Assessment of thiol/disulphide homoeostasis and ischaemia-modified albumin and their relationship with disease severity in children with chronic urticaria.

BACKGROUND: The pathogenesis chronic urticaria (CU) hasn't been fully understood. In recent years, it has been shown that thiol-disulphide homoeostasis, as an antioxidant system, plays important roles in both healthy individuals and various diseases. In different ischaemia-reperfusion states, high oxidative stress causes ischaemia-modified albumin (IMA) generation. AIM: To investigate thiol/disulphide balance and IMA level in children with CU and their association with disease severity. METHODS: Thirty children with CU and 20 healthy children as controls, aged 1-18 years, were included in this cross-sectional study. In all subjects, total thiol, native thiol, disulphide levels and IMA levels were measured in plasma by spectrophotometry. Disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios were calculated. The disease severity was rated by Urticaria Activity Score (UAS). RESULTS: In the children with CU, the levels of native thiol (375.56 ± 56.22 µmol/L) and total thiol (415.69 ± 54.75 µmol/L) were significantly lower than the control group (475.20 ± 71.87 and 511.20 ± 73.73 µmol/L, respectively) (p = 0.000, p = 0.000). The ratio of native/total thiol * 100 was lower in patients than the control group (p = 0.002). IMA was significantly higher in the patient group than control group (p = 0.000). No significant correlation was found between UAS and thiol/disulphide homoeostasis (p > 0.05). The disulphide levels, disulphide/native thiol and disulphide/total thiol levels were found to be higher in patients with positive family history for autoimmune disorders than those without (p < 0.05). CONCLUSION: In children with CU, impaired thiol/disulphide homoeostasis and increased IMA suggest that oxidative stress may play role in the disease pathogenesis.

Optimization of a fast screening method for the assessment of low molecular weight thiols in human blood and plasma suitable for biomonitoring studies.

An adequate level of low molecular weight thiols (LMW-SH, especially glutathione (GSH)) protects cellular macromolecules against toxic agents, and is used as a sensitive biomarker of exposure to toxic compounds. During sample collection, storage and preparation, non-enzymatic and enzymatic oxidation of LMW-SH can occur leading to analytical inaccuracy. The aim of this study was to optimize a fast and reliable screening method for the determination of LMW-SH, mainly GSH, in blood and plasma samples as well as to investigate the impact of storage conditions on the LMW-SH stability. Based on our results, the described spectrophotometric method allows fast and reliable determination of LMW-SH in blood and plasma samples. Results on incubation of samples at 37 °C imply that synthesis of LMW-SH (probably GSH) as well as dynamic interexchange among various thiols forms can be induced in blood cells in in vitro conditions. Importantly, the level of LMW-SH in blood and plasma stored at -20 °C was constant, indicating that they can be stored at -20 °C for at least 30 days. Therefore, the method is suitable for assessment of LMW-SH in long-term human biomonitoring as well as environmental field studies, especially those involving a large number of samples such as epidemiological studies.

Serum catalase, thiol and myeloperoxidase levels in children passively exposed to cigarette smoke.

BACKGROUND: Free radicals found in cigarette smoke can harm all tissues and cellular structures in the human body. Passive smoking increases free radical production, leads to the depletion of antioxidants and increases oxidative stress which causes lipid peroxidation. Many studies have been conducted to determine the effects of passive smoking on antioxidant enzymes and lipid levels in adults, but pediatric studies on this topic are few. In our study, we compared the levels of antioxidants, oxidants, total and LDL cholesterol in children exposed to passive cigarette smoking with a healthy control group that was not exposed to passive smoking. METHODS: A total of 41 children (4-17 years of age, 24 girls and 17 boys) exposed to passive smoking and 18 healthy girls and 12 healthy boys were included in this study. Secondhand smoking was confirmed via measurement of the cotinine/creatinine ratio. Various sociodemographic characteristics of patients were recorded. The levels of catalase, thiol, myeloperoxidase were measured to determine the antioxidant and oxidant levels in children, while the levels of total cholesterol and LDL cholesterol were measured to determine the alterations in lipid profile. RESULTS: The groups were similar in regard to demographic characteristics. Myeloperoxidase levels were significantly higher in the passive cigarette smoking group compared to the non-exposure group; however, catalase and thiol levels were similar. In regard to lipid profile, the levels of total cholesterol and LDL cholesterol were also similar in those with and without exposure to passive smoking. CONCLUSIONS: Our findings suggest that the effects of passive smoking initially influence oxidants (MPO), but not antioxidants (thiol and catalase). However, it is apparent that passive smoking adversely affects oxidative balance in children and this may lead to the development of various diseases which could cause significant morbidity and mortality.

