Pre-analytical factors affecting ascorbic and uric acid quantification in human plasma

We have recently described a new capillary electrophoresis assay to measure serum ascorbic and uric acids in which a baseline separation of peaks was obtained in less than 4 min by using a 60.2 cm x 75 microm uncoated capillary with a 100 mmol/L sodium borate running buffer pH 8. Since during sample preparation AA is rapidly oxidized, we employed our new capillary electrophoresis method to analyze the pre-analytical factors affecting its stability. In particular we evaluated how the standard mix preparation, the blood collection (plasma EDTA or serum) and the plasma protein precipitation influence the results of analysis. Our data suggest that standard ascorbate must be dissolved in a solution containing cysteine and EDTA in order to avoid oxidation and that EDTA blood collection is better than serum for AA measurement. Moreover, the type and the quantity of the precipitating compound are critical parameters to obtain a complete recovery of analytes. We performed AA and UA analysis in 32 healthy volunteers with the optimized experimental conditions by using our capillary electrophoresis method and a reference CE assay. Obtained data were compared to Bland-Altman test to verify the accuracy of our CZE method.     

The cincinnati lipid research clinic family study: Familial determinants of plasma uric acid

Summary Commingling analysis of plasma uric acid levels in a random sample of 160 nuclear families supports the hypothesis that there is a mixture of three distributions. Assuming one, two, and three components in the underlying distribution, we obtained the corresponding p-values (for power transformation) as 0.059, 1.040, and 1.643, respectively. Path analysis with p=0.059 gives genetic (h ²) and cultural (c ²) heritabilities as 0.256 and 0.199, without much support for intergenerational differences, assortative mating, or maternal effects. Complex segregation analysis with p=0.059 supports multifactorial inheritance, consistent with the findings of Gulbrandsen et al. (1979) and Morton (1979) in other populations. This study also fails to support a major locus hypothesis, contrary to earlier reports.This work was supported in part by N.I.H. and N.I.M.H. Grants GM 28719, and MH 31302, and by contract NO-1-HV-2-2914L from the National Heart, Lung, and blood Institute (Lipid Research Clinic's Program), General Clinical Research Center, and the CLINFO center Grant RR-00068-19     

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.     

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 acarbose on the increased plasma concentration of uric acid induced by sucrose ingestion

Sucrose is converted fructose and glucose, which may increase plasma uric acid concentration (pUA) through increased purine degradation and/or decreased uric acid (UA) excretion. To investigate effects of acarbose, an inhibitor of alpha-glucosidase, on the increased pUA from sucrose administration, we measured pUA and urinary UA excretion in 6 healthy subjects before and after administering sucrose, with and without co-administration of acarbose. Sucrose raised pUA by 10% (p < 0.01). However, excretion and fractional clearance of UA were unchanged. Sucrose and acarbose coadministration also increased pUA, but less than did sucrose alone (sucrose: 4.9 to 5.4 mg/dl; sucrose + acarbose, 4.7 to 4.9 mg/dl, p < 0.05) without changes in urinary excretion and fractional clearance of UA. Acarbose appears to attenuate the rise in pUA by sucrose ingestion by inhibiting sucrose absorption.     

Manipulation of plasma uric acid in broiler chicks and its effect on leukocyte oxidative activity

Birds have high metabolic rates, body temperatures, and plasma glucose concentrations yet physiologically age at a rate slower than comparably sized mammals. These studies were designed to test the hypothesis that the antioxidant uric acid protects birds against oxidative stress. Mixed sex broiler chicks (3 wk old) were fed diets supplemented or not with purines (0.6 mol hypoxanthine or inosine). Study 1 consisted of 18 female Cobb x Cobb broilers that were fed purines for 7 days, whereas study 2 consisted of 12 males in a 21-day trial. Study 3 involved 30 mixed sex broilers that were fed 40 or 50 mg allopurinol/kg body mass (BM) for 21 days, a drug that lowers plasma uric acid (PUA). PUA and leukocyte oxidative activity (LOA) were determined weekly for all studies. For study 2, pectoralis major shear force, relative kidney and liver sizes (RKS and RLS), and plasma glucose concentrations were also determined. In study 1, PUA concentration was increased three- and twofold (P < 0.001) in birds fed inosine or hypoxanthine, respectively, compared with control birds. LOA of birds supplemented with inosine was lower (P < 0.05) than that of control or hypoxanthine birds. In study 2, PUA concentrations were increased fivefold (P < 0.001) in birds fed inosine and twofold (P < 0.001) in birds fed hypoxanthine compared with control birds at day 21. RKS (g/kg BM) was greater (P < 0.001) for chicks fed purine diets compared with control chicks. Muscle shear value was lower (P < 0.05) in chicks fed purine diets. PUA concentration was decreased (P < 0.001) in birds consuming allopurinol diets, whereas LOA was increased (P < 0.01) in study 3. These studies show that PUA concentrations can be related to oxidative stress in birds, which can be linked to tissue aging.     

