Oxidative status in different settings and with different methodological approaches compared by Receiver Operating Characteristic curve analysis.

OBJECTIVES: To test the performance of different analytical approaches in highlighting the occurrence of deregulated redox status in various physio-pathological situations. DESIGN AND METHODS: 35 light and 61 heavy smokers, 19 chronic renal failure, 59 kidney transplanted patients, and 87 healthy controls were retrospectively considered for the study. Serum oxidative stress and antioxidant status, assessed by spectrophotometric Reactive Oxygen Metabolites (d-ROMs) and Total Antioxidant Capacity (TAC) tests, respectively, were compared with plasma free (F-MDA) and total (T-MDA) malondialdehyde, both quantified by isotope-dilution-gas chromatography-mass spectrometry (ID-GC-MS). Sensitivity, specificity and cut-off points of T-MDA, F-MDA, d-ROMs and TAC were evaluated by both Receiver Operating Characteristic (ROC) analyses and area under the ROC curve (AUC). RESULTS: Only T-MDA assay showed a clear absence of oxidative stress in controls and significant increase in all patients (AUC 1.00, sensitivity and specificity 100%). Accuracy was good for d-ROMs (AUC 0.87, sensitivity 72.8%, specificity 100%) and F-MDA (AUC 0.82, sensitivity 74.7%, specificity 83.9%), but not high enough for TAC to show in patients impaired antioxidant defense (AUC 0.66, sensitivity 52.0%, specificity 92.9%). CONCLUSIONS: This study reveals T-MDA as the best marker to detect oxidative stress, shows the ability of d-ROMs to identify modified oxidative status particularly in the presence of high damages, and evidences the poor TAC performance. d-ROMs and TAC assays could be useful for routine purposes; however, for an accurate clinical data evaluation, their comparison versus a "gold standard method" is required.

An encapsulated juice powder concentrate improves markers of pulmonary function and cardiovascular risk factors in heavy smokers.

OBJECTIVE: Cigarette smoking is associated with reduced pulmonary function and increased risk factors for cardiovascular disease. This randomized placebo-controlled double-blind study evaluated the effects of two different combinations of mixed fruit and vegetable juice powder concentrate (Juice Plus+, NSA, Collierville, TN) on heavy smokers. METHODS: At baseline (T 0) and after 3 months' supplementation (T 1), pulmonary function parameters and cardiovascular risk factors-that is, plasma total homocysteine (tHcy) with related B vitamins and cysteine (tCys) concentrations-were assessed in 75 apparently healthy smokers (aged 49.2 ± 10.6 years, >20 cigarettes/d, duration ≥10 years) randomized into 3 groups: placebo (P), fruit/vegetable (FV) and fruit/vegetable/berry (FVB). RESULTS: T 0: most smokers showed abnormalities in tHcy and tCys concentrations. T 1: respiratory function was unchanged in P and slightly, but not significantly, improved in FV, whereas FVB showed a significant improvement in forced expiratory flow at 25% (FEF25; p < 0.0001 vs P and FV) and significant improvement in CO diffusion lung/alveolar volume (DLCO/VA). FV and FVB (50%) showed significant reduction in tHcy and tCys compared to T 0 ( p < 0.0001) and P ( p < 0.0001). CONCLUSIONS: At T 1, both supplemented groups, but to a greater extent the FVB group, showed improvements in some pulmonary parameters, cardiovascular risk factors, and folate status. The beneficial effects of Juice Plus+ supplementation could potentially help smokers, even if smoking cessation is advisable.

Plasma total cysteine and cardiovascular risk burden: action and interaction.