A robust and versatile mass spectrometry platform for comprehensive assessment of the thiol redox metabolome.

Several diseases are associated with perturbations in redox signaling and aberrant hydrogen sulfide metabolism, and numerous analytical methods exist for the measurement of the sulfur-containing species affected. However, uncertainty remains about their concentrations and speciation in cells/biofluids, perhaps in part due to differences in sample processing and detection principles. Using ultrahigh-performance liquid chromatography in combination with electrospray-ionization tandem mass spectrometry we here outline a specific and sensitive platform for the simultaneous measurement of 12 analytes, including total and free thiols, their disulfides and sulfide in complex biological matrices such as blood, saliva and urine. Total assay run time is < 10 min, enabling high-throughput analysis. Enhanced sensitivity and avoidance of artifactual thiol oxidation is achieved by taking advantage of the rapid reaction of sulfhydryl groups with N-ethylmaleimide. We optimized the analytical procedure for detection and separation conditions, linearity and precision including three stable isotope labelled standards. Its versatility for future more comprehensive coverage of the thiol redox metabolome was demonstrated by implementing additional analytes such as methanethiol, N-acetylcysteine, and coenzyme A. Apparent plasma sulfide concentrations were found to vary substantially with sample pretreatment and nature of the alkylating agent. In addition to protein binding in the form of mixed disulfides (S-thiolation) a significant fraction of aminothiols and sulfide appears to be also non-covalently associated with proteins. Methodological accuracy was tested by comparing the plasma redox status of 10 healthy human volunteers to a well-established protocol optimized for reduced/oxidized glutathione. In a proof-of-principle study a deeper analysis of the thiol redox metabolome including free reduced/oxidized as well as bound thiols and sulfide was performed. Additional determination of acid-labile sulfide/thiols was demonstrated in human blood cells, urine and saliva. Using this simplified mass spectrometry-based workflow the thiol redox metabolome can be determined in samples from clinical and translational studies, providing a novel prognostic/diagnostic platform for patient stratification, drug monitoring, and identification of new therapeutic approaches in redox diseases.

Low-cost colorimetric assay of biothiols based on the photochemical reduction of silver halides and consumer electronic imaging devices.

This work describes a new approach for the determination of free biothiols in biological fluids that exploits some of the basic principles of early photographic chemistry - that was based on silver-halide recording materials - and uses broadly-available imaging devices (i.e. flatbed scanners) as detectors. Specifically, the proposed approach relies on the ability of biothiols to bind to silver ions and dissociate the silver halide crystals thus changing the photosensitivity of silver halide crystal suspension. The changes induced by biothiols on the light intensity transmitted through the silver halide suspension, after photochemical reduction, were measured with a simplified photometric approach that employs a flatbed scanner operating in transmittance mode. The overall analytical procedure for the determination of biothiols was easily executable, fast and could be applied with inexpensive and commercially available materials and reagents. What is more, physiologically relevant biothiol levels could be inspected even by the unattended eye. The developed assay was successfully applied to the determination of biothiols in urine and blood plasma samples with detection limits as low as 10µM, satisfactory recoveries (92-97%), good reproducibility (6.7-8.8%) and high selectivity against other major components of biological fluids. The utility of the method to the determination of reduced/oxidized thiol ratio's as well as its application under natural light illumination, without external energy sources, was also demonstrated and is discussed with regard to point-of need applications in facility-limited settings.

