Purine catabolism in advanced carotid artery plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Purine Catabolism in Advanced Carotid Artery Plaque

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.     

Effects of Allopurinol [4-Hydroxypyrazolo(3,4-d)Pyrimidine] on the Metabolism of Allantoin in Soybean Plants

Some studies on the effects of xanthine oxidase inhibitor allopurinol [4-hydroxypyrazolo(3,4-d)pyrimidine] on allantoin metabolism of soybean plants (Glycine max cv. Tamanishiki) are reported. Soybean seedlings, aseptically germinated for 96 hours on agar containing 1 millimolar allopurinol, contained only slight amounts of allantoin, allantoic acid, and urea as compared with controls. Analysis of purines and pyrimidines of the allopurinol-treated seedlings showed marked accumulation of xanthine both in the cotyledons and seedling axes. No hypoxanthine accumulation was found. Xanthine accumulation due to allopurinol treatment was relatively low after the cotyledons had fallen. For nodulated plants, allopurinol caused a significant drop in allantoin (+allantoic acid) in the stems and nodules, accompanied by a striking accumulation of xanthine in the nodules. The xanthine concentration in the nodules far exceeded that in the germinated seedlings. Allopurinol at a concentration of 50 micromolar strongly inhibited xanthine oxidase prepared from soybean nodules.

The results suggested that the main pathway of allantoin formation in soybean plants was through purine decomposition, via xanthine-uric acid. It was specially noted that a very active purine-decomposing system existed in soybean nodules.

     

Organization of purine degradation in the liver of a teleost (carp; cyprinus carpio L.). A study of its subcellular distribution

Summary Carp liver was fractionated by differential and density gradient centrifugation and assayed for enzymes of purine catabolism. While urate oxidase is an excusively peroxisomal enzyme, only a very small percentage of the enzymes xanthine oxidase, allantoinase and allantoicase is associated with subcellular or ganelle fractions. There is no general purine catabolizing subcellular compartment.There is some but not yet conclusive evidence for the assumption that urate oxidase is a membrane bound enzyme.     

Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage

Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential sigma(54)-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the sigma(54)-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced (14)CO(2) from minimal medium containing [(14)C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated (14)CO(2) production from [(14)C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.     

Purine requirements for the expression of Cape buffalo serum trypanocidal activity

Cape buffalo serum contains xanthine oxidase which generates trypanocidal H₂O₂ during the catabolism of hypoxanthine and xanthine. The present studies show that xanthine oxidase-dependent trypanocidal activity in Cape buffalo serum was also elicited by purine nucleotides, nucleosides, and bases even though xanthine oxidase did not catabolize those purines. The paradox was explained in part, by the presence in serum of purine nucleoside phosphorylase and adenosine deaminase, that, together with xanthine oxidase, catabolized adenosine, inosine, hypoxanthine, and xanthine to uric acid yielding trypanocidal H₂O₂. In addition, purine catabolism by trypanosomes provided substrates for serum xanthine oxidase and was implicated in the triggering of xanthine oxidase-dependent trypanocidal activity by purines that were not directly catabolized to uric acid in Cape buffalo serum, namely guanosine, guanine, adenine monophosphate, guanosine diphosphate, adenosine 3′:5-cyclic monophosphate, and 1-methylinosine. The concentrations of guanosine and guanine that elicited xanthine oxidase-dependent trypanocidal activity were 30–270-fold lower than those of other purines requiring trypanosome-processing which suggests differential processing by the parasites.     

Enzymes of purine catabolism in soybean plants

Remarkable formation and utilization of allantoin is observedin soybean (Glycine max variety A62-1). To study this, variousenzymes involved in purine catabolism (i.e., xanthine oxidase,uricase and allantoinase) were measured in different regionsof soybean plants during development. Uricase, which catalyzesthe direct formation of allantoin from uric acid, was studiedin detail. The activities of these three enzymes were highest in the rootnodules, indicating that the nodules are the major site of allantoinmetabolism. Radicles only showed appreciable activity about80 hr after the seeds were planted. Allantoinase activity wasdetected in all regions tested, showing that allantoin translocatedfrom the nodules can be metabolized in the roots, stem and leaves.In the nodules, xanthine oxidase was localized in the nuclearfraction, while uricase was mainly restricted to the mitochondrialfraction and allantoinase to the soluble fraction. Uricase was partially purified from the nodules and radicles,respectively. The pH optimum of enzyme from the nodules was9.5, whereas that of enzyme from the radicles was 7.0. The enzymefrom the nodules did not require a cofactor, while that fromthe radicles showed an absolute requirement for a cofactor,which was a low molecular substance easily separable from theapoprotein. Thus, the uricase in nodules differs in chemicalproperties from that in the host plant. The results are discussedin relation to change in the allantoin level in soybean tissues. (Received November 1, 1974; )     