We hypothesized that redox analysis could provide sensitive markers of the oxidative pathway associated to the presence of an increasing number of cardiovascular risk factors (RFs), independently of type. We classified 304 subjects without cardiovascular disease into 4 groups according to the total number of RFs (smoking, hypertension, hypercholesterolaemia, hyperhomocysteinaemia, diabetes, obesity, and their combination). Oxidative stress was evaluated by measuring plasma total and reduced homocysteine, cysteine (Cys), glutathione, cysteinylglycine, blood reduced glutathione, and malondialdehyde. Twenty-seven percent of subjects were in group 0 RF, 26% in 1 RF, 31% in 2 RF, and 16% in ≥ 3 RF. By multivariable ordinal regression analysis, plasma total Cys was associated to a higher number of RF (OR = 1.068; 95% CI = 1.027-1.110, P = 0.002). Total RF burden is associated with increased total Cys levels. These findings support a prooxidant effect of Cys in conjunction with RF burden, and shed light on the pathophysiologic role of redox state unbalance in preclinical atherosclerosis.

Simultaneous free and glycosylated pyridinium crosslink determination in urine: validation of an HPLC-fluorescence method using a deoxypyridinoline homologue as internal standard.

Pyridinoline (Pyr), deoxypyridinoline (D-Pyr), galactosyl-pyridinoline (Gal-Pyr) and glucosyl-galactosyl pyridinoline (GluGal-Pyr) are enzymatic mature pyridinium crosslinks. Generally, only total Pyr and D-Pyr urinary amounts (free+bound forms) are evaluated by HPLC as indices of bone resorption. This report describes the validation of an HPLC-fluorescence method for the simultaneous evaluation of free Pyr and D-Pyr, together with GluGal-Pyr and Gal-Pyr, in urine of healthy women (n=20, aged 27-41) and girls (n=20, aged 5-10). The use of an unnatural D-Pyr homologue, here proposed for the first time as internal standard, and of pure Pyr, D-Pyr, GluGal-Pyr and Gal-Pyr synthesized to be used as primary calibrators, guarantees method specificity and correct crosslink quantification. Urine, spiked with IS, was solid-phase extracted prior to HPLC analysis. Total Pyr and D-Pyr amounts were also evaluated after urine hydrolysis. The HPLC method was validated for selectivity, sensitivity, linearity, precision, accuracy, recovery and stability for all measured crosslinks. Both free and total Pyr and D-Pyr as well as GluGal-Pyr and Gal-Pyr amounts were significantly higher in girls than in women (p<0.0001), indicating an increased collagen turnover rather than only bone turnover. Gal-Pyr, for the first time evaluated in girls, was under its lower quantification limit (

Effects of encapsulated fruit and vegetable juice powder concentrates on oxidative status in heavy smokers.

OBJECTIVE: Long-term cigarette smoking has negative effects on oxidative status, promoting low-density lipoprotein (LDL) oxidation and formation of lipid peroxides. We evaluated the effects of 2 different encapsulated formulas, consisting primarily of mixed juice powder concentrate, on oxidative status compared with placebo. METHODS: This randomized, double-blind, placebo-controlled study was performed on 101 apparently healthy heavy smokers (>20 cigarettes/d, duration >10 years; median age 47 years, range 41-57 years; 54 M) before and after 3 months' supplementation. Subjects were randomized into 3 groups, well matched for sex and age: (1) placebo; (2) fruit/vegetable (FV); and (3) fruit/vegetable/berry (FVB). Analysis of oxidative status was performed on 75 (46 M) compliant subjects (>95% of assigned capsules). Changes in lipid panel parameters, oxidative-INDEX (Oxy-I, calculated on the basis of serum hydroperoxides and total antioxidant capacity measured by spectrophotometric methods), oxidized-LDL (ox-LDL; enzyme-linked immunosorbent assay [ELISA] method), and malondialdehyde (MDA; gas chromatography-mass spectrometry method) in free (fMDA), bound (bMDA), and total (tMDA = fMDA + bMDA) forms are reported. Statistical analysis was performed with R statistical software. RESULTS: After supplementation, compared with placebo, both FV and FVB groups showed a significant decrease in total cholesterol (p < 0.05), ox-LDL (p = 0.03), and fMDA levels (p = 0.004) accompanied by a slight increase in bMDA concentrations, possibly as the result of fMDA conjugation. Moreover, a significant decrease in Oxy-I was found in both active groups compared with placebo (p < 0.001). CONCLUSION: Intervention with both nutraceutical formulations resulted in improvement in some oxidative alterations attributed to long-term cigarette smoking.