Oxidative Stress Assessment in Response to Ultraendurance Exercise: Thiols Redox Status and ROS Production according to Duration of a Competitive Race.

Purpose. Response to an ultraendurance competitive race on thiols redox status, reactive oxygen species (ROS) production, and oxidative stress (OxS) was investigated according to duration. Methods. Twenty-four elite runners were examined: six completed 50 km and eighteen 100 km. Blood and urine samples were collected before and immediately after the race. Erythrocytes and plasma aminothiols by high-performance liquid chromatography, total antioxidant capacity (TAC), and OxS biomarkers (protein carbonyl (PC), thiobarbituric acid-reactive substances (TBARS), 8-isoprostane (8-iso-PGF2α), and 8-OH-2-deoxyguanosine (8-OH-dG)) by immunoenzymatic assays and ROS production by Electron Paramagnetic Resonance were assessed. Results. Significant increases (P between <0.05 and <0.0001) were recorded in plasma total and oxidized aminothiols concentration and TAC (P < 0.0001) only after 100 km: plasmatic (ROS production (+12 versus +29%), PC (+54 versus +115%), and TBARS (+28 versus +55%)) and urinary (8-OH-dG.creatinine(-1) (+71 versus +158%) and 8-iso-PGF2α.creatinine(-1) (+43 versus +135%)) concentrations for 50 and 100 km (duration 4 h 3' versus 8 h 42'), respectively. Conclusion. Very prolonged ultraendurance exercise causes an increase in ROS production and OxS depending on specific biomarker examined but always linearly and directly related to exercise duration. Redox status of erythrocytes was preserved. A relationship between running performance and both prerace ROS production and antioxidant-redox status was found in 100 km race.

Simultaneous assessment of endogenous thiol compounds by LC-MS/MS.

Biological thiol compounds are very important molecules that participate in various physiological events. Alteration in levels of endogenous thiols has been suggested as a biomarker of early stage of pathological changes. We reported a chemical derivatization- and LC-MS/MS-based approach to simultaneously determine thiol compounds including glutathione (GSH), cysteine (Cys), N-acetyl cysteine (NAC), homocysteine (Hcy), and cysteinylglycine (CysGly) in biological samples. Thiol-containing samples were derivatized with monobromobimane (mBrB) at room temperature, followed by LC-MS/MS analysis. Assessment of the analytes with baseline separation was completed within 10min, using a gradient elution on a C18 reversed-phase column. Excellent linearity was observed for all analytes over their concentration ranges of 1-400µM. The lowest limits of detection (S/N=3) in a range from 0.31fmol (for NAC) to 4.98fmol (for CysGly) were achieved. The results indicate that this approach was sensitive, selective, and well suited for high-throughput quantitative determination of the biological thiols.

A Novel Tool for the Assessment Oxidative Stress in Age-Related Macular Degeneration: Thiol/Disulfide Homeostasis Revisited.

