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Introduction: Rapid eye movement (REM) sleep behavior disorder (RBD) is associated with an increased risk of developing Parkinson's disease (PD). Low uric acid (UA) levels are associated with the risk of development and progression of PD. Allantoin is the major oxidation product of UA and is considered as a biomarker of oxidative stress. We aimed to compare serum levels of UA, allantoin, and allantoin/UA ratio in RBD patients with those in healthy controls, and to examine their associations with clinical severity., Methods: We evaluated serum levels of UA, allantoin, and allantoin/UA ratio in 38 RBD patients (one female, mean age 66.8 (SD 6.3) years) and in 47 controls (four females, 66.8 (7.6) years). All RBD patients were assessed according to an examination protocol, which included structured interview, Montreal Cognitive Assessment (MoCA), Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS), and dopamine transporter single-photon emission computed tomography (DAT-SPECT). The lower putaminal binding ratio from both hemispheres was used for analysis., Results: Mean serum allantoin concentration and allantoin/UA ratio were significantly increased in the RBD group compared to controls (2.6 (1.8) vs. 1.4 (0.7) μmol/l, p = 0.0004, and 0.008 (0.004) vs. 0.004 (0.002), p < 0.0001, respectively). There were no significant differences in UA levels between the two groups. No significant associations between any biochemical parameter and RBD duration, putaminal binding ratio on DAT-SPECT, MDS-UPDRS, or MoCA score were found., Conclusion: Serum allantoin and allantoin/UA ratio are increased in RBD patients in comparison to controls, which may reflect increased systemic oxidative stress in prodromal synucleinopathy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Allantoin is an excellent biomarker of oxidative stress in humans as the main product of uric acid oxidation by reactive oxygen species. Yet, allantoin determination is still not routinely performed in clinical laboratories. Therefore, we developed a fast, simple, selective, and sensitive UHPLC-MS/MS method for allantoin determination in human serum using an isotopically labeled internal standard. Our analytical protocol provided high sensitivity by mass spectrometry detection and high throughput by HILIC-MS/MS analysis within 4 min, with one-step serum sample preparation approximately within 7 min. Lastly, our protocol was fully validated to demonstrate its reliability in allantoin determination in human serum. The method showed an excellent linear range from 0.05 to 100 μM, with precision ranging from 1.8 to 11.3% (RSD), and with accuracy (relative error %) within ±6.0%. The method was then applied to analyze the concentration of allantoin in serum samples from 71 patients with chronic gout without treatment with xanthine oxidase inhibitors. The median serum allantoin concentration in the cohort was 2.8 μM (n = 71). Overall, our simple analytical protocol has the potential to be easily implemented in clinical routine practice for monitoring allantoin as a key oxidative stress biomarker., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In patients with gout, the serum uric acid (SUA) is usually lower during acute gouty attacks than during intercritical periods. It has been suggested that systemic inflammatory response can cause this phenomenon. The objective is to determine whether therapy with TNF inhibitors (TNFis) affects SUA levels in patients with systemic autoimmune rheumatic diseases (SARDs) and whether SUA changes correlate with pro-inflammatory cytokines or with the oxidative stress marker allantoin. In this study, SUA, CRP, creatinine, MCP-1, IFN-α2, IFN-γ, Il-1β, IL-6, IL-8, IL-10, IL-12, IL-17a, IL-18, IL-23, IL-33, TNF-α, and allantoin levels were measured prior to and after 3 months of TNFis treatment in patients with SARDs. The values obtained in the biochemical assays were then tested for associations with the patients' demographic and disease-related data. A total of 128 patients (rheumatoid arthritis, n = 44; ankylosing spondylitis, n = 45; psoriatic arthritis, n = 23; and adults with juvenile idiopathic arthritis, n = 16) participated in this study. Among the entire patient population, SUA levels significantly increased 3 months after starting treatment with TNFis (279.5 [84.0] vs. 299.0 [102.0] μmol/l, p < 0.0001), while the levels of CRP, IL-6, IL-8, and MCP-1 significantly decreased. Male sex was the most powerful baseline predictor of ΔSUA in univariate and multivariate models. None of the measured laboratory-based parameters had statistically significant effects on the magnitude of ΔSUA. 3 months of anti-TNF therapy increased the levels of SUA in patients with SARDs, but neither the measured pro-inflammatory cytokines nor the oxidation to allantoin appeared responsible for this effect.

Chronic kidney disease (CKD) involves significant metabolic abnormalities and has a high mortality rate. Because the levels of serum metabolites in patients with CKD might provide insight into subclinical disease states and risk for future mortality, we determined which serum metabolites reproducibly associate with mortality in CKD using a discovery and replication design. Metabolite levels were quantified via untargeted liquid chromatography and mass spectroscopy from serum samples of 299 patients with CKD in the Modification of Diet in Renal Disease (MDRD) study as a discovery cohort. Six among 622 metabolites were significantly associated with mortality over a median follow-up of 17 years after adjustment for demographic and clinical covariates, including urine protein and measured glomerular filtration rate. We then replicated associations with mortality in 963 patients with CKD from the African American Study of Kidney Disease and Hypertension (AASK) cohort over a median follow-up of ten years. Three of the six metabolites identified in the MDRD cohort replicated in the AASK cohort: fumarate, allantoin, and ribonate, belonging to energy, nucleotide, and carbohydrate pathways, respectively. Point estimates were similar in both studies and in meta-analysis (adjusted hazard ratios 1.63, 1.59, and 1.61, respectively, per doubling of the metabolite). Thus, selected serum metabolites were reproducibly associated with long-term mortality in CKD beyond markers of kidney function in two well characterized cohorts, providing targets for investigation., (Copyright © 2018 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Allantoin is the main product of uric acid oxidation and was found to be augmented in atherosclerotic plaque in human autopsy and in animal models of atherosclerosis. Uric acid is abundant in human plasma and is prone to oxidation in inflammatory conditions such as atherosclerosis. In this study, we found a significant increase in plasma uric acid (P=0.002) and allantoin (P=0.025) in participants of the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) that presented common carotid intima-media thickness (c-IMT) within the 75th percentile (c-IMT≥P75). Multiple linear regression showed an association of c-IMT with uric acid (β=0.0004, P=0.014) and allantoin (β=0.018, P=0.008). This association was independent of age, the traditional risk factor LDL/HDL ratio, and non-traditional risk factors: pulse pressure, neck circumference, and the inflammatory marker myeloperoxidase. The independent and strong association of allantoin with c-IMT shows that it might be a useful marker, along with other traditional risk factors, to evaluate an early stage of atherosclerosis.