Proteomics reveals novel oxidative and glycolytic mechanisms in type 1 diabetic patients' skin which are normalized by kidney-pancreas transplantation.

BACKGROUND: In type 1 diabetes (T1D) vascular complications such as accelerated atherosclerosis and diffused macro-/microangiopathy are linked to chronic hyperglycemia with a mechanism that is not yet well understood. End-stage renal disease (ESRD) worsens most diabetic complications, particularly, the risk of morbidity and mortality from cardiovascular disease is increased several fold. METHODS AND FINDINGS: We evaluated protein regulation and expression in skin biopsies obtained from T1D patients with and without ESRD, to identify pathways of persistent cellular changes linked to diabetic vascular disease. We therefore examined pathways that may be normalized by restoration of normoglycemia with kidney-pancreas (KP) transplantation. Using proteomic and ultrastructural approaches, multiple alterations in the expression of proteins involved in oxidative stress (catalase, superoxide dismutase 1, Hsp27, Hsp60, ATP synthase delta chain, and flavin reductase), aerobic and anaerobic glycolysis (ACBP, pyruvate kinase muscle isozyme, and phosphoglycerate kinase 1), and intracellular signaling (stratifin-14-3-3, S100-calcyclin, cathepsin, and PPI rotamase) as well as endothelial vascular abnormalities were identified in T1D and T1D+ESRD patients. These abnormalities were reversed after KP transplant. Increased plasma levels of malondialdehyde were observed in T1D and T1D+ESRD patients, confirming increased oxidative stress which was normalized after KP transplant. CONCLUSIONS: Our data suggests persistent cellular changes of anti-oxidative machinery and of aerobic/anaerobic glycolysis are present in T1D and T1D+ESRD patients, and these abnormalities may play a key role in the pathogenesis of hyperglycemia-related vascular complications. Restoration of normoglycemia and removal of uremia with KP transplant can correct these abnormalities. Some of these identified pathways may become potential therapeutic targets for a new generation of drugs.

Dimethylarginines in complicated type 1 diabetes: roles of insulin, glucose, and oxidative stress.

To investigate the roles of insulin, glucose, and oxidative stress on plasma asymmetric and symmetric dimethylarginine (ADMA, SDMA) levels in complicated diabetes, we studied patients with type 1 diabetes (T1D; n = 20), T1D + end-stage renal disease under hemodialysis (T1D + ESRD; n = 12), T1D + ESRD who received kidney transplant (KD; n = 16), and T1D + ESRD who received kidney-pancreas transplant (KP; n = 20) and healthy controls (n = 50). Levels of ADMA, SDMA, and free and total malondialdehyde (MDA) were increased in all patients, with the highest rises for SDMA and free MDA in T1D+ESRD. In KP, the normalized glycemia contributes to the recovery of ADMA, SDMA, and MDA levels toward normal values. From the covariance analyses, both glucose and insulin relate significantly to ADMA in T1D + ESRD (beta = +0.004, beta = -0.038, respectively) and in KP (beta = +0.032, beta = +0.032, respectively). Creatinine clearance and insulin relate to SDMA in all patient groups (beta = -0.006). Our results provide evidence for the effect of kidney-pancreas transplant on the recovery of ADMA, SDMA, and indexes of oxidative stress toward normal values. Only free MDA allows one to discriminate the magnitude of the oxidative status, as increased total MDA could also be attributable to a reduced renal function.

Effects of alpha-lipoic acid administration on plasma glucose levels, total malondialdehyde values and withdrawal signs in rats treated with morphine or morphine plus naloxone.

