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.     

Action of biologically-relevant oxidizing species upon uric acid. Identification of uric acid oxidation products

Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.     

Effect of allopurinol on the utilization of purine degradation pathway intermediates by tobacco cell cultures

Suspension cultured Nicotiana tabacum (tobacco) cells grow slowly on intermediates of the purine degradation pathway (hypoxanthine, xanthine, uric acid, allantoin, and urea) as their sole nitrogen source indicating that this degradation pathway is operative in these cells. The hypoxanthine analog, allopurinol inhibited tobacco cell growth on hypoxanthine but not uric acid. This helps confirm that the site of action of allopurinol is the conversion of hypoxanthine to uric acid by xanthine oxidase. Attempts to select cells which could grow in the presence of allopurinol with hypoxanthine as the nitrogen source were not successful.     

The metabolism of caffeine by a Pseudomonas putida strain

1) A bacterium capable of growing aerobically with caffeine (1,3,7-trimethylxanthine) as sole source of carbon and nitrogen was isolated from soil. The morphological and physiological characteristics of the bacterium were examined. The organism was identified as a strain of Pseudomonas putida and is referred to as Pseudomonas putida C1. 15 additional caffeine-degrading bacteria were isolated, and all of them were also identified as Pseudomonas putida strains. The properties of the isolates are discussed in comparison with 6 Pseudomonas putida strains of the American Type Culture Collection. 2) The degradation of caffeine by Pseudomonas putida C1 was investigated; the following 14 metabolites were identified: 3,7-dimethylxanthine (theobromine), 1,7-dimethylxanthine, 7-methylxanthine, xanthine, 3,7-dimethyluric acid, 1,7-dimethyluric acid, 7-methyluric acid, uric acid, allantoin, allantoic acid, ureidoglycolic acid, glyoxylic acid, urea, and formaldehyde. Formaldehyde has been demonstrated to be the product of oxidative N-demethylation mediated by an inducible demethylase. A pathway of caffeine degradation is proposed.     

The moose,purine degradation,and environmental adaptation

It is accepted that allantoin is the end-product of purine degradation in mammals, except that uricase activity has been lost during the evolution of humans in which uric acid protects the brain from oxidative damage. However, we have found that the moose Alces americanus excretes extremely low urinary concentrations of allantoin and high concentrations of uric acid very similar to those of humans. Exposure to extreme cold is known to cause oxidative damage, and we suggest that the retention of uric acid by the moose represents an adaptation enabling the species to survive at high latitudes.     

Catabolism of caffeine and related purine alkaloids in leaves ofCoffea arabica L.

In a study of purine alkaloid catabolism pathways in coffee,¹⁴C-labelled theobromine, caffeine, theophylline and xanthine were incubated with leaves ofCoffea arabica. Incorporation of label into¹⁴CO₂ was determined and methanol-soluble metabolites were analysed by high-performance liquid chromatography-radiocounting. The data obtained demonstrate catabolism of caffeine theophylline 3-methylxanthine xanthine. Xanthine is degraded further by the conventional purine catabolism pathway to CO₂ and NH₃ via uric acid, allantoin and allantoic acid. The conversion of caffeine to theophylline is the rate-limiting step in purine alkaloid catabolism and provides a ready explanation for the high concentration of endogenous caffeine found inC. arabica leaves. Although theobromine is converted primarily to caffeine, a small portion of the theobromine pool appears to be degraded to xanthine by a caffeine-independent pathway. In addition to being broken down to CO₂, via the purine catabolism pathway, xanthine is metabolised to 7-methylxanthine. Metabolism of [2-¹⁴C]xanthine byC. arabica leaves in the presence of 5 mM allopurinol results in very large increases in incorporation of radioactivity into 7-methylxanthine as degradation of the substrate via the purine catabolism pathway is blocked. The identity of 7-methylxanthine in these studies was confirmed by gas chromatography-mass spectrometry analysis.Abbreviations HPLC-RC high-performance liquid chromatography-radiocounting This work was supported by the British Council which provided H.A. with Japan-UK travel grants. F.M.G. was supported by a Biotechnology and Biological Sciences Research Council grant to A.C.     