Background and Aims: Chronic kidney disease (CKD) is characterized by increased oxidative stress (OS). In consideration of the well-known link between OS and DNA methylation we assessed DNA methylcytosine (mCyt) concentrations in CKD patients at baseline and during cholesterol lowering treatment., Methods and Results: DNA methylation and OS indices (malonyldialdehyde, MDA; allantoin/uric acid ratio, All/UA) were measured in 30 CKD patients randomized to three cholesterol lowering regimens for 12 months (simvastatin 40 mg/day, ezetimibe/simvastatin 10/20 mg/day, or ezetimibe/simvastatin 10/40 mg/day) and 30 age- and sex-matched healthy controls. DNA methylation was significantly lower in CKD patients vs. controls (4.06 ± 0.20% vs. 4.27 ± 0.17% mCyt, p = 0.0001). Treatment significantly increased mCyt DNA concentrations in all patients (4.06 ± 0.04% at baseline; 4.12 ± 0.03% at 4 months; 4.17 ± 0.03% at 8 months; and 4.20 ± 0.02% at 12 months, p = 0.0001 for trend). A trend for a greater effect on DNA methylation was observed with combined treatment ezetimibe/simvastatin 10/40 mg/day (+5.2% after one year treatment). The treatment-associated mCyt increase was significantly correlated with the concomitant reduction in MDA concentrations and All/AU ratios., Conclusion: Our results demonstrate that CKD patients have a lower degree of DNA methylation and that cholesterol lowering treatment restores mCyt DNA concentrations to levels similar to healthy controls. The treatment-associated increase in DNA methylation is correlated with a concomitant reduction in OS markers. The study was registered at clinicaltrials.gov (NCT00861731)., (Copyright © 2017 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background Oxidative stress (OS) represents the primary mediator of chronic heart failure (CHF) development and progression. It is well established that homocysteine is able to generate reactive oxygen species. Small amounts of allantoin in human serum result from free radical action on urate and may provide a stable marker for in vivo free radical activity. To investigate whether some easily measurable indexes such as antioxidants (uric acid, glutathione) and related molecules (allantoin, homocysteine and cysteine) can serve as OS biomarkers. Methods We investigated 75 stable CHF patients. Aminothiols and purine compound levels were determined by capillary electrophoresis. Results The homocysteine level was markedly elevated in CHF patients, whatever the aetiology. Parameters of the transsulfuration pathway and the investigated purine compounds were significantly increased. Conversely, total glutathione was decreased. The allantoin/uric acid ratio was significantly higher in CHF patients with an hyperhomocysteinaemia >17 μmol/L. All parameters of the transsulfuration and purine degadation pathways were significantly correlated, suggesting an OS in CHF patients. Conclusion Our data show an imbalance of serum aminothiols and purine compounds in these CHF patients on adapted therapy. We suggest that the evaluation and control of these new markers may help improve the OS that participates in the progression of the disease.

It is controversial that uric acid (UA) levels are related to the severity of hypertension in preeclampsia (PE). Our aim in this study was to determine whether UA, xanthine oxidase activity (XOA), allantoin and nitrite levels are related to arterial blood pressure (BP) in PE. We formed a control group (n = 20) and a PE group (n = 20) for the study. Their BPs and plasma UA, XOA, allantoin and nitrite levels were measured. The values from the control and PE pregnant women were assessed via a Wilcoxon matched-pairs test. A Pearson correlation test was also performed. In addition, the diagnostic value of these tests was evaluated via receiver operating characteristic (ROC) analysis. The BP, UA, XOA and allantoin levels in the PE patients were found to be higher when compared with those of the pregnant controls. The UA, XOA and allantoin levels showed high correlations with BP in cases of PE. However, there was no superiority among the correlations. No differences were observed between the groups in terms of nitrite levels and the relationship between nitrite and BP. UA, XOA and allantoin levels may be high due to placental cell death because of abnormal trophoblastic activity observed in PE. Moreover, the reactive oxygen products that are created during the genetic material degradation may explain how UA, XOA and allantoin levels are related to BP. According to ROC analysis, UA, XOA and allantoin assays are reliable predictors for the determination of PE.

Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM), which is a major public health problem in the world. To reveal the metabolic changes associated with DN, we analyzed the serum, urine, and renal extracts obtained from control and streptozotocin (STZ)-induced DN rats by (1)H NMR-based metabonomics and multivariate data analysis. A significant difference between control and DN rats was revealed in metabolic profiles, and we identified several important DN-related metabolites including increased levels of allantoin and uric acid (UA) in the DN rats, suggesting that disturbed purine metabolism may be involved in the DN. Combined with conventional histological and biological methods, we further demonstrated that xanthine oxidase (XO), a key enzyme for purine catabolism, was abnormally activated in the kidney of diabetic rats by hyperglycemia. The highly activated XO increased the level of intracellular ROS, which caused renal injury by direct oxidative damage to renal cells, and indirect inducing inflammatory responses via activating NF-κB signaling pathway. Our study highlighted that metabonomics is a promising tool to reveal the metabolic changes and the underlying mechanism involved in the pathogenesis of DN.

Background and Aim: Tryptophan (Trp) degradation via indoleamine (2,3)-dioxygenase (IDO), with consequent increased in kynurenine (Kyn) concentrations, has been proposed as marker of immune system activation. Oxidative stress (OS) might contribute to the pro-inflammatory state in chronic kidney disease (CKD) through the activation of NF-kB, with consequent activation and recruitment of immune cells., Methods and Results: Serum concentrations of Trp and Kyn, oxidative stress indices malondialdehyde (MDA) and allantoin/uric acid (All/UA) ratio and anti-oxidant amino acid taurine were measured in 30 CKD patients randomized to 40 mg/day simvastatin (group 1), ezetimibe/simvastatin 10/20 mg/day (group 2) or ezetimibe/simvastatin 10/40 mg/day (group 3) and treated for 12 months. Baseline Kyn and Kyn/Trp ratio were higher in CKD patients vs. healthy controls (1.67 ± 0.62 μmol/L vs 1.25 ± 0.40 μmol/L, p < 0.01 and 0.036 ± 0.016 vs 0.023 ± 0.010, p < 0.001 respectively). Both Kyn and Kyn/Trp ratio significantly decreased after cholesterol lowering treatment, to values comparable with healthy controls after one year treatment (1.67 ± 0.62 μmol/L vs 1.31 ± 0.51 μmol/L, p < 0.0001 and 0.036 ± 0.016 vs 0.028 ± 0.012 p < 0.0001, respectively). This was paralleled by a significant decrease in MDA (218 ± 143 nmol/L vs 176 ± 123 nmol/L, p < 0.01) and All/UA ratio (1.47 ± 0.72 vs 1.19 ± 0.51, p < 0.01) in CKD patients., Conclusions: Amelioration of both oxidative and inflammation status after cholesterol lowering treatment in CKD might be mediated by restoration of antioxidant taurine concentrations during therapy (from 51.1 ± 13.3 μmol/L at baseline to 63.1 ± 16.4 μmol/L, p < 0.001 by ANOVA), suggesting that improvement of both oxidative and inflammation status in CKD patients could be explained, at least partly, by the cholesterol lowering effects., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Uric acid is the final product of human purine metabolism. It was pointed out that this compound acts as an antioxidant and is able to react with reactive oxygen species forming allantoin. Therefore, the measurement of allantoin levels may be used for the determination of oxidative stress in humans. The aim of the study was to clarify the antioxidant effect of uric acid during intense exercise. Whole blood samples were obtained from a group of healthy subjects. Allantoin, uric acid, and malondialdehyde levels in plasma and erythrocytes were measured using a HPLC with UV/Vis detection. Statistical significant differences in allantoin and uric acid levels during short-term intense exercise were found. Immediately after intense exercise, the plasma allantoin levels increased on the average of 200 % in comparison to baseline. Plasma uric acid levels increased slowly, at an average of 20 %. On the other hand, there were no significant changes in plasma malondialdehyde. The results suggest that uric acid, important antioxidant, is probably oxidized by reactive oxygen species to allantoin. Therefore allantoin may be suitable candidate for a marker of acute oxidative stress.