Morphine (CAS 57-27-2) administration or its removal induces alterations in glucose levels and oxidative status or behaviour signs, which may be hypothetically closely related; if this is correct, controlling glucose changes may lead to modifications in peroxide levels and in behaviour profile. It therefore seems important to find a drug able to control alterations of glucose metabolism, peroxide generation and behaviour symptoms in morphine or morphine withdrawal animals. This paper describes the effects of morphine or morphine plus naloxone (CAS 51481-60-8) on the plasma levels of glucose, malondialdehyde (MDA) (CAS 100683-54-3) and behavioural signs in rats treated or not with alpha-lipoic acid (CAS 1077-28-7), known to interfere with glucose and peroxide levels. The administration of morphine or its removal by naloxone alters plasma glucose levels, increases MDA values, and also affects signs such as pain threshold values, fecal excretion and jumping behaviour. The injection of alpha-lipoic acid decreases glycemia in rats treated with morphine or morphine plus naloxone. This result may be due to the capacity of alpha-lipoic acid to facilitate glucose transport and its utilization. The administration of a-lipoic acid to rats given morphine or morphine plus naloxone lowers total MDA levels because of its peroxide scavenging capacity. In animals injected with morphine plus naloxone, which show altered pain thresholds, high fecal excretion and jumping behaviour, treatment with alpha-lipoic acid increases latency times, decreases fecal excretion and reduces jumping. These effects can be attributed to the capacity of alpha-lipoic acid to interfere with mediators or peroxides involved in the modified behaviour. The glycemia levels, MDA values and behavioural signs seem to be interconnected in the reported experiments. The administration of alpha-lipoic acid is demonstrated to control the alterations in plasma glucose levels, peroxide values or behavioural profile in animals receiving morphine or morphine plus naloxone.

Free and total plasma malondialdehyde in chronic renal insufficiency and in dialysis patients.

BACKGROUND: Available data about oxidative status in patients with end-stage renal disease (ESRD) or on dialysis are contradictory. The present cross-sectional study aimed to investigate the role of renal insufficiency and dialysis on lipid peroxidation. To separate the effects of uraemia from dialysis-induced stress, we enrolled 26 patients with renal insufficiency on conservative treatment (ESRD), 23 on peritoneal dialysis (PD), 30 on haemodialysis (HD) and 30 controls. METHODS: Plasma malondialdehyde (MDA) levels, both total (tMDA) and free (fMDA), were measured as indexes of oxidative stress by gas chromatography-mass spectrometry. Bound MDA (bMDA) levels were calculated as the difference between tMDA and fMDA. RESULTS: Total and bMDA concentrations were significantly higher in patients than in controls (ESRD > HD > PD). In PD and HD patients, fMDA levels were similar and significantly higher than in ESRD. Multivariate analysis, with tMDA, fMDA and bMDA as dependent variables, showed similar and significant tMDA and bMDA relations with residual renal function (t = -2.160, P = 0.035) and albumin (t = -2.049, P = 0.045). Erythropoietin dose affected only fMDA values (t = -2.178, P = 0.034). CONCLUSIONS: Free and bMDA concentrations identified different MDA patterns. Bound MDA, not excreted by kidneys, accounts alone for high tMDA concentrations in ESRD patients, while both fMDA and bMDA contribute to tMDA values in dialysis patients. These findings show that increased tMDA could be indicative not only of recent lipid peroxidation, and they also highlight the importance of evaluating free, bound and total MDA in patients with reduced renal function in order to assess their oxidative status.

Morphine or its withdrawal affects plasma malondialdehyde, vitamin E levels and absence or presence of abstinence signs in rats.