Evidence for an involvement of glutamine synthetase in regulation of nitrogenase activity in Rhodopseudomonas capsulata

In the presnet studies with whole cells and extracts of the photosynthetic bacterium Rhodopseudomonas capsulata the rapid inhibition of nitrogenase dependent activities (i.e. N₂-fixation acetylene reduction, or photoproduction of H₂) by ammonia was investigated. The results suggest, that the regulation of the nitrogenase activity by NH ₄ ⁺ in R. capsulata is mediated by glutamine synthetase (GS). (i) The glutamate analogue methionine sulfoximine (MSX) inhibited GS in situ and in vitro, and simultaneously prevented nitrogenase activity in vivo. (ii) When added to growing cultures ammonia caused rapid adenylylation of GS whereas MSX abolished the activity of both the adenylylated and unadenylylated form of the enzyme. (iii) Recommencement of H₂ production due to an exhaustion of ammonia coincided with the deadenylylation of GS. (iv) In extracts, the nitrogenase was found to be inactive only when NH ₄ ⁺ or MSX were added to intact cells. Subsequently the cells had to be treated with cetyltrimethylammonium bromide (CTAB). (v) In extracts the nitrogenase activity declined linearily with an increase of the ration of adenylylated vs. deadenylylated GS. A mechanism for inhibition of nitrogenase activity by ammonia and MSX is discussed.Abbreviations BSA bovin serum albumine - CTAB cetyltrimethylammonium bromide - GOGAT l-glutamine: 2-oxoglutarate amino transferase - GS glutamine synthetase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MSX l-methionine-d,l-sulfoximine     

Biosynthesis of Ureides from Purines in a Cell-free System from Nodule Extracts of Cowpea [Vigna unguiculata (L) Walp.

The synthesis of ¹⁴C-labeled xanthine/hypoxanthine, uric acid, allantoin, allantoic acid, and urea from [8-¹⁴C]guanine or [8-¹⁴C]hypoxanthine, but not from [8-¹⁴C]adenine, was demonstrated in a cell-free extract from N₂-fixing nodules of cowpea (Walp.). The ¹⁴C recovered in the acid/neutral fraction was present predominantly in uric acid and allantoin (88-97%), with less than 10% of the ¹⁴C in allantoic acid and urea. Time courses of labeling in the cell-free system suggested the sequence of synthesis from guanine to be uric acid, allantoin, and allantoic acid. Ureide synthesis was confined to soluble extracts from the bacteroid-containing tissue, was stimulated by pyridine nucleotides and intermediates of the pathways of aerobic oxidation of ureides, but was completely inhibited by allopurinol, a potent inhibitor of xanthine dehydrogenase (EC 1.2.1.37). The data indicated a purine-based pathway for ureide synthesis by cowpea nodules, and this suggestion is discussed.     

Diazotrophic growth of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata under microaerobic conditions in the dark

Diazotrophy of Rhodopseudomonas acidophila and Rhodopseudomonas capsulata was not obligatorily linked to photosynthesis. In the dark R. acidophila grew with dinitrogen as sole nitrogen source at a dissolved oxygen tension of 15 Torr (= 2.0 kPa); the doubling time was 8 h. Acetylene reduction by whole cells was more sensitive to oxygen in the light than in the dark. 16.5 mg N₂ were fixed per g lactic acid consumed. R. capsulata synthesized nitrogenase and fixed dinitrogen in the dark at a dissolved oxygen tension of less than one Torr (= 0.13 kPa). The doubling time of this bacterium was 16 h and 10.5 mg N₂ were fixed per g lactic acid consumed.Abbreviation kPa kilopascal     

The relationship between uric acid and its oxidative product allantoin: a potential indicator for the evaluation of oxidative stress in birds

Uric acid is the main nitrogenous waste product in birds but it is also known to be a potent antioxidant. Hominoid primates and birds lack the enzyme urate oxidase, which oxidizes uric acid to allantoin. Consequently, the presence of allantoin in their plasma results from non-enzymatic oxidation. In humans, the allantoin to uric acid ratio in plasma increases during oxidative stress, thus this ratio has been suggested to be an in vivo marker for oxidative stress in humans. We measured the concentrations of uric acid and allantoin in the plasma and ureteral urine of white-crowned sparrows (Zonotrichia leucophrys gambelii) at rest, immediately after 30 min of exercise in a hop/hover wheel, and after 1 h of recovery. The plasma allantoin concentration and the allantoin to uric acid ratio did not increase during exercise but we found a positive relationship between the concentrations of uric acid and allantoin in the plasma and in the ureteral urine in the three activity phases. In the plasma, the slope of the regression describing the above positive relationships was significantly higher immediately after activity. We suggest that the slope indicates the rate of uric acid oxidation and that during activity this rate increases as a result of higher production of free radicals. The present study demonstrates that allantoin is present in the plasma and in the ureteral urine of white-crowned sparrows and therefore might be useful as an indicator of oxidative stress in birds.     