Selective purine release from P388D1 macrophages injured by silica

Silica particles are toxic to primary cultures of macrophages or the P388D1 cell line in vitro. Loss of viability in these model systems is accompanied by depletion of ATP content within 3 to 6 hours. The mechanisms responsible for ATP depletion will be explored in this paper. After prelabeling for 1 hour with 3H-adenine, silica-treated cells released 60-80% of their labeled acid-soluble pool into the culture medium. This release did not occur after phagocytosis of nontoxic titanium dioxide particles and was specific for purines. ATP depletion was accompanied by purine catabolism: inosine, hypoxanthine, xanthine, and uric acid were detected in the culture medium using thin layer or high-performance liquid chromatography. The final xanthine oxidase step in purine catabolism generates reactive oxygen metabolites. Silica toxicity was not prevented by the xanthine oxidase inhibitor allopurinol nor exogenous purines. It is concluded that adenine nucleotide depletion and purine catabolism are not solely responsible for irreversible injury in silica toxicity. It is hypothesized that purine catabolism and release from injured macrophages may lead to generation of reactive oxygen species, injury to surrounding tissue, and fibrosis.     

The genomes of the South American opossum (Monodelphis domestica) and platypus (Ornithorhynchus anatinus) encode a more complete purine catabolic pathway than placental mammals

The end product of purine catabolism varies amongst vertebrates and is a consequence of independent gene inactivation events that have truncated the purine catabolic pathway. Mammals have traditionally been grouped into two classes based on their end product of purine catabolism: most mammals, whose end product is allantoin due to an ancient loss of allantoinase (ALLN), and the hominoids, whose end product is uric acid due to recent inactivations of urate oxidase (UOX). However little is known about purine catabolism in marsupials and monotremes. Here we report the results of a comparative genomics study designed to characterize the purine catabolic pathway in a marsupial, the South American opossum (Monodelphis domestica), and a monotreme, the platypus (Ornithorhynchus anatinus). We found that both genomes encode a more complete set of genes for purine catabolism than do eutherians and conclude that a near complete purine catabolic pathway was present in the common ancestor of all mammals, and that the loss of ALLN is specific to placental mammals. Our results therefore provide a revised history for gene loss in the purine catabolic pathway and suggest that marsupials and monotremes represent a third class of mammals with respect to their end products of purine catabolism.     

Antioxidant Status and Purine Bases in Carotid Artery Plaque

Free radical excess and oxidative stress are implicated in the formation and progression of atherosclerotic plaque through actions on susceptible vascular cells, such as by activating xanthine oxidase. Purine bases and other antioxidant compounds could play important protective roles in atherogenesis, as could nonenzymatic low molecular weight thiol defenses, not previously evaluated in carotid artery plaque. Therefore, we measured purine catabolites (hypoxanthine, xanthine, uric acid, allantoin) and antioxidant compounds (total sulphydryl groups, homocysteine, cysteine, and glutathione) in advanced carotid artery plaque and found a high ratio of allantoin to uric acid, suggesting a ongoing local oxidative stress.     

Antioxidant status and purine bases in carotid artery plaque

Free radical excess and oxidative stress are implicated in the formation and progression of atherosclerotic plaque through actions on susceptible vascular cells, such as by activating xanthine oxidase. Purine bases and other antioxidant compounds could play important protective roles in atherogenesis, as could nonenzymatic low molecular weight thiol defenses, not previously evaluated in carotid artery plaque. Therefore, we measured purine catabolites (hypoxanthine, xanthine, uric acid, allantoin) and antioxidant compounds (total sulphydryl groups, homocysteine, cysteine, and glutathione) in advanced carotid artery plaque and found a high ratio of allantoin to uric acid, suggesting a ongoing local oxidative stress.     

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.     