OBJECTIVES: Various experimental observations show that morphine treatment generates reactive oxygen species, and that its discontinuation leads to signs of withdrawal. We therefore investigated plasma malondialdehyde and vitamin E levels under both conditions to verify the occurrence of any alterations in oxidative metabolism, and whether these are associated with behavioural changes. METHODS: We investigated the effects of morphine or morphine plus naloxone on plasma malondialdehyde, vitamin E levels and withdrawal signs such as jumping, wet dog shakes and faecal excretion in rats. Furthermore, isopropylnoradrenaline was injected in rabbits to verify its effects on plasma malondialdehyde levels. KEY FINDINGS: Morphine treatment increased free malondialdehyde and decreased vitamin E levels. The elevation in malondialdehyde levels were exacerbated by the abrupt removal of morphine by naloxone, which also led to the appearance of withdrawal signs. The increased malondialdehyde values can be attributed to the interactions of reactive oxygen species with unsaturated fatty acids, and the lowered levels of vitamin E to its interactions with reactive oxygen species. CONCLUSIONS: A connection seems to exist between altered peroxide status and withdrawal signs in abstinent animals.

Plasma malondialdehyde levels and opiate withdrawal signs observed in rats treated with morphine plus naloxone: effects of alpha-lipoic acid administration.

A number of experimental studies have found that reactive oxygen species are involved during morphine treatment or withdrawal. The aims of this study were to analyse whether morphine administration and/or removal are related to peroxide generation and/or signs of withdrawal in rats, and whether the changes in antioxidant status induced by the administration of an antioxidant may modify peroxide levels and behavioural signs. We injected morphine or morphine and naloxone into rats and evaluated the plasma levels of peroxide malondialdehyde (MDA) and the appearance of withdrawal signs. We also investigated the effects on these parameters induced by the administration of the antioxidant alpha-lipoic acid (LA). Morphine treatment increased MDA levels. Abrupt naloxone-induced morphine withdrawal caused a further and significant increase in MDA, and the appearance of withdrawal signs such as abnormal fecal excretion, shortened latency times and jumping. The administration of LA lowered MDA levels in the rats treated with morphine or morphine plus naloxone, and also decreased MDA values and abstinence signs in the animals treated with morphine plus naloxone. The effects of LA were attributed to its capacity to scavenge peroxides and interfere with the biogenesis of the arachidonic acid metabolites involved in the expression of abstinence symptoms.

Anesthetic propofol enhances plasma gamma-tocopherol levels in patients undergoing cardiac surgery.

BACKGROUND: Propofol (2,6-diisopropylphenol) is an anesthetic drug with antioxidant and antiinflammatory properties, documented both in vitro and in experimental models of ischemia-reperfusion injury and septic shock. These properties have been related to the similarity of its chemical structure to that of endogenous tocopherols, which are phenol-containing radical scavengers. This study evaluated the effects of propofol on alpha- and gamma-tocopherol (alpha- and gamma-T) levels and on selected markers of oxidant-antioxidant and inflammatory status in patients undergoing cardiac surgery. METHODS: Patients were randomly assigned for anesthesia with either propofol (propofol group, n = 22) or sevoflurane (control group, n = 21). Plasma levels of alpha- and gamma-T, individual antioxidant capacity, malondialdehyde, and interleukin 10 were measured before, during, and after anesthesia. In addition, levels of the proinflammatory prostaglandin E2 as a marker of cyclooxygenase-2 activity and those of interleukin 10 were measured in whole blood cultured with bacterial lipopolysaccharide. RESULTS: Gamma-T levels increased significantly during surgery in propofol group (P < 0.0001 vs. control group). By contrast, alpha-T similarly decreased in both groups. Malondialdehyde and interleukin 10 increased markedly and individual antioxidant capacity decreased, without differences between groups. Prostaglandin E2 levels measured 24 h after anesthesia induction were significantly lower in the propofol than in the control group. In vitro studies highlighted the different capacity of gamma- and alpha-T to impair prostaglandin E2 synthesis by human monocytes challenged with bacterial lipopolysaccharide. CONCLUSIONS: The antiinflammatory properties of propofol that may be linked to its effect on gamma-T levels could be relevant in controlling the inflammatory response that accompanies tissue injury during reperfusion.