PURPOSE: To investigate thiol/disulfide status using a novel automated assay in patients with age-related macular degeneration (AMD) compared to age-matched healthy controls. METHODS: A total of 64 AMD patients [51 (79%) non-exudative, 13 (21%) exudative AMD] and 21 age-matched healthy control subjects were enrolled in this study. Plasma total thiol, native thiol, disulfide levels were measured and native thiol/disulfide ratio (TDR) was calculated using a novel spectrophotometric assay. RESULTS: Patients with AMD had significantly lower levels of total thiol (434.8 ± 7.0 µmol/L vs. 472.2 ± 7.9 µmol/L, p < 0.001), native thiol (393.6 ± 6.5 µmol/L vs. 437.5 ± 7.1 µmol/L, p = 0.004) compared to healthy controls. However, plasma disulfide levels were higher in AMD patients (20.6 ± 0.9 µmol/L vs. 17.3 ± 1.3 µmol/L, p = 0.113) compared to healthy controls. The TDR was not statistically different between the early AMD group and healthy controls (24.2 ± 2.3 vs. 29.5 ± 3.1, p = 0.345). However, intermediate and advanced stage AMD groups had significantly lower levels of TDR compared to healthy controls (21.6 ± 2.6 vs. 29.5 ± 3.1, p = 0.023 and 20.3 ± 1.2 vs. 29.5 ± 3.1, p = 0.005, respectively). Native TDR was significantly lower in patients with exudative and non-exudative AMD (19.9 ± 2.3 vs. 29.5 ± 3.1, p = 0.024 and 21.8 ± 1.14 vs. 29.47 ± 3.1 respectively, p = 0.011). CONCLUSION: A greater extent of thiol consumption occurred in AMD patients compared to age-matched healthy controls. However, despite the similar levels of total thiol levels between several grades of AMD, the plasma native TDR value was decreased in accordance with the severity of the disease, which reflected the disease grade better.

Serum thiols and cardiovascular risk scores: a combined assessment of transsulfuration pathway components and substrate/product ratios.

BACKGROUND: Serum thiols have shown associations with surrogate markers of cardiovascular disease. However, little information is available on their combined association with validated cardiovascular risk scores for primary prevention at population level. We sought to determine whether individual serum thiol concentrations and substrate/product ratios within the transsulfuration pathway are independently associated with such scores. METHODS: Data on clinical and demographic characteristics, serum thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione and cysteinylglycine) and high-sensitivity C-reactive protein (CRP) were collected from a sample of the Hunter Community Study without previous cardiovascular events [n=350, median age (IQR) = 62 (59-66) years]. Five-year absolute cardiovascular risk score for each subject was calculated using the Framingham Risk Equation. RESULTS: Median risk score was 7% (IQR 4-10). After adjusting for body mass index, estimated glomerular filtration rate and physical activity regression analysis showed independent associations between cardiovascular risk scores and a) higher serum homocysteine (B 0.066, 95% CI 0.040 to 0.091, P<0.001) and lower cysteine (B -0.003, 95% CI -0.005 to -0.001, P=0.003) and glutathione (B -0.029, 95% CI -0.056 to -0.003, P=0.03) concentrations; and b) higher homocysteine/cysteine (B 0.114, 95% CI 0.066 to 0.161, P<0.001) and lower glutathione/cysteinylglycine (B -1.145, 95% CI -2.030 to -0.260, P=0.011) ratios. No significant associations were observed between cardiovascular risk scores, taurine and CRP. CONCLUSIONS: Serum homocysteine, cysteine and glutathione are independently associated with cardiovascular risk scores at population level. Enzymatic pathways involved in reduced bioconversion of homocysteine into cysteine and increased glutathione degradation might play an important role in such associations.

Assessment of nitric oxide, advanced oxidation protein products, malondialdehyde, and thiol levels in patients with restless legs syndrome.

OBJECTIVES: We aimed to determine the importance of oxidative stress in the pathogenesis of restless legs syndrome (RLS) by quantification of advanced oxidation protein products and total thiol levels (as markers of oxidative protein damage), nitric oxide levels (as an antioxidant and endothelial function), and malondialdehyde levels (as a marker of lipid peroxidation) in patients with RLS. DESIGN AND METHODS: A total of 22 patients with primary RLS were enrolled in the study and 20 age-and-gender-matched healthy subjects were enrolled as a control group. Serum nitric oxide, malondialdehyde, thiol levels, and plasma advanced oxidation protein products levels were determined by spectrophotometric methods. RESULTS: Serum nitric oxide and thiol levels were lower in the patient group than in controls (p = 0.007 and p = 0.017, respectively). Plasma advanced oxidation protein products levels and serum malondialdehyde levels were found to be higher in patients with RLS than in controls (p = 0.017 and p = 0.008, respectively). Serum malondialdehyde level was found to be positively correlated with plasma advanced oxidation protein products levels (p = 0.039). Serum thiol level was found to be negatively correlated with plasma advanced oxidation protein products levels (p = 0.030). CONCLUSIONS: Increased advanced oxidation protein products, malondialdehyde levels, and decreased thiol and nitric oxide levels, may suggest that patients with RLS are under oxidative stress. Although both lipid peroxidation and protein oxidation may have a role in atherosclerosis in RLS, those factors may be related to the pathogenesis of RLS.

