Potent Xanthine Oxidase Inhibitor Research Articles

Measurement of endothelial cell free radical generation: evidence for a central mechanism of free radical injury in postischemic tissues.

Oxygen free radicals have been demonstrated to be important mediators of postischemic reperfusion injury in a broad variety of tissues; however, the cellular source of free radical generation is still unknown. In this study, electron paramagnetic resonance measurements with the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) demonstrate that bovine endothelial cells subjected to anoxia and reoxygenation become potent generators of superoxide and hydroxyl free radicals. A prominent DMPO-OH signal aN = aH = 14.9 G is observed on reoxygenation after 45 min of anoxic incubation. Quantitative measurements of this free radical generation and the time course of radical generation are performed. Both superoxide dismutase and catalase totally abolish this radical signal, suggesting that O2 is sequentially reduced from O2-. to H2O2 to OH.. Addition of ethanol resulted in trapping of the ethoxy radical, further confirming the generation of OH.. Endothelial radical generation was shown to cause cell death, as evidenced by trypan blue uptake. Radical generation was partially inhibited and partially scavenged by the xanthine oxidase inhibitor allopurinol. Marked inhibition of radical generation was observed with the potent xanthine oxidase inhibitor oxypurinol. These studies demonstrate that endothelial cells subjected to anoxia and reoxygenation, conditions observed in ischemic and reperfused tissues, generate a burst of superoxide-derived hydroxyl free radicals that in turn cause cell injury and cell death. Most of this free radical generation appears to be from the enzyme xanthine oxidase. Thus, endothelial cell free radical generation may be a central mechanism of cellular injury in postischemic tissues.

Effects of flavonoids on xanthine oxidation as well as on cytochrome c reduction by milk xanthine oxidase.

That flavonoids inhibit xanthine oxidase from cow milk was confirmed by measuring oxygen consumption with an oxygen electrode. In contrast, flavonoids did not inhibit glucose oxidase, another oxygen consuming enzyme. Among the flavonoids tested, quercetin, kaempferol, myricetin, chrysin, quercitrin, and morin were potent inhibitors of xanthine oxidase; their inhibition rates (%) were 80, 70, 69, 62, 59, and 51 at 100 microM (except chrysin at 50 microM), respectively. The xanthine oxidase-inhibiting activities of the flavonoids were not always well correlated with the suppressive activities of the flavonoids on cytochrome c reduction by a xanthine-xanthine oxidase system. The inhibition of xanthine oxidase by quercetin was not affected by cupric ion. The partition rates of the flavonoids between n-butanol and a buffer solution seemed to account for some of the inhibition.

Effect of allopurinol treatment in premature infants with idiopathic respiratory distress syndrome.

A randomized prospective study of the effectiveness of allopurinol (Ap), a potent and specific inhibitor of the enzyme xanthine oxidase, was performed in premature infants endangered by hypoxia. The drug was given at a dose of 20 mg/kg/day orally for 3 days. In the Ap-treated group the expected decrease in the serum concentration and urinary excretion of uric acid was accompanied by a decrease in the mortality rate of infants with idiopathic respiratory distress syndrome. In these patients a concomitant improvements in renal function, as indicated by an increased urinary flow rate and creatinine output, was also obvious. It is suggested that the observed beneficial effect is due to the specific inhibition of xanthine oxidase associated with Ap therapy leading to reduced generation of superoxide radicals and decreased urinary loss of purine.

Inhibition of mammalian xanthine oxidase by folate compounds and amethopterin.

We have examined the effects of folate compounds and the folate analog amethopterin (methotrexate) as inhibitors of mammalian xanthine oxidase and have found that they offer potent inhibition of the enzyme. We have compared the inhibitory potency of folic acid and its coenzyme derivative tetrahydrofolic acid to that of allopurinol, a known inhibitor of xanthine oxidase, and have demonstrated that folic acid and tetrahydrofolic acid are severalfold more potent than allopurinol as inhibitors of xanthine oxidase. Comparative inhibition constants calculated were 5.0 X 10(-7) M for folic acid. 1.25 X 10(-6) M for tetrahydrofolic acid, and 4.88 X 10(-6) M for allopurinol. Incubation of xanthine oxidase with folic acid at a concentration of 10(-6) M abolished 94% of the enzymic activity within 1 min of incubation with the enzyme. At the same concentration, allopurinol was almost ineffective as an inhibitor of xanthine oxidase. The substrate xanthine protected the enzyme against total inhibition by folic acid. Reversibility of the enzymic inhibition by folic acid was demonstrated. Folic acid-inactivated enzyme was totally regenerated either by filtration through Sephadex G-200 or by precipitation with ammonium sulfate. 2-Amino-4-hydroxypteridine was a poor substrate for the enzyme but a potent inhibitor for the oxidation of xanthine by the enzyme. The inhibition constant calculated was 1.50 X 10(-6) M. In the presence of an excess of xanthine oxidase, neither folic acid nor tetrahydrofolic acid and allopurinol exhibited any change in intensity of their absorbance or in the wavelength of their maximal absorbance that might have been suggestive of substrate utility. The folate analog amethopterin was also determined a potent inhibitor of mammalian xanthine oxidase. The inhibition constant calculated was 3.0 X 10(-5) M.