Plasma glutathione levels are independently associated with gamma-glutamyltransferase activity in subjects with cardiovascular risk factors.

To investigate whether GGT (gamma-glutamyltransferase) is associated to specific redox patterns. GGT, total and reduced aminothiols and malondialdehyde, were measured in 150 subjects (83 males, 48 (39-56) years), with none, one or more risk factors. By univariable analysis GGT was positively associated with age (p =0.001), male gender (p <0.001), risk factor number (p <0.001), ACE-inhibitors (p =0.008), anti-platelet agents (p =0.029), atherothrombotic events (p =0.001), glucose (p =0.013), malondialdehyde (p =0.029), plasma total cysteine (p =0.046) and inversely associated with plasma total glutathione (p =0.001). By multivariable analysis only male gender (p <0.001), risk factor number (p <0.001) and glutathione (p <0.001) were independently associated with GGT activity. These findings suggest that an ongoing redox imbalance, in terms of decreased plasma glutathione, is associated with raised GGT activity in subjects with a greater risk factor burden.

Aminothiol redox alterations in patients with chronic heart failure of ischaemic or non-ischaemic origin.

OBJECTIVE: Oxidative stress plays a role in the progression of chronic heart failure (CHF), but whether and how ischaemic heart disease (IHD) or non-IHD aetiology may account for differential redox alterations is currently unclear. We assessed the relation between thiol redox state and lipid peroxidation, as a marker of oxidative stress, in patients with CHF of ischaemic or non-ischaemic origin. METHODS: Blood reduced glutathione, plasma total and reduced cysteine, cysteinylglycine, homocysteine, glutathione, plasma alpha-tocopherol, ascorbic acid, and free malondialdehyde were assessed in 43 CHF heart transplant candidates (24 IHD and 19 non-IHD) and 30 controls matched for age, gender and number of atherosclerotic risk factors. RESULTS: Reduced cysteine was increased in CHF patients compared with controls. The highest levels were found in IHD versus non-IHD patients versus controls. Malondialdehyde levels were significantly higher in IHD patients than in controls, whereas antioxidant vitamins did not differ among the three groups. CONCLUSIONS: Specific abnormalities in the thiol pattern are associated with heart failure aetiology in CHF patients. Our findings point to the possible role of reduced cysteine in the progression of chronic IHD to heart failure status, as an additional pro-oxidant stimulus for worsening oxidative stress.

Pre-operative redox state affects 1-month survival in patients with advanced heart failure undergoing left ventricular assist device implantation.

BACKGROUND: Left ventricular assist device (LVAD) implantation has proven effective as a bridge to transplantation in end-stage heart failure patients (ESHFPs), although survival during device support is critical. Oxidative stress has been implicated in the development of heart failure, but the influence of redox state on in-hospital post-LVAD outcome has not been clarified. METHODS AND RESULTS: In this report we describe the oxidant/anti-oxidant profiles of 15 ESHFPs before LVAD placement, 5 of whom did not survive to 1 month, and in 30 subjects without cardiac disease, representing the control group. CONCLUSIONS: Preliminary findings suggest that adequate activity of the GPx-1-based anti-oxidant system before device placement is associated with patient survival up to 1 month, despite comparable baseline oxidative stress in patients who both survived and died (within 2 weeks post-LVAD).

Glutamate-cysteine ligase polymorphism, hypertension, and male sex are associated with cardiovascular events. Biochemical and genetic characterization of Italian subpopulation.