Aluminium phosphide poisoning and oxidative stress: serum biomarker assessment.

According to previous animal studies, aluminium phosphides (AlPs) may induce oxidative stress leading to generation of free radicals and alteration in antioxidant defense system. This study was conducted to evaluate the existence and degree of oxidative stress in patients with acute AlP ingestion. A total of 44 acute AlP ingested patients as well as 44 age- and sex-matched controls were included. All patients had acute poisoning symptoms with AlP at the time of presentation and had blood samples analyzed for lipid peroxidation, total antioxidant capacity and total thiol. Our findings showed that there is a significant increase in lipid peroxidation in AlP ingested group along with a reduction in total antioxidant capacity and total thiols groups. These clinical data confirm previous experimental models that showed AlP exposure might significantly augment lipoperoxidative damage with simultaneous alterations in the antioxidant defense system. Hence, our findings might justify use of antioxidants in treatment of acute AlP poisoning which needs to be clarified by additional clinical trials.

Assessment of paraoxonase 1, xanthine oxidase and glutathione peroxidase activities, nitric oxide and thiol levels in women with polycystic ovary syndrome.

OBJECTIVE: To investigate whether there is any relation between oxidative stress and the antioxidant system in the development of polycystic ovary syndrome (PCOS) by measuring serum nitric oxide (NO) levels and xanthine oxidase (XO) activity (a generator of reactive oxygen species) and antioxidant status by measuring serum thiol levels and glutathione peroxidase (GSHPx) and paraoxonase 1 (PON1) activities. DESIGN: Prospective case-control study. SETTING: University hospital in Turkey. SAMPLE: Thirty women with polycystic ovary syndrome and 20 age- and sex-matched healthy control subjects were included. METHODS: Serum XO, PON1 and GSHPx activity and NO and thiol levels were determined by spectrophotometric methods. MAIN OUTCOME MEASURES: Activity of serum XO, PON1 and GSH, as well as NO and thiol levels. RESULTS: Serum XO activities were higher in women with PCOS than in the control women (p<0.001). The PON1 activity was lower in women with PCOS than in the control women (p<0.001). No significant difference was found between NO and thiol levels and GSHPx activities of women with PCOS and the control women (p>0.05). Serum PON1 activities were negatively correlated with serum XO activities and NO levels. CONCLUSION: Increased oxidant XO activity and decreased lipid antioxidant PON1 activity, along with the observed negative correlation between these parameters, suggests that women with PCOS are under oxidative stress and that there is XO-mediated lipid peroxidation, which may be related to increased atherosclerosis seen in later life in such women.

Asoxime (HI-6) impact on dogs after one and tenfold therapeutic doses: assessment of adverse effects, distribution, and oxidative stress.

Asoxime (HI-6) is a well known oxime reactivator used for counteracting intoxication by nerve agents. It is able to reactivate acetylcholinesterase (AChE) inhibited even by sarin or soman. The present experiment was aimed to determine markers of oxidative stress represented by thiobarbituric acid reactive substances and antioxidants represented by ferric reducing antioxidant power, reduced and oxidized glutathione in a Beagle dog model. Two groups of dogs were intramuscularly exposed to single (11.4 mg/kg.b.wt.) or tenfold (114 mg/kg.b.wt.) human therapeutically doses of HI-6. HI-6 affinity for AChE in vitro was evaluated in a separate experiment. Complete serum biochemistry and pharmacokinetics were also performed with significant alteration in blood urea nitrogen, creatine phosphokinase, glucose and triglycerides. Blood samples were collected before HI-6 application and after 30, 60, and 120 min. The overall HI-6 impact on organism is discussed.