Modulation by fumarate of a P i -insensitive pyruvate kinase from Rhodopseudomonas capsulata

Cold lability was found to be responsible for the initial failure to detect pyruvate kinase activity in extracts of the facultative phototroph, Rhodopseudomonas capsulata. Taking advantage of the reversal of cold inactivation by high concentrations of monovalent cations, the enzyme could be partially purified by (NH₄)₂SO₄-precipitation and gelfiltration. In contrast to the enzyme from Rhodospirillum rubrum, the pyruvate kinase from R. capsulata is nearly insensitive to inorganic phosphate. Instead, it is susceptible to allosteric inhibition by fumarate. Adenosinemonophosphate and sugar phosphates as activators prevent the inhibitory action of fumarate.     

Purine metabolites in serum of higher primates,including man

Using a new method for simultaneous quantification of hypoxanthine, xanthine, uric acid, and allantoin by means of high-pressure liquid chromatography, purine metabolites of 18 species of higher primates, including man, have been determined. The data thus produced indicate that the serum concentrations of purine metabolites in primates are influenced by nutrition, sexual hormones, and the procedures used in catching the animals for venipuncture. In the Callitrichidae examined, serum concentrations of purines differ significantly from species to species. The results of a nutritional test show that Callithrix jacchus possesses an efficient system for degradation of dietary purines.     

微生物来源的尿酸氧化酶的研究进展及应用前景

尿酸氧化酶是一种重要的医药用酶,它催化嘌呤代谢途径中的尿酸氧化生成尿囊素和过氧化氢,因而被广泛用于治疗痛风,检测血液尿酸浓度,预防和治疗由于肿瘤化学治疗引起的高尿酸血症。综述了尿酸氧化酶的来源、酶学性质、基因克隆与表达及其用途,并对其在应用中存在的问题和前景作了展望。     

Carbon assimilation and extracellular antigens of some yeast-like fungi

Many yeast-like fungi assimilated n-hexadecane, butylamine and putrescine as sole carbon sources. Methanol was not assimilated. This points to a physiological similarity to endomycetous, hydrocarbon-utilizing yeasts. Stephanoascus ciferrii assimilated uric acid, adenine and allantoin as sole source of carbon and nitrogen. All strains of Geotrichum candidum and many other yeast-like fungi assimilated acetoin and butan-2,3-diol. Assimilation tests for adenine, uric acid, allantoin, acetoin and butan-2,3-diol were found to be suitable for taxonomic purposes.Extracellular antigens immunologically related to those produced by Geotrichum candidum were detected in the cell-free culture liquids of several yeast-like fungi. The extracellular antigen excreted by Stephanoascus ciferrii was species-specific.     

Linkage analysis with an interbreed backcross maps Dalmatian hyperuricosuria to CFA03

Dalmatians, like humans, excrete uric acid in their urine. All other dogs and most mammals excrete allantoin, a water-soluble compound that is further along the purine degradation pathway. Excretion of uric acid at high concentrations (hyperuricosuria) predisposes Dalmatians to the formation of urinary urate calculi. Hyperuricosuria (huu) is found in all Dalmatians tested and is inherited as an autosomal recessive trait. A genome scan and linkage analysis performed on a Dalmatian × Pointer interbreed backcross detected a single linked marker, REN153P03, located on CFA03. Haplotype analysis of the region around this marker defined a 3.3-Mb interval flanked by single recombination events. This interval, which contains the huu mutation, is estimated to include 24 genes. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Biosynthesis and metabolism of purine alkaloids in leaves of cocoa tea (Camellia ptilophylla)

The biosynthesis and metabolism of purine alkaloids in leaves ofCamellia ptilophylla (cocoa tea), a new tea resource in China, have been investigated. The major purine alkaloid was theobromine, with theophylline also being present as a minor component. Caffeine was not accumulated in detectable quantities. Theobromine was synthesized from [8-¹⁴C] adenine and the rate of its biosynthesis in the segments from young and mature leaves from flush shoots was approximately 10 times higher than that from aged leaves from 1-year old shoots. Neither cellfree extracts nor segments fromC. ptilophylla leaves could convert theobromine to caffeine. A large quantity of [2-¹⁴C] xanthine taken up by the leaf segments was degraded to¹⁴CO₂ via the conventional purine catabolic pathway that includes allantoin as an intermediate. However, small amounts of [2-¹⁴C] xanthine were also converted to theobromine. Considerable amounts of [8-¹⁴C] caffeine exogenously supplied to the leaf segments ofC. ptilophylla was changed to theobromine. These results indicate that leaves ofC. ptilophylla exhibit unusual purine alkaloid metabolism as i) they have the capacity to synthesize theobromine from adenine nucleotides, but they lack adequate methyltransferase activity to convert of theobromine to caffeine in detectable quantities, ii) the leaves have a capacity to convert xanthine to theobromine, probably via 3-methylxanthine.     

