Immunohistochemical localization of xanthine oxidase in human cardiac and skeletal muscle

The generation of a monoclonal antibody specific to xanthine oxidase and its use in the distribution of the enzyme in human tissue is described. Xanthine oxidase was purified from human and bovine milk by a rapid method, allowing for minimal proteolytic degradation, and the purified enzyme preparations were used for the immunization of BALB/c mice as well as for the subsequent selection of hybridomas. The hybridoma clone X1–7, IgG (2a, -light chain) was selected for further analysis and demonstrated to precipitate xanthine oxidase from human liver and skeletal muscle extracts. As determined by SDS-polyacrylamide gel electrophoresis of eluates from affinity chromatography, the X1–7 antibody bound to a main protein of 155 kDa, from human milk and skeletal muscle, and to proteins of 155, 143 and 95 kDa from human liver. Immunohistochemical studies, using two of the monoclonal antibodies with differing epitope specificity, revealed xanthine oxidase to be localized mainly in the vascular smooth muscle cells but also in a proportion of endothelial cells of capillaries and smaller vessels in both human cardiac and skeletal muscle. Immunoreactivity was additionally observed in human macrophages and mast cells. The results of the present study confirm previous reports of the presence of xanthine oxidase in capillary endothelial cells, but also demonstrates additional localization of the enzyme in vascular smooth muscle cells, macrophages and mast cells. The current findings verify that the distribution of xanthine oxidase in human tissue includes cardiac and skeletal muscle.     

Effect of acetyl-l-carnitine on lipid peroxidation and xanthine oxidase activity in rat skeletal muscle

It has been reported that acetyl-l-carnitine (AcCn) can reduce the degenerative processes in the central nervous system of rats, modify the fluidity of membranes and decrease the accumulation of lipofuscins in neurones. In light of these considerations we have assayed the in vitro effect of acetyl-l-carnitine on spontaneous and induced lipoperoxidation in rat skeletal muscle; in addition, the effect of AcCn on XD/XO ratio was evaluated. The presence of AcCn (10–40 mM) in incubation medium significantly reduced MDA and conjugated diene formation in rat skeletal muscle; moreover, a significant decrease in induced MDA levels was observed when microsomal preparation where incubated in the presence of 10–40 mM AcCn. Since a significant reduction of XO activity was detected in the presence of 10–80 mM AcCn, the reduced lipid peroxidation by AcCn seems to be due to an inhibition of XO activity.     

Inhibition of xanthine oxidase reduces hyperglycemia-induced oxidative stress and improves mitochondrial alterations in skeletal muscle of diabetic mice

Reactive oxygen species (ROS) have been widely implicated in the pathogenesis of diabetes and more recently in mitochondrial alterations in skeletal muscle of diabetic mice. However, so far the exact sources of ROS in skeletal muscle have remained elusive. Aiming at better understanding the causes of mitochondrial alterations in diabetic muscle, we designed this study to characterize the sites of ROS production in skeletal muscle of streptozotocin (STZ)-induced diabetic mice. Hyperglycemic STZ mice showed increased markers of systemic and muscular oxidative stress, as evidenced by increased circulating H(2)O(2) and muscle carbonylated protein levels. Interestingly, insulin treatment reduced hyperglycemia and improved systemic and muscular oxidative stress in STZ mice. We demonstrated that increased oxidative stress in muscle of STZ mice is associated with an increase of xanthine oxidase (XO) expression and activity and is mediated by an induction of H(2)O(2) production by both mitochondria and XO. Finally, treatment of STZ mice, as well as high-fat and high-sucrose diet-fed mice, with oxypurinol reduced markers of systemic and muscular oxidative stress and prevented structural and functional mitochondrial alterations, confirming the in vivo relevance of XO in ROS production in diabetic mice. These data indicate that mitochondria and XO are the major sources of hyperglycemia-induced ROS production in skeletal muscle and that the inhibition of XO reduces oxidative stress and improves mitochondrial alterations in diabetic muscle.     

The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase

A di-(carboxamidomethyl) derivative of molybdopterin, the organic component of the molybdenum cofactor, has been prepared under conditions favoring retention of all of the structural features of the molecule. The specific radioactivity of [1-14C]iodoacetamide incorporated relative to the amount of phosphate indicated two alkylation sites per pterin. Energy-dispersive x-ray analysis of the derivative showed the presence of 2 sulfurs in the derivative. An exact mass corresponding to the molecular formula C14H18N7O5S2 was obtained for the MH+ ion of the alkylated, dephosphorylated compound by fast atom bombardment mass spectroscopy. 1H NMR spectra of the phosphorylated and dephosphorylated forms of alkylated molybdopterin, in conjunction with the other data, have provided strong corroboration of the validity of the proposed structure of molybdopterin (Johnson, J. L., and Rajagopalan, K. V. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6856-6860) as a 6-alkylpterin with a 4-carbon side chain containing an enedithiol on C-1' and C-2', a secondary alcohol on C-3', and a phosphorylated primary alcohol on C-4'. As isolated, the di-(carboxamido-methyl)molybdopterin was found to be a 5,6,7,8-tetrahydropterin.     

ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe-XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O(2)*-) and hydroxyl radical (OH*), but also alkyl or alkoxyl radicals (R*) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe-XO/HX system. The generated amount of R* was dependent on the kind and concentration of saccharides added into the Fe-XO/HX system and no R* were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe-XO/HX system and saccharide molecules are essential for generating R* in the Fe-XO/HX system. It is expected that the toxicity of R* would be greater than of hydrophilic O(2)*- and OH* because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O(2)*- and OH*. The R* generated from the saccharide containing Fe-XO/HX can be effectively scavenged by selenium containing abzyme (Se-abzyme), indicating Se-abzyme is a promising antioxidant.     

Elevated aspartate transcarbamylase and dihydroorotase activities in erythrocytes from patients with hypoxanthine guanine phosphoribosyltransferase deficiency.

Activities of aspartate transcarbamylase and dihydroorotase are increased 6 to 8-fold in erythrocytes from individuals with hypoxanthine guanine phosphoribosyltransferase deficiency. The increased enzyme activities do not appear to be due to enzyme activation.     

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.     

The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase

Methods have been devised to examine the spectral properties and state of reduction of the pterin ring of molybdopterin (MPT) in milk xanthine oxidase and the Mo-containing domain of rat liver sulfite oxidase. The absorption spectrum of the native pterin was visualized by difference spectroscopy of each protein, denatured anaerobically in 6 M guanidine hydrochloride (GdnHCl), versus a sample containing the respective apoprotein and other necessary components. The state of reduction of MPT was also probed using 2,6-dichlorobenzenoneindophenol (DCIP) to measure reducing equivalents/MPT, after anaerobic denaturation of the protein in GdnHCl in the presence or absence of Hg2+. In the case of xanthine oxidase the data indicate that the terminal sulfide ligand of Mo causes the reduction of a native dihydro form of MPT to the tetrahydro level. This reduction does not occur if Hg2+ is added prior to denaturation of the protein. Based on its observed behavior, the native MPT in the Mo cofactor of xanthine oxidase is postulated to exist as a quinonoid dihydropterin. Quantitation of DCIP reduction by MPT of Mo fragment of sulfite oxidase showed a two-electron oxidation of MPT, even when the Mo fragment was denatured in the presence of Hg2+ to prevent internal reduction reactions due to sulfhydryls or sulfide. Difference spectra of DCIP-treated versus untreated Mo fragment showed that MPT had been fully oxidized. These data indicate that the native MPT in sulfite oxidase must be a dihydro isomer different from that in xanthine oxidase.     

Inhibition of xanthine oxidase improves myocardial contractility in patients with ischemic cardiomyopathy

Reactive oxygen species, in particular superoxide, have been closely linked to the underlying pathophysiology of ischemic cardiomyopathy: superoxide not only mediates mechanoenergetic uncoupling of the myocyte but also adversely impacts on myocardial perfusion by depleting endothelial-derived nitric oxide bioavailability. Xanthine oxidase generates superoxide upon oxidation of hypoxanthine and xanthine and has been detected in cardiac myocytes and coronary endothelial cells of patients with ischemic heart disease. Here we investigated the effects of oxypurinol, a xanthine oxidase inhibitor, on myocardial contractility in patients with ischemic cardiomyopathy. Twenty patients (19 males, 66+/-8 years) with stable coronary disease, severely suppressed systolic function (left ventricular ejection fraction 22+/-2%), and nonelevated uric acid plasma levels received a single intravenous dose of oxypurinol (400 mg). Cardiac MRI studies, performed before and 5.2+/-0.9 h after oxypurinol administration, revealed a reduction in end-systolic volumes (-9.7+/-4.2%; p=0.03) and an increase in left ventricular ejection fraction (+17.5+/-5.2%; p=0.003), whereas 6 patients (6 males, 63+/-3.8 years, ejection fraction 26+/-5%) who received vehicle only did not show significant changes in any of the parameters studied. Oxypurinol improves left ventricular function in patients with ischemic cardiomyopathy. These results underscore the significance of reactive oxygen species as important pathophysiological mediators in ischemic heart failure and point toward xanthine oxidase as an important source of reactive species that serve to modulate the myocardial redox state in this disease.     