Assessment of oxidative stress in lymphocytes with exercise.

This study investigated whether changes in the cellular composition of blood during exercise could partly account for observations of exercise-induced changes in lymphocyte oxidative stress markers. Markers of oxidative stress were assessed before and after 60 min of intense treadmill running. Samples were collected from 16 men (means ± SD: age 33 ± 13 yr; body mass index 23.8 ± 2.5 kg/m(2); maximal oxygen uptake 59.7 ± 5.2 ml·kg(-1)·min(-1)). Peripheral blood lymphocytes were assayed for protein carbonyl concentration, and plasma was assessed for lipid peroxides and antioxidant capacity. In a separate study, intracellular thiol concentration was determined in lymphocyte subsets from eight characteristically similar men by flow cytometry, of which T-cell memory populations were further identified on the basis of CD27, CD28, and CD45RA expression. Total lymphocyte protein carbonyls were transiently increased with exercise and returned to baseline within 15 min (P < 0.001). This change was accompanied by an increase in plasma lipid peroxides (P < 0.05) and total antioxidant capacity (P < 0.001). Correlation analyses showed that lymphocyte protein carbonyl content was not related to changes in the cellular composition of peripheral blood during exercise. Natural killer cells (CD3(-)CD56(+)) and late-differentiated/effector memory cells (CD4(+)/CD8(+)CD27(-)CD28(-)/CD45RA(+)), which mobilized most with exercise, showed high intracellular thiol content (P < 0.001). High thiol content suggests a lower oxidative load carried by these lymphocytes. Thus vigorous exercise resulted in a transient increase in lymphocyte oxidative stress. Results suggest this was unrelated to the alterations in the cellular composition of peripheral blood.

Assessment of serum paraoxonase and arylesterase activity in patients with endometrial cancer.

PURPOSE OF INVESTIGATION: Serum paraoxonase (PON 1) is one of the most important enzymatic antioxidants that hydrolyzes lipid peroxidation, an indicator of carcinogenic activity. The aim of this study was to compare the serum levels of paraoxonase and arylesterase activity in patients with endometrial cancer to those of healthy controls. METHODS: Serum paraoxonase and arylesterase activities, total free sulphydryl (-SH) groups and lipid hydroperoxide (LOOH) levels were measured in patients with endometrial cancer (n = 20) and controls (n = 23). RESULTS: Serum paraoxonase, arylesterase activities and total -SH group levels were significantly lower in patients compared to controls (p < 0.05, p < 0.05 and p < 0.001; respectively), while LOOH levels were significantly higher (p < 0.001). Among patients, serum paraoxonase and arylesterase activities were inversely correlated with LOOH levels (r = -0.680, p < 0.05; r = -0.708, p < 0.001; respectively), while these were positively correlated with the total -SH group (r = 0.526, p < 0.05; r = 0.508, p < 0.05; respectively). CONCLUSION: Reduced serum PON 1 activity might contribute to an impaired antioxidant defense system which plays a critical role in carcinogenesis in patients with endometrial cancer.

Assessment of paraoxonase activities in patients with knee osteoarthritis.

The objective of this study was to investigate serum paraoxonase and arylesterase activities, and lipid hydroperoxide (LOOH) and total thiol (total free sulfhydryl groups, -SH) levels along with lipid parameters in patients with knee osteoarthritis. Thirty-six patients with knee osteoarthritis and 30 healthy individuals were enrolled in the study. Serum paraoxonase and arylesterase activities were measured spectrophotometrically. LOOH levels were measured by ferrous oxidation with xylenol orange assay (FOX-2). Serum high-density lipoprotein-cholesterol (HDL-C), -SH levels, paraoxonase and arylesterase activities were significantly lower in the patient group than those in the controls (P < 0.05, for all), while LOOH and low-density lipoprotein (LDL) levels were significantly higher. In conclusion, paraoxonase and arylesterase activities were decreased significantly in patients with knee osteoarthritis. Lower serum paraoxonase-1 activity and lower level of HDL-C seem to be related to increased oxidative stress and inflammatory condition in these patients. It is known that paraoxonases reduce oxidative stress in serum and tissues thereby protecting against cardiovascular disease, particularly atherosclerosis. Thus, decreased paraoxonase and arylesterase activities play a role in the pathogenesis of atherosclerosis through increased susceptibility to lipid peroxidation in patients with osteoarthritis.

