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.     

Blood uric acid levels during hyperthermic stress.

Non-diabetic and streptozotocin diabetic adult male Sprague-Dawley derived rats were divided into normothermic and hyperthermic groups. Hyperthermia was achieved in an environmental chamber. Blood uric acid and lactate levels were performed on all animals. Significant increases in blood uric acid levels were found in all animals during hyperthermia. Blood lactic acid was also increased during hyperthermia indicating tissue hypoxia. It has been shown that increases in the breakdown of purine nucleotides occurs during hypoxia. This study indicates that increases in blood uric acid levels is an expected response to hyperthermic stress and is probably due to tissue hypoxia.     

Degradation of uric acid to urea and glyoxylate in peroxisomes.

The distribution of enzymes involved in purine degradation in fish and crustaceous liver was examined by centrifugation in a sucrose density gradient. In mackerel, yellow mackerel, and prawn liver and mantis club hepatopancreas, uricase and allantoinase were located only in the peroxisomes and in the soluble fraction from broken peroxisomes, and allantoicase was located only in the peroxisomes. Uricase and allantoinase seem to be located in the peroxisomal matrix and allantoicase in the peroxisomal membrane. Adenase, guanase, and xanthine oxidase were present only in the soluble fraction of mackerel liver.     

Histochemical studies of uric acid in some insects. 3. Excretion of uric acid by the malpighian tubules in Calliphora vicina and Rhodnius prolixus

In adult Calliphora uric acid is excreted throughout the Malpighian tubules. Histochemical preparations for the light microscope show uric acid passing through the cells and forming crystalline spheres in immediate contact with the microvilli. Uric acid appears to be synthesized and discharged into the haemolymph by the fat body cells. In Rhodnius there is no visible uric acid in the cells or lumen of the upper segment of the tubule (two-thirds of the total length of the tubule) apart from occasional deposits in the basal lamina. All uric acid excretion depends on the lower segment. Electron micrographs after argentaffin staining show high concentration of uric acid in the cytoplasm below the basal lamina (which also contains uric acid deposits). Uric acid is visible throughout the cell, particularly aroand the mitochondria; it is absent from the infolded plasma membrane and from all vacuoles. At the lumen there is a concentrated deposit of uric acid immediately beyond the plasma membrane. The uric acid particles unite with particles of unstained matrix material to form crystalline spheres. The fat body shows active synthesis of uric acid which is discharged by the cells into the intercellular channels and so to the basal lamina through which it passes into the haemolymph. As judged by histochemical preparations the haemolymph contains a high concentration of uric acid, very variable in different sites. Likewise large variations in uric acid secretion occur in different parts of the fat body.     

Investigation of the enzymic and electrochemical oxidation of uric acid derivatives.

The electrochemical oxidation of a number of N-methylated uric acids at the pyrolytic graphite and gold electrodes has been compared to their enzymic oxidation with type VIII peroxidase and H2O2. Spectral, electroanalytical and kinetic evidence supports the conclusion that for all compounds the electrochemical and enzymic reactions proceed by identical mechanisms.     

Discrete analysis of serum uric acid with immobilized uricase and peroxidase.

Commercially available uricase and peroxidase have been immobilized onto alkylamine glass and arylamine glass beads respectively. A discrete method has been developed to determine uric acid in serum using immobilized uricase and peroxidase. The method is based on generation of H2O2 from serum uric acid by immobilized uricase and its measurement by a colour reaction catalyzed by immobilized peroxidase. The minimum detection limit of the method was 8 microg/0.1 ml sample. The mean analytical recovery of added uric acid in serum was 87.5%. The within and between assay coefficient of variation (C.V.) were <6.58% and <10.77% respectively. The serum uric acid in apparently healthy adults and persons suffering from different disease was found to be 25-55 microg/ml, 32+/-2.25 (range, mean+/-S.D.) and 55-200 microg/ml; 52+/-6.4 (range, mean+/-S.D.) respectively by our method. A good correlation (r = 0.8170) was obtained between the serum urate values by this method and with those obtained by commercial Enzo-kit method.     

Effect of KC1 on renal uric acid excretion.