Mesure d'une activité métabolique in vivo. Cas du catabolisme purique chez le Pharbitis nil

In vivo measurement of a metabolic activity. — Purine catabolism in Pharbitis nil cotyledons. To study the influence of light and darkness on the level of purine degradation, an in vivo method of measuring this catabolic pathway has been developed. Cotyledon discs of Pharbitis nil were incubated in an aqueous solution of hypoxanthine-8-¹⁴C. This purine base was first oxidized by a NAD⁺ dependent xanthine dehydrogenase. The metabolic flow was expressed either by the quantity of products (xanthine + uric acid + allantoin + allantoic acid) or by the rate of hypoxanthine degradation in the discs. This easy, reproducible and very sensitive technique of measurement has been investigated with respect to several variables including tissue quantity, length of incubation time, hypoxanthine and NAD⁺ concentrations, and pH. The quantity of products varied proportionally with the tissue quantity but the catabolic rate was not linked to this. The best conditions of measurement were to incubate discs for at least 60 min in distilled water containing only hypoxanthine-8-¹⁴C at low concentrations (below 0.25 mM). The values obtained represent the actual in vivo level of the studied metabolic pathway. The NAD⁺ concentration within the tissue does not seem to be a limiting factor for hypoxanthine degradation, the hypoxanthine concentration itself appears to be the only limiting factor in endogenous purine catabolism.     

Identification of Products from Oxidation of Uric Acid Induced by Hydroxyl Radicals

The aim of the present study was to separate and characterise products formed by oxidation of uric acid by hydroxyl radicals with a view to probing for these products in vivo in clinical contexts. Aerated solutions of 200 μM uric acid, or its oxidation products, allantoin or parabanic acid, were exposed to gamma radiolysis, (52.0 Gy/min), as a source of HO- radicals, at pH 3.4 and 7.4. Aliquots were taken every 5 minutes for 20 minutes and oxidation products were separated by HPLC and analysed with a diode array detector. Identities of oxidation products were confirmed on the basis of similarity of retention times and absorbance spectra and peak purity parameters of known standards. Hydroperoxides were measured by tri-iodide formation in the 20 minute sample. Exposure of uric acid to such HO fluxes produced a net loss of the parent compound with formation of a complex mixture of products with allantoin and parabanic acid being the predominant products at pH 3.4. The rate of uric acid degradation at physiological pH was slower and the distribution of oxidation products was different. A small but significant amount of uric acid hydroperoxide was detected at both pHs. A mechanism for uric acid oxidation under these conditions is presented.     

Ureide biogenesis and the enzymes of ammonia assimilation and ureide biosynthesis in nitrogen fixing pigeonpea (Cajanus cajan) nodules

Allantoic acid production from IMP, XMP, inosine, xanthosine, hypoxanthine, xanthine, uric acid and allantoin was investigated by incubating each of these substrates withCajanus cajan cytosol and bacteroid fractions separately in the presence and absence of NAD+ and allopurinol. Allantoic acid synthesis by bacteroid fraction could only be observed with uric acid and allantoin as substrates. Addition of NAD⁺ or allopurinol to the reaction mixtures had no effect. However, with cytosol fraction, allantoic acid was produced by each of these substrates, with maximum rate with allantoin. With NAD⁺ or with allopurinol, allantoic acid was produced only with uric acid and allantoin as substrates. NADH production with cytosol fraction could again be observed with all the substrates. Except with uric acid and allantoin, allopurinol completely inhibited NADH formation. Regardless of the presence or absence of allopurinol, none of the substrates exhibited significant activity with bacteroid fraction. Based on the activities of glutamine synthetase, glutamate synthase, glutamate dehydrogenase, aspartate aminotransferase, asparagine synthetase, nucleotidase, nucleosidase, xanthine de-hydrogenase, uricase and allantoinase and their intracellular localisation in various nodule fractions, a probable pathway for the biogenesis of ureides in pigeonpea nodules has been proposed