Background: Allantoin in human plasma is a specific biomarker of oxidative stress. We describe a sensitive method to measure plasma allantoin using isocratic liquid chromatography and mass spectrometry (LC-MS/MS)., Methods: Direct injection of deproteinized plasma into the LC-MS/MS system was performed. The method was technically evaluated. Results on 200 healthy and 35 Type 2 diabetic Chinese subjects were compared., Results: Dose-response of allantoin was linear to at least 21 pmol (20 μmol/l in plasma); LOD was 0.16 pmol; recovery 99.2-100.2% at 1-5 μmol/l; accuracy, 98.5-100.8%; within-day and between-day CVs (n=6), <4.0% (at 5.00-40 μmol/l) and <2.0% (at 1-5 μmol/l), respectively. Plasma allantoin in diabetic patients was ~8-fold higher than in healthy subjects; mean (SD): 8.82 (7.26) and 1.08 (0.86) μmol/l, respectively (p<0.0001). Allantoin was slightly higher in healthy men than in age- and BMI-matched women: 1.21 (0.99) μmol/l, n=88 compared to 0.97 (0.74) μmol/l, n=112; p<0.001. No association with age was seen. Gender difference was also seen in the diabetes patients: men, n=14, 11.57 (8.57) μmol/l; women, n=21, 6.99 (5.75) μmol/l, p<0.05., Conclusions: Based on 95th percentiles of the healthy subjects, plasma allantoin of >2.2 μmol/l in women and >3.1 μmol/l in men indicates increased oxidative stress. Allantoin in diabetes subjects is clearly and markedly increased. The method will facilitate future studies of oxidative stress in human biomonitoring studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lipid-lowering therapy has been reported to reduce several oxidative stress (OS) markers in hypercholesterolemia. Since OS is frequently associated with renal dysfunction, we aimed to investigate the effect of hypolipidemic drugs on oxidative stress and plasma taurine (Tau), a sulfur amino acid with a marked antioxidant effect, in chronic kidney disease (CKD). We enrolled 30 CKD randomized to receive three different hypolipidemic regimens for 12 months: simvastatin alone (40 mg/day) or ezetimibe/simvastatin combined therapy (10/20 or 10/40 mg/day). Low molecular weight (LMW) thiols including homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine in their reduced and total form and oxidative stress indices as malondialdehyde (MDA) and allantoin/uric acid (All/UA) ratio were also evaluated. Tau concentration significantly increased throughout the therapy. The rise of taurine was more striking for the group with the concomitant administration of ezetimibe/simvastatin 10/40 mg/day (+31.6% after 1 year of therapy). A significant decrease of both MDA and All/UA ratio was observed during therapy for all patients (-19% for both MDA and All/UA ratio) with a more pronounced effect in patients treated with ezetimibe/simvastatin 10/40 mg/day (-26% for MDA and -28% for All/UA ratio). Besides, an increase of thiols reduced forms was found (+20.7% of LMW thiols redox status) with a greater effect in subjects treated with ezetimibe/simvastatin 10/40 mg/day (+24.7%). Moreover, we demonstrated that oxidative stress improvement during therapy was correlated with increased taurine levels. We hypothesize that taurine may be responsible for the oxidative stress improvement observed during lipid-lowering treatment through the reduction of superoxide anion production at the respiratory chain activity level.

Background: Although hyperuricemia is suggested to increase allantoin production in both pro- and antioxidant manners, it remains undetermined whether it increases the serum concentration of allantoin. In addition, since uric acid has both pro- and antioxidant actions, a decrease in the serum concentration of uric acid may have an effect on the pro-oxidant-antioxidant balance., Methods: To examine whether serum allantoin is correlated with serum urate, we measured those levels as well as other parameters in 63 healthy subjects. In addition, to determine whether serum allantoin is correlated with reactive oxygen species (ROS) biomarkers, we measured 8-hydroxy deoxyguanosine and 15-F2t-isoprostane, markers of ROS, in urine samples from 30 gout patients before and 1 year after benzbromarone treatment (50 mg/d)., Results: The serum concentration of allantoin was correlated with that of urate in healthy subjects (R = 0.27, p < 0.05). Benzbromarone treatment in the patients decreased the concentrations of allantoin and urate in serum by 17% (p < 0.05) and 49% (p < 0.05), respectively, and the benzbromarone-induced change in serum allantoin was correlated with that in serum urate (R = 0.39, p < 0.05). However, benzbromarone treatment did not change the ratios of 8-hydroxydeoxyguanosine/creatinine or 15-F2t-isoprostane/creatinine in urine., Conclusions: Our findings suggest that hyperuricemia contributes to an increase in serum concentration of allantoin, though they do not indicate that hyperuricemia is a major factor for controlling oxidative stress in vivo.

Objective: Previous studies on various enzymosomes (functional lipid vesicles encapsulating an enzyme) have been mostly carried out in vitro and have focused on preserving catalytic activity and improving the stability of the enzyme. Until now, few studies have focused on their in vivo fate. Similarly, although we have previously reported the increased in vitro uricolytic activity (about 2.2 times higher than that of free uricase, or three times higher than that of PEGylated uricase, Puricase(®), under physiological pH and temperature) and improved stability of the novel alkaline enzymosomes (functional lipid vesicles encapsulating uricase from Candida utilis: uricase-containing lipid vesicles, UOXLVs), it is still necessary to study the biological properties and hypouricemic effects of UOXLVs in vivo., Methods: The enzyme kinetics, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary safety of UOXLVs were evaluated., Results: The Michaelis constant (K(m)) value of the UOXLVs was slightly lower than that of the free enzyme. The enzyme release from the UOXLVs lasted over 12 hours and their circulation half-life was about sevenfold longer than that of the free uricase. Meanwhile, the UOXLVs had a 22-fold increase in the area under the curve compared with the free uricase. Furthermore, it took less than 3 hours for the UOXLVs to lower the plasma uric acid concentration from a high to a normal level, compared with 6 hours for the free uricase. In addition, the UOXLVs had much less immunogenicity than free uricase and were well tolerated by all animals throughout the observation period., Conclusion: The UOXLVs markedly improved the biological properties and enhanced the hypouricemic effects of uricase in vivo.

A comparison of the efficacy of the copper chelator, trientine, with combined renin angiotensin system (RAS) blockade on the progression of glomerular pathology in the diabetic (mREN-2)27 rat is reported. Animals were treated for 2 months with trientine, combined RAS blockers, combined trientine plus RAS blockers or none. Treatments began after inducing diabetes with streptozotocin. Physiological data were recorded monthly and light microscopic glomerular features were scored. Plasma allantoin and both plasma and renal protein carbonyls were measured as markers of oxidative stress. Trientine and RAS blockade decreased proteinuria and albuminuria and prevented an increase in creatinine clearance and kidney weight. Both reduced the diabetes-related glomerular features of mesangiolysis and glomerular segmental hypocellularity and trientine prevented severe tuft-to-capsule adhesion and reduced tubularization. Hypertension-related severe mesangial matrix expansion and global hypercellularity were increased by both treatments, which may reflect repair of mesangiolysis. Trientine reduced plasma but not renal protein carbonyls or plasma allantoin. In this model, trientine prevented the development of many diabetes-specific features similarly to RAS blockade. Amelioration of oxidative stress and features commonly observed in human diabetic nephropathy (DN), support a diabetes-related defect in copper (Cu) metabolism. The addition of Cu(II) chelation may improve current DN therapy., (© 2011 The Authors. Pathology International © 2011 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.)