Assessment of oxidative stress in chronic pancreatitis patients.

AIM: To assess the levels of antioxidant capacity and oxidative damage in blood of chronic pancreatitis (CP) patients in comparison with those in healthy control subjects, by using several different analytical techniques. METHODS: Thirty-five CP patients and 35 healthy control subjects were investigated prospectively with respect to plasma levels of thiols, ferric reducing ability of plasma (FRAP, i.e. antioxidant capacity), levels of protein carbonyls and thiobarbituric acid reactive substances (TBARS). Additionally, we evaluated the production of reactive oxygen species (ROS) in whole blood. RESULTS: The antioxidative thiols including cysteine, cysteinylglycine and glutathione were significantly lower in CP patients. In addition, the non-enzymatic antioxidant capacity was significantly lower in CP patients, which correlated with the amount of oxidative protein (protein carbonyls) and the extent of lipid damage (TBARS), both were significantly higher in CP patients. The ROS production in whole blood after stimulation with phorbol 12-myritate 13-acetaat, demonstrated a strong tendency to produce more ROS in CP patients. CONCLUSION: Oxidative stress may contribute to the pathogenesis of chronic pancreatitis by decreasing antioxidant capacity and increasing oxidative damage in CP patients may be a rationale for intervention with antioxidant therapy.

Global assessment of serum antioxidant status in hemodialysis patients.

BACKGROUND: Since chronic renal failure (CRF) was described as a state of oxidant/antioxidant imbalance augmented after hemodialysis (HD) initiation, we assessed the total antioxidant activity and single antioxidants in sera from uremic patients dialyzed or not, compared to subjects with normal renal function. METHODS: Serum total antioxidant activity (TAA--measured as Trolox equivalent antioxidant capacity; mmol/L), total plasma free thiols (Pt-SH; micromol/g protein), albumin (g/dL) and uric acid (mg/dL) were determined in 19 hemodialyzed patients, 15 CRF non-dialyzed patients (serum creatinine (Cr) = 4.4 +/- 2.7 mg/dL) and in 16 healthy controls. The "antioxidant gap" (mmol/L), as a measure of the combined activity of plasma antioxidants other than albumin and uric acid, was calculated. RESULTS: TAA and the "antioxidant gap" were higher in HD patients (1.21 +/- 0.12 vs. 0.96 +/- 0.13 in the non-HD group, p<0.001, and vs. 0.9 +/- 0.14 in controls, p<0.001, respectively, for TAA; 0.46 +/- 0.15 vs. 0.2 +/- 0.15, p<0.001, and vs. 0.21 +/- 0.16, p<0.001, respectively, for residual antioxidant activity). However, no differences existed in major plasma antioxidant levels (albumin and uric acid) among uremic patients, hemodialyzed or not. Pt-SH were reduced in nondialyzed patients as compared to controls (6.21 +/- 1.1 vs. 7.33 +/- 0.83, p=0.002), but were elevated in HD patients (11.9 +/- 1.1). CONCLUSIONS: These results suggest that HD patients appear to have improved plasma antioxidant status, hyperuricemia not being the sole contributor. Therefore, it seems reasonable to speculate that other antioxidants (thiols or some as yet unrecognized substances) could also be contributors. However, more reliable assays for extracellular antioxidant defense evaluation are required to validate this hypothesis.