The effect of two days' KC1 administration per os (total amount 11 g, i.e. 140 mEq potassium) on renal uric acid excretion was studied in healthy subjects under conditions of water diuresis. A significant increase in the uric acid excretory fraction (CUA/CCr..100) was found, from 8.04% in the control test to 10.31% under experimental conditions. Elevated renal uric excretion led to a significant drop in the plasma uric acid level from 4.9 mg% to 4.2 mg% after the administration to KC1. The findings suggest that KC1 influences the tubular transport of uric acid, but the mechanism of its action is still obscure.     

血尿酸与代谢性风险

越来越多研究表明,高血尿酸与代谢综合征密切相关。传统观点认为尿酸水平仅是代谢综合征的一个生物标志而不能预测代谢综合征,这种观点可能被修改。降低血尿酸可能是预防糖尿病及多种慢性疾病的一种新的治疗方法。     

Electron-paramagnetic-resonance spectroscopy of complexes of xanthine oxidase with xanthine and uric acid.

Rat fibrinogen was purified from rat plasma by using lysine–Sepharose chromatography, repeated precipitation with 25%-satd. (NH₄)₂SO₄ and gel chromatography on Sepharose 6B. To minimize proteolytic activity, rats were injected intravenously with Trasylol before bleeding and the collected blood was treated with Trasylol and di-isopropyl phosphorofluoridate. A 95%-clottable preparation was obtained in 70–75% yield; it proved to be free of factor XIII and plasminogen. It showed a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and on disc electrophoresis in 8m-urea. Alanine was the only detectable N-terminal amino acid. After reduction and modification of the thiol groups, the material could be separated into three distinct chains (Aα, Bβ and γ) by pore-limit polyacrylamide slab-gel electrophoresis in the presence of sodium dodecyl sulphate. The amino acid compositions of the whole fibrinogen and of the separated modified chains were determined. The molecular weights were 61000, 58000 and 51000 for Aα-, Bβ- and γ-chains respectively. Our results for the chains are in contrast with previous reports on rat fibrinogen [Bouma & Fuller (1975) J. Biol. Chem. 250, 4678–4683; Stemberger & Jilek (1976) Thromb. Res. 9, 657–660], in which no separation between Aα- and Bβ-chains was achieved on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis for 3h. Evidence is presented that this is probably due to Aα-chain degradation as a result of incomplete inhibition of proteolytic enzymes during the purification. Complete inhibition of proteolytic activities is essential in all steps of the present purification procedure.     

Reaction of uric acid with peroxynitrite and implications for the mechanism of neuroprotection by uric acid

Peroxynitrite, a biological oxidant formed from the reaction of nitric oxide with the superoxide radical, is associated with many pathologies, including neurodegenerative diseases, such as multiple sclerosis (MS). Gout (hyperuricemic) and MS are almost mutually exclusive, and uric acid has therapeutic effects in mice with experimental allergic encephalomyelitis, an animal disease that models MS. This evidence suggests that uric acid may scavenge peroxynitrite and/or peroxynitrite-derived reactive species. Therefore, we studied the kinetics of the reactions of peroxynitrite with uric acid from pH 6.9 to 8.0. The data indicate that peroxynitrous acid (HOONO) reacts with the uric acid monoanion with k = 155 M(-1) s(-1) (T = 37 degrees C, pH 7.4) giving a pseudo-first-order rate constant in blood plasma k(U(rate))(/plasma) = 0.05 s(-1) (T = 37 degrees C, pH 7.4; assuming [uric acid](plasma) = 0.3 mM). Among the biological molecules in human plasma whose rates of reaction with peroxynitrite have been reported, CO(2) is one of the fastest with a pseudo-first-order rate constant k(CO(2))(/plasma) = 46 s(-1) (T = 37 degrees C, pH 7.4; assuming [CO(2)](plasma) = 1 mM). Thus peroxynitrite reacts with CO(2) in human blood plasma nearly 920 times faster than with uric acid. Therefore, uric acid does not directly scavenge peroxynitrite because uric acid can not compete for peroxynitrite with CO(2). The therapeutic effects of uric acid may be related to the scavenging of the radicals CO(*-)(3) and NO(*)(2) that are formed from the reaction of peroxynitrite with CO(2). We suggest that trapping secondary radicals that result from the fast reaction of peroxynitrite with CO(2) may represent a new and viable approach for ameliorating the adverse effects associated with peroxynitrite in many diseases.