Objective: There is considerable controversy about what constitutes optimal zinc intakes in patients with type 2 diabetes mellitus. Several studies suggest that higher zinc intakes improve vascular function and decrease oxidative damage. We aimed to assess the effects of zinc supplementation using a range of reliable biomarkers of oxidative damage and vascular function in patients with type 2 diabetes., Methods: Forty male type 2 diabetic patients were supplemented either with 240 mg/day of zinc as zinc gluconate (n=20) or with placebo (n=20) for 3 months. Blood and spot urine samples were taken at baseline, days 3 and 7, months 1, 2 and 3 during supplementation and 1 month after cessation. Serum zinc, reliable biomarkers of oxidative damage (F(2)-isoprostanes, neuroprostanes, cholesterol oxidation products, allantoin) as well as hydroxyeicosatetraenoic acid products and vascular-related indices (augmentation index, pulse wave velocity and aortic pressure) were measured., Results: Despite significantly higher levels of serum zinc in the treatment group, markers of oxidative damage, levels of hydroxyeicosatetraenoic acid products and vascular indices were unchanged by zinc supplementation during the four-month study period., Conclusion: Improving the zinc status in patients with type 2 diabetes with normal zinc levels did not have any impact on oxidative damage and vascular function, and such supplementation may not be generally beneficial in these individuals., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Cigarette smoking predisposes to the development of multiple diseases involving oxidative damage. We measured a range of oxidative damage biomarkers to understand which differ between smokers and nonsmokers and if the levels of these biomarkers change further during the act of smoking itself. Despite overnight abstinence from smoking, smokers had higher levels of plasma total and esterified F(2)-isoprostanes, hydroxyeicosatetraenoic acid products (HETEs), F(4)-neuroprostanes, 7-ketocholesterol, and 24- and 27-hydroxycholesterol. Levels of urinary F(2)-isoprostanes, HETEs, and 8-hydroxy-2'-deoxyguanosine were also increased compared with age-matched nonsmokers. Several biomarkers (plasma free F(2)-isoprostanes, allantoin, and 7β-hydroxycholesterol and urinary F(2)-isoprostane metabolites) were not elevated. The smokers were then asked to smoke a cigarette; this acute smoking elevated plasma and urinary F(2)-isoprostanes, plasma allantoin, and certain cholesterol oxidation products compared to presmoking levels, but not plasma HETEs or urinary 8-hydroxy-2'-deoxyguanosine. Smokers showed differences in plasma fatty acid composition. Our findings confirm that certain oxidative damage biomarkers are elevated in smokers even after a period of abstinence from smoking, whereas these plus some others are elevated after acute smoking. Thus, different biomarkers do not measure identical aspects of oxidative stress., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The allantoin/uric acid (All/UA) ratio and malondialdehyde (MDA) plasma levels have been proposed as important markers for monitoring oxidation triggered by the action of free radicals (FR). Here, we describe an easy field amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of All, UA, and free MDA in human plasma. The plasma samples were simply filtered through centrifugation membrane tubes for protein elimination and directly injected on a capillary without complex cleanup and/or sample derivatization procedures. The use of a run buffer composed of 300 mmol/L sodium borate at pH 10 with 50 mmol/L of N-methyl-D: -glucamine and an overimposed pressure/voltage of 0.1 psi during the electrophoretic run allows basline resolution of the analytes within 17 min. The electrokinetic injection allows a detection limit of 15 nmol/L for All, 20 nmol/L for UA and 10 nmol/L for MDA in a plasma sample, thus significantly improving the LOD of previous described methods based on capillary electrophoresis. Precision tests indicate a good repeatability of our method both for migration times (CV = 1.85%) and areas (CV = 2.87%). Moreover, a good reproducibility of intra- and inter-assay tests was obtained (CV = 4.63% and CV = 6.59% respectively). The suitability of the method was tested by measuring analyte levels in 40 healthy volunteers.

Uromodulin storage diseases are characterized by hyperuricemia of underexcretion type and renal insufficiency. Although these diseases are caused by mutations in the UMOD gene that encodes the kidney-specific glycoprotein uromodulin, the effect of uromodulin mutation on the kidney has not been clearly established. In this study, we investigated the effect by comparing transgenic mice expressing human uromodulin with and without mutation. Change in the intracellular localization of human uromodulin protein was shown in the kidney of transgenic mice expressing mutant human uromodulin by a deglycosylation experiment. Then, we determined by microarray technology and quantitative real-time PCR that the strongly induced gene in the kidney of these mice was 5-α-reductase 2, an enzyme that converts testosterone into the more potent androgen. Moreover, the expressions of androgen-induced genes β-glucuronidase, ornithine decarboxylase structural 1, and cytochrome P450 4a12a were increased. The increase in mRNA levels of urate reabsorptive transport system urate transporter 1 could be investigated, but the changes in its protein level and renal urate handling could not be demonstrated. Therefore, it is suggested that a uromodulin mutation may be responsible for the enhancement of renal androgen action.

Objective: To assess the influence of a 12-week training programme on plasmatic levels of allantoin, an in vivo marker for oxidative stress, in adolescents with Down syndrome. This finding would be of great interest, since oxidative damage has been proposed as a pathogenic mechanism of several pathologies in this population., Materials: To reach this goal, 31 male adolescents with Down syndrome (16.3 (1.1) years; 155.2 (5.7) cm; 70.8 (4.5) kg) performed a 12-week training programme, three sessions per week, consisting of warm-up (15 min) followed by a main part (20-35 min (increasing 5 min each 3 weeks)) at a work intensity of 60-75% of peak heart rate (increasing by 5% each 3 weeks) and then a cool-down period (10 min). According to previous studies, it should be emphasised that the maximal heart rate for individuals with Down syndrome was predicted by the equation HRmax = 194.5-(0.56 age). The control group included seven age-, sex- and BMI-matched adolescents with trisomy 21 that did not perform any training programme. The levels uric acid and allantoin were assayed in plasma by HPLC. This protocol was approved by an institutional ethics committee., Results: When compared with baseline, plasmatic levels of allantoin were decreased significantly (22.09 (1.62) vs 18.74 (1.38) micromol/l; p<0.001) after being exercised. Furthermore, the allantoin/uric acid ratio was decreased significantly (0.071 (0.006) vs 0.059 (0.004); p<0.05). On the contrary, no changes were reported in controls., Conclusion: A 12-week aerobic programme significantly reduced oxidative damage expressed in terms of plasmatic allantoin content in adolescents with Down syndrome. Further studies on this topic are required.

