system. In the absence of saccharides no R were detected, indication that there is an interaction between the saccharide molecules and the free radicals generated from the iron containing hypoxanthine/xanthine oxidase system. 

Flow cytometric analyses demonstrated that pretreatment of Chinese hamster ovary cell line AS52 with 50 iM ascorbic acid before exposure to the hypoxanthine-xanthine oxidase radical generating system enhanced cell cycle arrest at the G2/M DNA damage checkpoint when compared to cells treated with hypoxanthine-xanthine oxidase without premedication (Bijur et al. 1999). Ascorbic acid had no effect on cell cycle progression in the absence of oxidative stress. 

The role of reactive oxygen species generated by hypoxanthine/xanthine oxidase in the induction of apoptosis was studied by Gansauge et al. (1997). In human fibroblast cell line WL38 after 48 h incubation with 1 mM hypoxanthine/xanthine and 0.05 U xanthine oxidase/ml apoptosis but not necrosis was observed in proliferating fibroblasts. Catalase hin-... 

The reaction of a tryptophan derivative, N- tert-butoxycarbonyl)-L-tryptophan, with hypoxanthine/ xanthine oxidase/Fe(III)-EDTA mainly resulted in the oxygenation of the pyrrole ring of the indole nucleus (Itakura etal. 1994). 2-[(ferf-Butoxy-carbonyl)-amino]-3-(3-indolyl)propionic acid and N- (fer f-butoxycarb onyl) -hT-formylkynurenine were identified as the major products. 

Holtzman s method (54) circumvents the problems associated with the Billiar method by including catalase in the hypoxanthine-xanthine oxidase reaction chain according to the following scheme ... 

Hydroxyl radical was generated from Fenton reaction with the mixing of FeSO (2.27 iM) and H2O2 (227 iM). Superoxide radical was generated from a hypoxanthine-xanthine oxidase (HPX-XOD) reaction system. Both hydroxyl radical and superoxide were trapped by 5,5-dimethyl-1-pyrroline-l-oxide (DMPO). 

Resveratrol can also prevent the initial events of atherosclerosis in endothelial cells by inhibition of the enzymatic systems producing reactive oxygen species, such as NADPH oxidase and hypoxanthine/xanthine oxidase, and by the inhibition of both the expression of adhesion molecules and the monocyte adhesion to endothelial cells [167]. Moreover, red wine polyphenols, in particular E-resveratrol, have been shown to inhibit the proliferation and the migration of vascular smooth muscle cells in intima, notably involved in the formation of atherosclerotic plaques [173]. In fact, resveratrol specifically blocks the mTOR pathway, which is activated by oxidized LDL, and regulates the proliferation of smooth muscle cells [174]. Other stilbenoids have been shown to exert an activity on this target as well. For example, pterostilbene inhibits vascular smooth muscle cell proliferation... 

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