Stereochemistry of nucleic acids and their constituents. XXIX. Crystal and molecular structure of allopurinol, a potent inhibitor of xanthine oxidase

Stereochemistry of nucleic acids and their constituents. XXIX. Crystal and molecular structure of allopurinol, a potent inhibitor of xanthine oxidase

Selective Inhibition of Xanthine Oxidase by 3-Hydroxy-1-nitrophenyl-1H-pyrazolo[4,3-c]pyridines

Some 3-hydroxy-1-nitrophenyl-1H-pyrazolo[4,3-c]-pyridines were synthesized and characterized. The ability of the compounds to inhibit purine-catabolizing enzymes was evaluated. These pyrazolopyridines were found to exhibit selective inhibitory effects on xanthine oxidase, while adenosine deaminase, guanosine deaminase, and guanine deaminase were unaffected. 3-Hydroxy-1-(2',4'-dinitrophenyl)-1H-pyrazolo[4,3-c]pyridine was the most potent inhibitor of xanthine oxidase.

Reduction of niridazole by rat liver xanthine oxidase

The soluble fraction of liver contains an enzyme that catalyzes the reduction of niridazole by either NADH or hypoxanthine. The enzyme is probably xanthine oxidase because niridazole reduction is blocked by potent inhibitors of xanthine oxidase and, on purification of the enzyme, xanthine oxidase specific activity approximately parallels the niridazole reductase activity. Moreover, milk xanthine oxidase also catalyzed the reduction of niridazole by hypoxanthine.

Depletion of erythrocyte phosphoribosylpyrophosphate in man.

Allopurinol, a potent inhibitor of xanthine oxidase, is widely used for the treatment of hyperuricemia. When this drug was given to nine patients with gout, the concentration of phosphoribosylpyrophosphate (PRPP) in red cells was reduced significantly (p<0.01). Studies in vivo and in vitro showed that this depletion of PRPP results from the enzymatic conversion of allopurinol to allopurinol ribonucleotide, a process that consumes PRPP. The findings that allopurinol depletes PRPP and is apparently converted to its ribonucleotide in vivo suggest that this widely used drug may have a variety of biochemical effects in addition to xanthine oxidase inhibition. The observed changes provide several likely mechanisms to account for the previously documented inhibitory effect of allopurinol on purine synthesis de novo.

Potent xanthine oxidase inhibitors—4(or 5)-diazoimidazole-5(or 4)-carboxamide and two related compounds

Potent xanthine oxidase inhibitors—4(or 5)-diazoimidazole-5(or 4)-carboxamide and two related compounds

Some Effects of Organic Solvents on the Reaction Kinetics of Milk Xanthine Oxidase

Abstract The rate of reduction of 2,6-dichlorobenzenoneindophenol by xanthine oxidase plus its aldehydic or purine substrates is strikingly stimulated by the admixture of water-miscible organic solvents. In addition to acting as the electron acceptor in these assays, 2,6-dichlorobenzenoneindophenol is found to be a potent competitive inhibitor of xanthine oxidase with a KI of 1 x 10-6 M. The leuco form of this dye is a much less effective inhibitor, with a KI of 1.5 x 10-5 M. Dimethoxyethane and other water-miscible organic solvents reduce the inhibitory potency of 2,6-dichlorobenzenoneindophenol and thus give the appearance of activating the enzyme in this dye reduction assay. The inhibition of the enzyme by several other competitive inhibitors was not affected by dimethoxyethane. Dimethoxyethane, per se, is an inhibitor of xanthine oxidase. Its effects on the kinetics of this enzyme are notably complex. The reversal of the 2,6-dichlorobenzenoneindophenol inhibition by organic solvents appears best explained in terms of their effects on the solvent properties of the medium.

PREVENTION OF HYPERURICEMIA IN LEUKEMIA AND LYMPHOMA: USE OF ALOPURINOL, A XANTHINE OXIDASE INHIBITOR.

Allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine], a potent inhibitor of xanthine oxidase, was given orally to patients with leukemia and lymphoma in whom marked hyperuricemia was present or was to be expected as a result of cytolytic therapy. There was a decrease in serum uric acid in each patient, and a decrease in urine uric acid excretion in each of the ten patients in whom it was measured. This was accompanied by only a moderate increase in urinary oxypurines and no evidence of renal, hematologic, gastrointestinal, or hepatic toxicity.

Enzymic cleavage of folic acid by extracts from human blood cells: I. Preparation and co-factor requirements of the enzyme system

1. 1. Preparation is described of an enzyme system from human blood which can degrade folic acid to yield p-aminobenzoylglutamic acid and 2-amino-4-hydroxy-6-formyl-pteridine. 2. 2. The enzyme system requires ATP, Mn ++ and glutathione as cofactors and shows optimum activity at pH 5. 3. 3. The enzyme is apparently present in blood in an inactive state. Activity is liberated on treatment with hydrochloric acid. 4. 4. Evidence is given for the presence of heat-labile inhibitors which may be separated from the active fraction by (NH 4) 2SO 4 fractionation. These inhibitors depress the cleavage of folic acid when the cofactors requirements are supplied by extracts prepared from blood but not when ATP, Mn ++ and glutathione are present. 5. 5. Thiol compounds like cystein or thioglycollic acid cannot replace glutathione as a cofactor for the enzyme. Coenzyme A can partially serve this purpose. 6. 6. The suggestion is made that these active mechanisms for cleavage of folio acid in animal tissues may be important for the metabolic disposal of folic acid, as also for the biosynthesis of biologically important unconjugated pteridines like 2-amino-4-hydroxy-6-formyl-pteridine which is a very potent inhibitor of xanthine oxidase.