Myocardial ischemia (MI) is a worldwide epidemic. Compound Danshen Tablets (CDTs), an herbal compound preparation, are widely used to treat MI in China. In this study, we aimed to explore novel biomarkers to increase the understanding of MI and investigate therapeutic mechanisms of CDT by using a metabolomic approach. Plasma extracts from sham, MI model, CDT- and western medicines (isosorbide dinitrate, verapamil, propranolol, captopril, and trimethazine)-treated rats were analyzed by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The orthogonal partial least square (OPLS) model was built to find metabolites expressed in significantly different amounts between MI and sham rats. Meanwhile, partial least squares discriminant analysis (PLS-DA) was used to investigate CDT's protective effects. The results showed that CDT presented protective effects on MI by reversing potential biomarkers to sham levels, especially for the four metabolites in the pathway of purine metabolism (hypoxanthine, xanthine, inosine and allantoin)., (Copyright 2009 Elsevier B.V. All rights reserved.)

The purpose of this study was to determine the effects of allopurinol (AL) on xanthine oxidoreductase (XOR) activity and uric acid (UA) levels in chickens. Thirty 5-week-old broilers were divided into three groups and fed 0 (control), 25 (AL25) or 50 (AL50) mg AL per kg of body mass for 5 weeks. Chicks were weighed twice weekly and leukocyte oxidative activity (LOA) and plasma purine levels were determined weekly in five birds per group. Chicks were sacrificed after 2 or 5 weeks, and samples from tissues were taken for analysis of XOR activity. Plasma UA concentrations were lower (P<0.001) and xanthine and hypoxanthine concentrations were greater (P<0.001) in AL25 and AL50 birds compared to controls, whereas no differences (P=0.904) were detected in allantoin concentrations. By week 5, body mass was reduced (P<0.001) to 84.0 and 65.1% of that in controls for AL25 and AL50 broilers, respectively, and LOA was 4.1 times greater (P<0.05) in AL25 compared to control birds. Liver XOR activity was increased by 1.1 and 1.2 times in AL25 and AL50 birds, but there was no change (P>0.05) in XOR activity in the pancreas and intestine. These results suggest that AL effect on XOR activity is tissue dependent.

Background: Most epidemiological studies have reported a significant association between elevated serum levels of uric acid (UA) and increased cardiovascular disease. On the other hand, UA is the most abundant antioxidant in the human body. We hypothesized that UA levels would change noticeably in association with the degree of oxidative stress in acute ischemic stroke., Methods: We analyzed consecutive patients with acute ischemic stroke in the anterior circulation system within 24 h of symptom onset, confirmed by diffusion-weighted imaging (DWI), and with a modified NIH Stroke Scale (mNIHSS) score of 5 or greater. Baseline demographics, serial clinical scores, serial UA and allantoin (a nonenzymatic metabolite of UA) levels, UA change (baseline UA - UA at 48 h), and DWI lesion volumes were compared between 45 patients with recanalized vessels (RV) and 43 patients with nonrecanalized vessels (NV) in follow-up imaging., Results: The RV (vs. NV) patients were more likely to receive thrombolytic treatment (p = 0.005), achieve a reduction in day-14-mNIHSS scores (p = 0.001), and greater changes in UA (p = 0.024) and allantoin levels (p = 0.003). The UA levels dropped at 48 h and gradually increased in a U-shaped pattern. UA change (r = 0.360; p = 0.001) rather than baseline UA (r = 0.044; p = 0.681) was significantly correlated with infarct volume. In the RV (vs. NV) patients, there was a stronger association between infarct volume and UA change (r = 0.483; p = 0.001) or allantoin levels (r = 0.466; p = 0.017)., Conclusion: Our results suggest that UA might be a consumptive and reproducible antioxidant in acute ischemic stroke, and this pattern appears to be influenced by recanalization success and infarct volume size., (Copyright 2010 S. Karger AG, Basel.)

Background: To investigate the possible relationship between homocysteine and allantoin levels in hemodialyzed patients, serum levels of thiols and purine compounds were analyzed before and after dialysis sessions., Methods: 16 clinically stable non-diabetic patients hemodialyzed on polysulfone membranes were compared with 36 control subjects. Serum samples were collected before and after hemodialysis sessions. Total homocysteine, cysteine, glutathione, cysteinylglycine, uric acid, hypoxanthine, and allantoin were measured by capillary electrophoresis., Results: Pre-dialysis homocysteine, allantoin, and uric acid were significantly elevated in dialysis patients as compared to controls. Cysteine, glutathione, and hypoxanthine levels were similar in both groups. Homocysteine significantly decreased, but did not normalize after dialysis sessions. Glutathione and cysteinylglycine levels remained unchanged after dialysis sessions, whereas cysteine decreased. Uric acid, hypoxanthine, and allantoin levels were significantly reduced by dialysis sessions. The allantoin/uric acid ratio was higher in dialyzed patients before hemodialysis (0.049 +/- 0.023 vs. 0.016 +/- 0.012 in controls; p < 0.001), and became elevated after a dialysis session (0.084 +/- 0.033; p = 0.002)., Conclusions: Despite the use of biocompatible membranes, homeostasis of thiols and purine compounds is disturbed in hemodialysed patients. We suggest that allantoin could be used as a marker for oxidative stress in hemodialyzed patients.

Urate is the terminal product of purine metabolism in primates, including humans. Urate is also an efficient scavenger of oxidizing species and is thought to be an important antioxidant in human body fluids. Allantoin, the major oxidation product of urate, has been suggested as a candidate biomarker of oxidative stress because it is not produced metabolically. Although urate is converted to allantoin under strongly alkaline pH, such conditions have been used in the past to facilitate extraction of allantoin. We evolved a method for the determination of allantoin concentrations in human plasma and serum by gas chromatography-mass spectrometry without such artifact. With this method, we show that alkaline conditions do indeed cause breakdown of urate, leading to significant overestimation of allantoin concentration in human samples. By using our alternative method, serum samples from 98 volunteers were analyzed, and allantoin levels were found to be significantly lower than was previously reported. The in vivo utility and sensitivity of our method was further evaluated in human nasal-lining fluids. We were able to demonstrate an ozone-induced increase in allantoin, in the absence of increases in either ascorbate or glutathione oxidation products.

This study investigated the effect of a single dose of tomato sauce on healthy male volunteers in a randomized crossover study. Healthy male subjects (n = 10) were enrolled. Placebo (rice and olive oil) or tomato (tomato sauce, rice and olive oil) meals were provided to the volunteers. Blood and urine samples were taken before consumption of meal (0 h) and 2, 4, 6, 24 and 48 h after meal. Consumption of tomato sauce increased plasma lycopene level by 5-22%, with a maximum level at 24 h (p<0.01) after the meal. Levels of plasma F(2)-isoprostanes, hydroxyeicosatetraenoic acid products, allantoin and urinary 8-hydroxy-2'-deoxyguanosine did not change after either meal, but urinary F(2)-isoprostanes (p<0.05) significantly decreased at 48 h compared to 0 h after the tomato sauce meal. This study showed that a single dose of tomato sauce meal had only a limited antioxidant effect in vivo.