Pharmacokinetics and pharmacodynamics of febuxostat (TMX-67), a non-purine selective inhibitor of xanthine oxidase/xanthine dehydrogenase (NPSIXO) in patients with gout and/or hyperuricemia

The diurnal change of sUA and the effect of febuxostat on this change were investigated in 10 patients with gout and/or hyperuricemia. The diurnal sUA change after the last dose during the 4-week treatment phase (20 mg, QD) was almost the same as the pre-treatment value. Considering the dose, the AUC(obs) and Cmax of unchanged drug in patients with gout and/or hyperuricemia were estimated to be similar to those of healthy male adults. The results show that a 6-week treatment with febuxostat is safe and well-tolerated in the target patient population for this drug.     

Inhibition of xanthine oxidase reduces oxidative stress and improves skeletal muscle function in response to electrically stimulated isometric contractions in aged mice

Oxidative stress is a putative factor responsible for reducing function and increasing apoptotic signaling in skeletal muscle with aging. This study examined the contribution and functional significance of the xanthine oxidase enzyme as a potential source of oxidant production in aged skeletal muscle during repetitive in situ electrically stimulated isometric contractions. Xanthine oxidase activity was inhibited in young adult and aged mice via a subcutaneously placed time-release (2.5 mg/day) allopurinol pellet, 7 days before the start of in situ electrically stimulated isometric contractions. Gastrocnemius muscles were electrically activated with 20 maximal contractions for 3 consecutive days. Xanthine oxidase activity was 65% greater in the gastrocnemius muscle of aged mice compared to young mice. Xanthine oxidase activity also increased after in situ electrically stimulated isometric contractions in muscles from both young (33%) and aged (28%) mice, relative to contralateral noncontracted muscles. Allopurinol attenuated the exercise-induced increase in oxidative stress, but it did not affect the elevated basal level of oxidative stress that was associated with aging. In addition, inhibition of xanthine oxidase activity decreased caspase-3 activity, but it had no effect on other markers of mitochondrial-associated apoptosis. Our results show that compared to control conditions, suppression of xanthine oxidase activity by allopurinol reduced xanthine oxidase activity, H₂O₂ levels, lipid peroxidation, and caspase-3 activity; prevented the in situ electrically stimulated isometric contraction-induced loss of glutathione; prevented the increase in catalase and copper-zinc superoxide dismutase activities; and increased maximal isometric force in the plantar flexor muscles of aged mice after repetitive electrically evoked contractions.     

Catalase,carbonic anhydrase and xanthine oxidase activities in patients with mycosis fungoides

Abstract

Mycosis fungoides (MF) is the most common form of cutaneous T-cell lymphoma. In several studies the relationship between catalase (CAT), human cytosolic carbonic anhydrases (CA; hCA-I and hCA-II) and xanthine oxidase (XO) enzyme activities have been investigated in various types of cancers but carbonic anhydrase, catalase and xanthine oxidase activities in patients with MF have not been previously reported. Therefore, in this preliminary study we aim to investigate CAT, CA and XO activities in patients with MF. This study enrolled 32 patients with MF and 26 healthy controls. According to the results, CA and CAT activities were significantly lower in patients with mycosis fungoides than controls (p?<?0.001) (p?<?0.001). There was no significant difference in XO activity between patient and control group (p?=?0.601). Within these findings, we believe these enzyme activity levels might be a potentially important finding as an additional diagnostic biochemical tool for MF.     

Metabolism of xanthine and hypoxanthine in the tea plant (Thea sinensis L.).

1. The metabolism of xanthine and hypoxanthine in excised shoot tips of tea was studied with micromolar amounts of [2(-14)C]xanthine or [8(-14)C]hypoxanthine. Almost all of the radioactive compounds supplied were utilized by tea shoot tips by 30 h after their uptake. 2. The main products of [2(-14)C]xanthine and [8(-14)C]hypoxanthine metabolism in tea shoots were urea, allantoin and allantoic acid. There was also incorporation of the label into theobromine, caffeine and RNA purine nucleotides. 3. The results indicate that tea plants can catabolize purine bases by the same pathways as animals. It is also suggested that tea plants have the ability to snythesize purine nucleotides from glycine by the pathways of purine biosynthesis de novo and from hypoxanthine and xanthine by the pathway of purine salvage. 4. The results of incorporation of more radioactivity from [8(-14)C]hypoxanthine than from [2(-14)C]xanthine into RNA purine nucleotides and caffeine suggest that hypoxanthine is a more effective precursor of caffeine biosynthesis than xanthine. The formation of caffeine from hypoxanthine is a result of nucleotide synthesis via the pathway of purine salvage.