Allantoin,the oxidation product of uric acid is present in chicken and turkey plasma

Urate oxidase is not present in birds yet allantoin, a product of this enzyme, has been measured in birds. Studies were designed to compare the concentrations of plasma purine derivatives in chickens and turkeys fed inosine-supplemented diets. The first study consisted of 12 male chicks that were fed diets supplemented with 0.6 mol inosine or hypoxanthine per kilogram diet from 3- to 6-week-old. Study 2 consisted of 12 turkey poults (toms) fed inosine-supplemented diets (0.7 mol/kg) from 6- to 8-week-old. Plasma allantoin and oxypurines concentrations were measured weekly using high performance liquid chromatography. Plasma uric acid (PUA) in chickens fed inosine-supplemented diets increased from 0.31 to 1.34 mM (P<0.05) at the end of week 2. In turkeys, those fed control diet had 0.17 mM PUA concentration compared to 0.3 mM in those fed the inosine diet at week 2 (P<0.05). Allantoin concentration increased in chickens from week 1 to 2 while a decrease was observed in turkeys (P<0.005) for both treatments. These data show that allantoin is present in turkey and chicken plasma. The presence of allantoin in avian plasma is consistent with uric acid acting as an antioxidant in these species.     

Plasmodium-induced inflammation by uric acid

Infection of erythrocytes with the Plasmodium parasite causes the pathologies associated with malaria, which result in at least one million deaths annually. The rupture of infected erythrocytes triggers an inflammatory response, which is induced by parasite-derived factors that still are not fully characterized. Induced secretion of inflammatory cytokines by these factors is considered a major cause of malaria pathogenesis. In particular, the inflammatory cytokine tumor necrosis factor (TNF) is thought to mediate most of the life-threatening pathologies of the disease. Here we describe the molecular characterization of a novel pathway that results in the secretion of TNF by host cells. We found that erythrocytes infected by Plasmodium accumulate high concentrations of hypoxanthine and xanthine. Degradation of Plasmodium-derived hypoxanthine/xanthine results in the formation of uric acid, which triggers the secretion of TNF. Since uric acid is considered a "danger signal" released by dying cells to alert the immune system, Plasmodium appears to have co-evolved to exploit this warning system. Identifying the mechanisms used by the parasite to induce the host inflammatory response is essential to develop urgently needed therapies against this disease.     

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.     

Free radical metabolite of uric acid

Uric acid has previously been shown to act as a water-soluble antioxidant. Although the antioxidant activity of uric acid has been attributed to its ability to scavenge free radicals, the one-electron uric acid oxidation product of such a scavenging reaction has not been detected. It order to determine whether a free radical metabolite of uric acid could be formed via one-electron redox processes, we oxidized uric acid with potassium permanganate, horseradish peroxidase/hydrogen peroxide, and hematin/hydrogen peroxide systems. With the use of the rapid-mixing, continuous-flow electron spin resonance technique, we were able to detect the urate anion free radical in all three radical-generating systems. Based on N15-isotopic-labeling experiments, we show that the unpaired electron of this radical is located primarily on the five-membered ring of the purine structure. We were also able to demonstrate that this radical could be scavenged by ascorbic acid.     

Analysis of excretion fraction of uric acid

Excretion fraction of uric acid (EFUA), is one of the most important hallmarks for diagnosis of familial juvenile hyperuricemic nephropathy (FJHN) and hereditary renal hypouricemia. EFUA was measured in 20 patients with FJHN. However, low excretion fraction (<6%) was found also in healthy FJHN family members and healthy controls (ref. ranges EFUA: men 6-12%, women 6-20%). Similar finding of low EFUA was reported recently. Distribution of EFUA was further studied in 2,416 healthy controls, which were selected from 6,000 samples and divided according to age. In conclusion, finding of low EFUA in family members is a risk factor for renal damage and indication for purine metabolic investigations with subsequent molecular biology analysis. As EFUA could be found also in healthy controls--it should be interpreted with care and other features of FJHN (such as hyperuricemia, progressive renal disease in family) should be taken to account.