Current conventional measurement of allantoin levels in human serum uses an HPLC method. However, performing this assay is time-consuming and sample-intensive, and it requires expensive equipment. We have developed a novel enzyme cycling method for measuring allantoin concentrations in human serum. In the first step, serum allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5), and endogenous ammonia is simultaneously removed by the action of glutamine synthetase II (EC 6.3.1.2). In the second step, l-methionine sulfoximine is used to inhibit glutamine synthetase II, and ammonia is liberated from allantoate by the activity of allantoate amidohydrolase (EC 3.5.3.9). In the final step, the ammonia is then converted to NAD by NAD synthetase (EC 6.3.1.5). Subsequent action of glucose dehydrogenase (EC 1.1.1.47) and diaphorase (EC 1.6.99.2) in the presence of glucose and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1) acts to cycle the formed NAD between its oxidized and reduced forms, resulting in the production of WST-1 formazan, which is monitored at 450 nm. The assay standard curve is linear from 0 to 70 muM allantoin. The level of allantoin in healthy subjects was measured to be 8.2+/-3.1 microM (n=30).

Background: Uric acid is the final product of purine metabolism in humans. It was determined that this compound has important antioxidative properties and it may be oxidized to allantoin by various reactive oxygen species. Therefore, the measurement of allantoin may be useful for the determination of oxidative stress in humans., Methods: We measured allantoin and uric acid in human plasma and erythrocytes obtained from patients with chronic renal failure before hemodialysis (n=30) and blood donors (n=30). We used a method based on selective isolation of allantoin from deproteinized plasma and erythrocyte lysate samples on AG 1-X8 resin and its derivatization to glyoxylate-2, 4-dinitrophenylhydrazone. Separation of glyoxylate-2, 4-dinitrophenylhydrazone from interfering substances was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. Uric acid was determined by reversed phase HPLC with UV/VIS detection at 292 nm., Results: We found significant differences in allantoin and uric acid concentration between the patients with chronic renal failure and the control group both in plasma (20.5+/-6.5 micromol/L and 323.9+/-62.9 micromol/L vs. 2.1+/-1.1 micromol/L and 270.1+/-62.3 micromol/L, p<0.05) and erythrocytes [82.8+/-39.1 nmol/g hemoglobin (Hb) and 110.7+/-28.8 nmol/g Hb vs. 20.1+/-6.1 nmol/g Hb and 82.1+/-23.7 nmol/g Hb, p<0.05]., Conclusions: Significant higher levels of allantoin in both plasma and erythrocytes of patients with chronic renal failure indicate that allantoin may be used as a good marker of oxidative stress.

Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with mental retardation and Alzheimer-like dementia, characteristic change of the individual's phenotype and premature ageing. Oxidative stress is known to play a major role in this pathology since a gene dose effect leads to elevated ratio of superoxide dismutase to catalase/glutathione peroxidase compared to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of DS. Hyperuricemia is another feature of DS that has an interesting relationship with oxidative stress since uric acid represents an important free radical scavenger. However its formation is connected to the conversion of Xanthine dehydrogenase (XDH) to Xanthine oxidase (XO) which leads to concomitant production of free radicals. Here we report that plasma samples from DS patients in pediatric age, despite an increased total antioxidant capacity, largely due to elevated Uric acid content (UA), present significantly elevated markers of oxidative damage such as increased allantoin levels. Moreover DS plasma samples do not differ from healthy control ones in terms of Coenzyme Q10 and susceptibility to peroxidative stimuli. On the contrary, lymphocyte and platelet CoQ10 content was significantly lower in DS patients, a fact that might underlie oxidative imbalance at a cellular level.

Allantoin (All) is an oxidative end product of purines in mammals. The small amount of All present in human plasma or serum results from free radical action on urate and may provide a stable marker of in vivo free radical activity. Because free radicals have been implicated in the development and progression of atherosclerosis, this study focused on the metabolic compounds of the All pathway. We propose a new fast CE (CE/UV) method for the simultaneous determination of All, uric acid (UA), hypoxanthine (HX), and xanthine (X) in human plasma. These products were quantified in the plasma of patients with chronic renal failure before hemodialysis (n = 6), patients with chronic heart failure (n = 6) and controls (n = 6). The filtered plasma were diluted ten-fold before the direct injection in CE/UV (195 nm), which allows separating the four compounds in less than 13 min. The metabolites were detectable at concentrations of 0.3-0.6 micromol/L. The method was linear over the range 0.5-150 micromol/L for All, HX, and X and 10-1500 micromol/L for UA (r > 0.99). The analytical performance of this method is satisfactory with intra-assay CV < 3.4%, inter-assay CV < 5% (HX and X < 7%), and recovery (93-101%). The proposed CE-UV method appears to be a useful tool for studying physiological and pathological changes of HX, UA, and All levels in plasma samples, the latter being a possible indicator of free radical damage in vivo.

A new enzymatic assay for specifically measuring allantoin concentration in serum has been developed. The currently used methods for allantoin analysis are time consuming and nonspecific or depend on the use of expensive equipment. In our method, allantoin is converted to allantoate by the action of allantoinase (EC 3.5.2.5). The allantoate produced is hydrolyzed to ureidoglycine and ammonia by the action of allantoate amidohydrolase (EC 3.5.3.9). Nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (EC 1.4.1.4) subsequently acts on the ammonia produced, resulting in a change in absorbance at 340nm due to the consumption of reduced nicotinamide adenine dinucleotide phosphate. The amount of allantoin present is related to the change in the absorbance. The standard curve is linear up to at least 1mM allantoin. The procedure is simple, rapid, and accurate. The method has been used to measure serum allantoin levels after oral administration of purine nucleotides to experimental animals, including rats that have uricase catalyzing the conversion of urate to allantoin.

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.

A historical review of allantoin research is presented. An increased allantoin level in the trophoblast and serum of pregnant women has been demonstrated. Allantoin concentration decreased in placental tissues and increased in the serum in developing placental insufficiency.

Objective: Homocysteine increases the damage to the cardiovascular system in different ways, one of them is the formation of reactive oxygen species resulting from the auto-oxidation of homocysteine. At the same time, uric acid is one of the major antioxidants in the plasma and protects the cells towards increased ROS activity. In humans, allantoin is only formed from non-enzymatic oxidation of uric acid by free radicals. We aimed to determine the levels of homocysteine, uric acid and allontoin in patients with coronary artery diseases, and to evaluate the possible correlation between homocysteine and allantoin., Methods and Results: Plasma total homocysteine, uric acid and allantoin levels of 50 patients with coronary artery diseases and 23 healthy controls were determined by HPLC methods. Commercial diagnostic kits were used for the determination of other biochemical parameters. We obtained higher homocysteine, uric acid and allantoin levels in patients than in controls (p < 0.0001). Homocysteine levels were positively correlated with uric acid (r = 0.435, p < 0.0001) and allantoin (r = 0.583, p < 0.0001) levels in the whole study population. This correlation was persistent between allantoin and homocysteine after adjustment of these parameters for age, sex and creatinine. We accepted 15.0 micromol/l as a cut-off value between normal and mildly elevated homocysteine levels for patients and controls. Twenty-five patients showed moderate hyperhomocysteinaemia. The mean allantoin and uric acid values of the moderate hyperhomocysteinaemic group were significantly higher than that of the group having lower homocysteine levels than this cut-off value (p < 0.0001 for allantoin, p < 0.02 for uric acid)., Conclusion: Results imply that there is increased allantoin production resulting from uric acid oxidation by free radicals in hyperhomocysteinaemic patients with coronary artery disease. The possible significance of the relationship between homocysteine and allantoin warrants further study.