Regulation of Purine Metabolism in Intact Leaves of Coffea arabica

The capacity of Coffea arabica leaves (5- x 5-mm pieces) to synthesize de novo and catabolize purine nucleotides to provide precursors for caffeine (1,3,7-trimethylxanthine) was investigated. Consistent with de novo synthesis, glycine, bicarbonate, and formate were incorporated into the purine ring of inosine 5[prime]-monophosphate (IMP) and adenine nucleotides ([sigma]Ade); azaserine, a known inhibitor of purine de novo synthesis, inhibited incorporation. Activity of the de novo pathway in C. arabica per g fresh weight of leaf tissue during a 3-h incubation period was 8 [plus or minus] 4 nmol of formate incorporated into IMP, 61 [plus or minus] 7 nmol into [sigma]Ade, and 150 nmol into caffeine (the latter during a 7-h incubation). Coffee leaves exhibited classical purine catabolism. Radiolabeled formate, inosine, adenosine, and adenine were incorporated into hypoxanthine and xanthine, which were catabolized to allantoin and urea. Urease activity was demonstrated. Per g fresh weight, coffee leaf squares incorporated 90 [plus or minus] 22 nmol of xanthine into caffeine in 7 h but degraded 102 [plus or minus] 1 nmol of xanthine to allantoin in 3 h. Feedback control of de novo purine biosynthesis was contrasted in C. arabica and Cucurbita pepo, a species that does not synthesize purine alkaloids. End-product inhibition was demonstrated to occur in both species but at different enzyme reactions.     

A new spectrophotometric assay method of xanthine oxidase in crude tissue homogenate

A new spectrophotometric assay method of xanthine oxidase applicable to the crude tissue homogenate containing uricase was presented in this paper. By adding potassium 2,4-dihydroxy-6-carboxy-1,3,5-triazine (potassium oxonate) (0.1 mm) to the crude xanthine oxidase reaction system, uric acid was stoichiometrically formed from xanthine and detectable allantoin was not formed and the formation of uric acid was not influenced by uricase.Distribution of xanthine oxidase in various rat tissues was measured by this method, and it was shown that the activity was high in the liver, the small intestine, and the spleen. Uricase was shown to distribute mainly in the liver of rats.     

Acetaminophen-induced oxidation of protein thiols. Contribution of impaired thiol-metabolizing enzymes and the breakdown of adenine nucleotides

The administration of a hepatotoxic dose of acetaminophen (250 mg/kg) to mice induced the loss of protein thiols in mouse liver. Our data suggest that a significant portion of this loss was due to protein thiol oxidation. The administration of the nonhepatotoxic regioisomer, 3'-hydroxyacetanilide (600 mg/kg) did not produce a similar decrease in liver protein thiols despite producing similar levels of covalent binding. Mice treated with acetaminophen exhibited decreased glutathione peroxidase activity, decreased thioltransferase activity, and decreased adenine nucleotide concentrations in the liver. The increase in urinary allantoin after the administration of acetaminophen suggests that the decrease in adenine nucleotides was due to their degradation in the liver. Acetaminophen also promoted the conversion of the enzyme xanthine dehydrogenase to the oxidase form, and pretreatment of mice with allopurinol, an inhibitor of xanthine oxidase, significantly decreased acetaminophen-mediated hepatotoxicity. The conversion of xanthine dehydrogenase to the oxidase form may lead to a transient increase in the production of activated oxygen species. The increase in activated oxygen species coupled with decreases in glutathione peroxidase and thioltransferase activity may be responsible in part for the increased levels of oxidized protein thiols observed following acetaminophen administration.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.     

Allantoin excretion in snakes

Allantoin, which is generally regarded as absent in the excreta of snakes, has been demonstrated in the urinary deposits of 7 species of snakes by a sensitive paper chromatographic method. It appears that allantoin is the end product of purine catabolism in these animals.     

Age-related changes in xanthine oxidase activity and lipid peroxidation,as well as in the correlation between both parameters,in plasma and several organs from female mice

Xanthine oxidase, a purine catabolism enzyme, has been implicated as an important source of oxidant production and plays an essential role in several inflammatory and oxidative stress-related diseases. It is known that the increasing levels of oxidants cause the chronic oxidative stress characteristic of the ageing process. The aim of the present work was to determine the changes in xanthine oxidase activity and oxidative damage to lipids in several organs (liver, kidney, spleen, lung and two different brain areas, namely cerebral cortex and brainstem) and plasma from two different age groups of BALB/c female mice: adult (7-month-old) and old (18-month-old) mice, as well as to analyse the possible correlation between both parameters. Xanthine oxidase activity was significantly increased in liver, cerebral cortex and plasma from old mice in comparison with adults. Similar results were obtained in the lipid peroxidation levels, in which old mice showed a high increment in liver and cerebral cortex. Moreover, the results show a significant and positive correlation between xanthine oxidase activity and lipid peroxidation levels in cerebral cortex. The age-related increase in the xanthine oxidase activity and lipid peroxidation in liver and cerebral cortex of mice seems to suggest that the xanthine oxidase plays a role in the acceleration of the oxidative damage in these organs with age and its possible contribution to the pathophysiological changes associated to the process of ageing.