BACKGROUND: Glutathione (GSH) is an important intravascular scavenger that protects endothelial cells from atherosclerosis. However, it is still unknown whether cardiovascular (CV) events are associated with metabolic and genetic factors, linked to GSH synthesis in an Italian subpopulation, and if a glutamate-cysteine ligase polymorphism within the catalytic subunit (GCLC) could affect blood and plasma GSH concentrations. METHODS: One hundred subjects, with or without CV risk factors, were enrolled to evaluate plasma and erythrocyte redox status (GSH, homocysteine, cysteine, cysteinylglycine), antioxidant vitamins (alpha-tocopherol and ascorbate), malondialdehyde, a lipid peroxidation product, and the presence of the GCLC-129 C/T polymorphism; an experimental hyperhomocysteinemia after methionine-induced stimulation of transsulfuration pathway was performed in 91% of enrolled subjects. Clinical, biochemical, and genetic variables were correlated with the presence of CV events (myocardial infarction, transient ischemic attacks, and stroke). RESULTS: By multiple logistic regression analysis, male sex (P = .027), hypertension (P = .001), and GCLC C/T genotype (P = .009) were the only variables associated with events. Plasma alpha-tocopherol content decreased postmethionine in the T allele subjects compared with wild type (P for time x group interaction = .001). Plasma-reduced GSH level was higher in C/T than in C/C genotype subjects at both time points (P for group = .03), whereas intracellular GSH concentration did not differ between the 2 genotype groups either at baseline or postmethionine. CONCLUSIONS: GCLC T allele, together with hypertension and male sex, is associated with CV events in our study population. Moreover, after stimulation of transsulfuration, intracellular GSH content is preserved in T allele subjects, probably by increases in GSH turnover and export, and consumption of alpha-tocopherol.

Low plasma glutathione levels after reperfused acute myocardial infarction are associated with late cardiac events.

OBJECTIVE: To clarify whether an altered redox state persists in the subacute phase of myocardial infarction and if specific redox patterns are associated with later cardiac events. METHODS: Ninety-seven patients [80 men, median 63 (interquartile range, 53, 69) years] with a first acute myocardial infarction, with (53%) or without ST segment elevation, treated with successful percutaneous interventions, were tested at 5-6 days after admission for plasma alpha-tocopherol, ascorbic acid, total and reduced homocysteine, cysteine, glutathione, cysteinylglycine and blood-reduced glutathione, all assessed by high-pressure liquid chromatography. Free malondialdehyde was evaluated by gas chromatography. A subgroup of 14 patients had adjunctive blood samples within 1 h and at 72 h after angioplasty. Blood samples from 44 patients matched for age, sex, and risk factors served as controls. Patients were followed up for median 15 (interquartile range, 9, 17) months for cardiac events. RESULTS: All plasma-reduced aminothiols, vitamins and plasma total glutathione were significantly lower in myocardial infarction at 5-6 days than in controls. In the 14 myocardial infarction patients sampled repeatedly, plasma-reduced glutathione, cysteinylglycine, total glutathione, and alpha-tocopherol significantly decreased, whereas blood-reduced glutathione, total homocysteine, and cysteine significantly increased over time. During follow-up, 20 of 97 (21%) patients had adverse cardiac events. Multivariate analysis revealed that only plasma-reduced glutathione was independently associated with events (hazard ratio 0.42, 95% confidence interval 0.18-0.99, P=0.04). CONCLUSIONS: Acute myocardial infarction patients have an altered redox state at 5-6 days after successful reperfusion with respect to controls. Low plasma levels of reduced glutathione at discharge are associated with cardiac events at follow-up.

Blood glutathione as independent marker of lipid peroxidation in heart failure.