Uric acid is the end product of purine metabolism in humans. It has been pointed out that uric acid acts as an antioxidant and is capable to react with biologically relevant oxidants to form allantoin. Therefore, measurement of allantoin in humans was proposed as a marker of oxidative stress. We estimated allantoin in human plasma obtained from the patients with chronic renal failure before hemodialysis (n=30), patients with non-insulin dependent diabetes mellitus (n=30) and blood donors (n=30) using a method based on selective isolation of allantoin from deproteinized plasma samples on AG 1-X8 resin and its derivatization to glyoxylate-2,4-dinitrophenylhydrazone. The method is free from urate and glyoxylate interferences. Separation of glyoxylate-2,4-dinitrophenylhydrazone from other hydrazones was achieved on reversed phase HPLC with gradient elution and UV/VIS detection at 360 nm. The analytical performance of this method is satisfactory with intra-assay CV 5.7%, inter-assay CV 8.3% and recovery 94.1%. We have determined other parameters of oxidative stress (malondialdehyde, total antioxidant status, superoxide dismutase and glutathione peroxidase) too. The preliminary reference range of allantoin in a group of blood donors is 4.76+/-2.99 micromol/L. In the patients with chronic renal failure and the patients with non-insulin dependent diabetes mellitus we found allantoin levels in plasma (27.1+/-13.8) micromol/L and (11.08+/-5.90) micromol/L, respectively. It seems that allantoin is a possible indicator of free radical damage in vivo.

The increased production of reactive oxygen species (ROS) from activated neutrophils in Behçet's disease (BD) and recurrent aphthous stomatitis (RAS) may result in increased oxidative stress. Uric acid can react rapidly with neutrophil-derived ROS to form allantoin. The purpose of the study was to evaluate the serum levels of allantoin as a new marker of oxidative stress in BD compared with malondialdehyde (MDA) levels as a well-known marker. Blood samples were obtained from 23 BD patients, 22 RAS patients as positive controls, and 21 healthy controls. When compared to the healthy controls, we found higher allantoin and MDA levels in the BD patients and higher MDA levels in the RAS patients. Serum ascorbic acid levels in the BD patients were significantly lower than in the controls. Increased allantoin and MDA levels suggest the possible involvement of free radicals in BD. As allantoin is only a product of uric acid oxidation by reactive oxygen and nitrogen species, it may also be used as a marker of oxidative stress in BD.

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.

Objective: Patients with systemic lupus erythematosus (SLE) experience excess morbidity and mortality due to coronary artery disease (CAD) that cannot be fully explained by the classical CAD risk factors. Among emerging CAD risk factors, oxidative stress is currently being emphasized. We evaluated the effects of longterm antioxidant vitamins on markers of oxidative stress and antioxidant defense and endothelial function in 39 patients with SLE., Methods: Patients were randomized to receive either placebo or vitamins (500 mg vitamin C and 800 IU vitamin E daily) for 12 weeks. Markers of oxidative stress included malondialdehyde (MDA) and allantoin. Antioxidants measured included erythrocyte superoxide dismutase and glutathione peroxidase, plasma total antioxidant power (as FRAP value), and ascorbic acid and vitamin E concentrations. Endothelial function was assessed by flow-mediated dilatation (FMD) of the brachial artery and plasma concentration of von Willebrand factor (vWF) and plasminogen activator inhibitor type 1 (PAI-1). Primary outcome of the study included the change in lipid peroxidation as revealed by MDA levels. Secondary outcomes included changes in allantoin and antioxidant levels and change in endothelial function., Results: After treatment, plasma ascorbic acid and alpha-tocopherol concentrations were significantly (p < 0.05) increased only in the vitamin-treated group, associated with a significant decrease (p < 0.05) in plasma MDA. Other oxidative stress markers and antioxidant levels remained unchanged in both groups. FMD and vWF and PAI-1 levels remained unchanged in both groups., Conclusion: Combined administration of vitamins C and E was associated with decreased lipid peroxidation, but did not affect endothelial function in patients with SLE after 3 months of therapy.

The effect of Wen-Pi-Tang extract on influenza virus infection in mice was investigated. The administration of Wen-Pi-Tang extract at a dose of 100mg/kg body wt. for 8 consecutive days to influenza virus-infected mice reversed the lack of body wt. gain and prevented the increase in lung weight caused by the infection in comparison with uninfected mice, while allopurinol, a xanthine oxidase (XOD) inhibitor, did not show these effects. The serum levels of uric acid and allantoin in influenza virus-infected mice were reduced by Wen-Pi-Tang extract administration. Moreover, Wen-Pi-Tang extract reduced the uric acid level more as the dose increased, although it exerted lower activity than allopurinol. The XOD activity of the lungs was elevated by influenza virus infection, but Wen-Pi-Tang extract administration inhibited this activity, indicating prevention of lung damage by oxygen free radicals generated by XOD. After the administration of Wen-Pi-Tang extract to influenza virus-infected mice, the lung superoxide dismutase activity was not significantly different from that of uninfected mice, whereas lung catalase activity was lower in the former than the latter, but slightly higher than that of influenza virus-infected mice, suggesting that Wen-Pi-Tang extract may prevent the generation of highly toxic hydroxyl radicals in the lung. In addition, the administration of both Wen-Pi-Tang extract and allopurinol reduced the degree of lung consolidation caused by influenza virus infection. In particular, Wen-Pi-Tang extract reduced the consolidation score in a dose-dependent manner and more markedly than allopurinol did. This study suggests that Wen-Pi-Tang extract could improve pathological conditions of the lungs induced by influenza virus infection.

Free radicals are implicated in many diseases including atherosclerosis, cancer and also in rheumatoid arthritis. Reaction of uric acid with free radicals, such as hydroxyl radical and hypochlorous acid (HOCl) results in allantoin production. In this study, we measured the serum allantoin levels, oxidation products of uric acid, as a marker of free radical generation in rheumatoid arthritis. Fasting blood samples were obtained from 21 rheumatoid patients and 15 healthy controls. In this study, the serum allantoin and uric acid levels were measured by a gas chromatography-mass spectrometry method and the ratios were calculated. The mean allantoin and uric acid levels and ratios in the patient group were 22.1 +/- 11.3, 280.5 +/- 65.0 and 8.0 +/- 3.7 microM, while in the control group they were 13.6 +/- 6.3, 278.3 +/- 53.6 and 4.9 +/- 2.1 microM, respectively. The effects of gender, age, menopausal status, duration of disease and medications on serum allantoin and uric acid levels of the patient and control groups were studied. Our results suggest that uric acid acts as a free radical scavenger and thus is converted to allantoin. Increased allantoin levels suggest the possible involvement of free radicals in rheumatoid arthritis.

Background: Down syndrome (DS) is a chromosomal abnormality (trisomy 21) leading to mental retardation, to the characteristic change of individual's phenotype and to the pathological features of Alzheimer disease. Patients with DS have elevated ratio of superoxide dismutase to (catalase plus glutathione peroxidase) with respect to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of accelerated aging., Results: We report that persons with DS have elevated uric acid levels compared with controls, 348.56+/-22.78 versus 284.00+/-20.86 micromol/l (p=0.018). The levels of hypoxanthine and xanthine in DS children (6.35+/-0.31 and 1.02+/-0.23 micromol/l) were significantly lower than in controls (7.83+/-0.59 and 2.43+/-0.66 micromol/l). This result suggests increased conversion of hypoxanthine and xanthine to uric acid with subsequent free radical-dependent oxidation of uric acid to allantoin, mechanisms potentiated by the oxidative stress in DS. Allantoin is a nonenzymatic oxidative product of uric acid in human. In DS individuals, the levels of allantoin were significantly higher than those in healthy controls (18.58+/-2.27 and 14.07+/-1.07 micromol/l, respectively, p=0.03)., Conclusions: Our data supported the presumption of increased oxidative stress in DS.

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.

To study the uricolytic properties of urate oxidase in granivorous and carnivorous birds, experiments were conducted in pigeons (Columba livia domestica) and Red-tailed Hawks (Buteo jamaicensis). Plasma concentrations of allantoin and uric acid were determined at various times in experimental groups before and after receiving 100, 200, and 600 U/kg urate oxidase once daily and were compared with controls. All regimens caused a significant decrease in plasma uric acid concentrations within 2 days after the first administration, when compared with controls. Furthermore, both doses used in Red-tailed Hawks (200 and 100 U/kg) caused a significant postprandial suppression of plasma uric acid concentrations. Plasma allantoin concentrations were significantly higher when compared with controls. The ability of urate oxidase to prevent the physiological postprandial hyperuricaemia in Red-tailed Hawks and to reduce plasma uric acid concentrations to undetectable levels, shows the great potential of this drug for treating avian hyperuricaemia.

Background: In patients with chronic heart failure (CHF), hyperuricemia is a common finding and is associated with reduced vasodilator capacity and impaired peripheral blood flow. It has been suggested that the causal link of this association is increased xanthine oxidase (XO)-derived oxygen free radical production and endothelial dysfunction. We therefore studied the effects of XO inhibition with allopurinol on endothelial function and peripheral blood flow in CHF patients after intra-arterial infusion and after oral administration in 2 independent placebo-controlled studies., Methods and Results: In 10 CHF patients with normal serum uric acid (UA) levels (315+/-42 micromol/L) and 9 patients with elevated UA (535+/-54 micromol/L), endothelium-dependent (acetylcholine infusion) and endothelium-independent (nitroglycerin infusion) vasodilation of the radial artery was determined. Coinfusion of allopurinol (600 microg/min) improved endothelium-dependent but not endothelium-independent vasodilation in hyperuricemic patients (P<0.05). In a double-blind, crossover design, hyperuricemic CHF patients were randomly allocated to allopurinol 300 mg/d or placebo for 1 week. In 14 patients (UA 558+/-21 micromol/L, range 455 to 743 micromol/L), treatment reduced UA by >120 micromol/L in all patients (mean reduction 217+/-15 micromol/L, P<0.0001). Compared with placebo, allopurinol improved peak blood flow (venous occlusion plethysmography) in arms (+24%, P=0.027) and legs (+23%, P=0.029). Flow-dependent flow improved by 58% in arms (P=0.011). Allantoin, a marker of oxygen free radical generation, decreased by 20% after allopurinol treatment (P<0.001). There was a direct relation between change of UA and improvement of flow-dependent flow after allopurinol treatment (r=0.63, P<0.05)., Conclusions: In hyperuricemic CHF patients, XO inhibition with allopurinol improves peripheral vasodilator capacity and blood flow both locally and systemically.

The objectives of the present study were to investigate the recovery of [14C]allantoin in urine of sheep dosed intravenously and degradation of allantoin by rumen micro-organisms. The recovery of [14C]allantoin in the urine of eight sheep was measured during three periods in two experiments. Individual values of [14C]allantoin recovery varied from 66 to 95 % (mean value 83 (se 1.6) %). The recovery of [14C]allantoin showed no relation to the level of feed intake. There was some evidence that glomerular filtration rate was an important factor affecting the amount of urinary allantoin recovered in one experiment. Incomplete recovery of plasma [14C]allantoin in the urine indicated losses of plasma [14C]allantoin via non-renal routes. This is supported by the disappearance of 14C from rumen contents incubated in vitro with [14C]allantoin for 48 h (88 %) and the presence of 14C in saliva in vivo from sheep sampled after dosing with [14C]allantoin. However, the amount of 14C activity in the saliva was very low (equivalent to only 1.5 % of the total dose in sheep producing saliva at a rate of 15 litres/d). The proportion of renal and non-renal excretion of purine derivatives was found to be unpredictable both between and within individual animals. The factors responsible for this variability need to be identified, and existing models of excretion of purine derivatives may need to be modified accordingly to improve their accuracy of prediction. A single intravenous injection of [4,5-14C]allantoin provides a simple alternative to infusion methods used to measure the proportion of plasma allantoin excreted in the urine of sheep. Using this method it may be feasible to validate PD excretion models in other ruminant livestock.

This study was performed to examine the effects of several kinds of dietary fibers (DF) with different physical properties on dietary RNA metabolism. Male Wistar strain rats, 4 wk old, were fed diets with or without a 3% yeast RNA and a 5% DF (cellulose, chitin, chitosan, inulin, and xanthan gum) for 20 d (Experiment 1) or 5 d (Experiment 2). Feeding DF tested lowered the serum uric acid and allantoin concentrations and the urinary excretions of their compounds and increased the amount of RNA excreted into the feces compared with fiber-free. The water-holding capacity and nucleotide adsorption of chitin and chitosan in acidic solutions were higher than those of cellulose. The digestion rate of RNA by RNase A in vitro was found to be lower in the DF tested than in fiber-free. The decrease was remarkable in chitosan and xanthan gum. The uptakes of 14C-labeled adenosine and adenosine 5'-monophosphate (5'-AMP) in the rat jejunum were markedly decreased in regard to chitosan and xanthan gum in comparison with the fiber-free. These phenomena suggest that DF with high viscosity is more strongly associated with the suppression of RNA digestion by RNase A and the depression of the uptake of purine compounds to jejunum. The present results reveal that the elevation of serum uric acid concentration induced by dietary RNA can be suppressed by DF in rats.

Background: The small amount of allantoin present in human serum results from free radical (FR) action on urate and may provide a stable marker of free radical activity in vivo. We describe a gas chromatography-mass spectrometry (GC-MS) assay for serum allantoin and report a reference range in healthy individuals., Methods: Fasting blood samples were obtained from 134 healthy middle-aged volunteers (56 men, mean age 55, range 45-72; 78 women, mean age 55, range 50-72) Allantoin was assayed using 15N(2) allantoin as an internal standard. After isolation from aqueous standards or serum by extraction onto an anion exchange column (AG-MP1), allantoin was derivatised with N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA). Derivatives were injected onto an HP-1 column and analysed using a Mass Selective Detector with Single Ion Monitoring at 398 and 400 m/z., Results: The distribution of serum allantoin concentrations in men and women was non-Gaussian and log transformation was used for the analysis of data. Women (10.8 +/- 1.7 micromol/l (mean +/- S.D.)) had significantly lower serum allantoin levels than men (13.4 +/- 1.6 micromol/l, p=0.015). Reference ranges (95% CI) for middle-aged healthy subjects were 7.4-46.8 micromol/l (men) and 3.7-31.2 micromol/l (women)., Conclusion: Gas chromatography-mass spectrometry provides a reliable and accurate method for the determination of serum allantoin.