BACKGROUND: Aminothiols have a critical function as intracellular redox buffers and constitute furthermore an important extracellular redox system. Lipid peroxidation is increased in chronic heart failure (CHF), but the contribution of each thiol to oxidative stress in this syndrome has not been evaluated. AIM: To assess the correlation between blood and plasma concentrations of aminothiols and lipid peroxidation as marker of oxidative stress in CHF patients. METHODS: Blood reduced glutathione (GSH), plasma total and reduced cysteine, cysteinylglycine, homocysteine, GSH, alpha-tocopherol, ascorbic acid, and free malondialdehyde (MDA) were assessed in samples obtained from 26 CHF heart transplant candidates and 26 age- and gender-matched controls with atherosclerotic risk factors and no history of cardiovascular disease. Results are expressed as median and interquartile range (I-III). RESULTS: MDA levels were significantly higher in CHF patients than in controls [1.03 (0.56-1.60) microM vs. 0.70 (0.40-0.83) microM, p=0.006]. Blood reduced GSH concentrations were significantly higher [662 (327-867) microM vs. 416 (248-571) microM, p=0.016], while alpha-tocopherol levels were significantly lower [15 (13-19) microM vs. 21 (17-32) microM, p=0.001] in CHF patients than in controls. By multivariate logistic regression analysis, the only independent predictors of lipid peroxidation, as expressed by MDA levels > or = 1.00 microM, were increased blood GSH concentrations (OR 1.003 per unit, 95% CI 1.001 to 1.006, p=0.008), ischemic (OR 20, 95% CI 2.6 to 155, p=0.004) and non ischemic CHF etiology (OR 11, 95% CI 1.3 to 99, p=0.026). CONCLUSIONS: Abnormalities in intracellular GSH cycling are associated to increased lipid peroxidation in CHF.

Methionine challenge paradoxically induces a greater activation of the antioxidant defence in subjects with hyper- vs. normohomocysteinemia.

To determine whether hyperhomocysteinemia induced post-methionine loading (PML) is associated with different response in the aminothiol redox state and oxidative stress vs. normohomocysteinemia, we assessed PML plasma thiols, vitamins, free malondialdehyde (MDA), and blood reduced glutathione (GSH) in 120 consecutive subjects (50 [35-56] years, 83 males), divided into two groups according to PML plasma total Hcy < 35 microM (Group 1, n = 65) or > or = 35 microM (Group 2, n = 55). In the group as a whole, plasma reduced cysteine and cysteinylglycine, blood reduced GSH (all p for time = 0.0001) and plasma total GSH (p for time = 0.001) increased from baseline to PML. MDA values were unchanged. Group 1 and 2 differed in blood reduced GSH (p for group = 0.004, higher in Group 2), and MDA levels (p for group = 0.024, lower in Group 2). The oxidative stress induced by methionine challenge seems to be opposed by scavenger molecules activation, namely GSH, and lipid peroxidation does not increase. This mechanism paradoxically appears to be more efficient in hyperhomocysteinemic subjects.

Effect of homocysteine lowering by 5-methyltetrahydrofolate on redox status in hyperhomocysteinemia.

The endothelial dysfunction induced by hyperhomocysteinemia can be reversed by 5-methyltetrahydrofolate (5-MTHF) via homocysteine (Hcy) lowering. An additive antioxidant action of 5-MTHF has been suggested to ameliorate endothelial dysfunction through increased nitric oxide production and superoxide radical scavenging, independent of Hcy lowering. The aim of the study was to assess whether 5-MTHF affects the redox state in hyperhomocysteinemia. We examined the effect of 3 months of oral 5-MTHF treatment (15 mg/day) on the redox pattern in 48 hyperhomocysteinemic subjects compared to 24 untreated hyperhomocysteinemic subjects. By analysis of variance with repeated measures in the 72 subjects, 5-MTHF markedly decreased plasma total Hcy (p-tHcy; P = 0.0001) and blood-total glutathione (GSH; b-tGSH; P = 0.002). By multivariate linear regression in the treated subjects, p-tHcy changes from baseline to 3 months (adjusted by baseline p-tHcy levels) correlated only with changes in reduced cysteinylglycine (P = 0.001). The effects of treatment on Hcy lowering and GSH metabolism were greater in medium than in moderate hyperhomocysteinemia. In conclusion, high-dose 5-MTHF treatment for 3 months ensures marked Hcy lowering to normal values even in subjects with high Hcy levels, and should be the treatment of choice in medium hyperhomocysteinemia. Furthermore, 5-MTHF shows a favorable interaction with GSH metabolism.