Antioxidative effects of sulfurous mineral water: protection against lipid and protein oxidation.

OBJECTIVES: To investigate the antioxidative properties of sulfurous drinking water after a standard hydropinic treatment (500 ml day(-1) for 2 weeks). SUBJECTS/METHODS: Forty apparently healthy adults, 18 men and 22 women, age 41-55 years old. The antioxidant profile and the oxidative condition were evaluated in healthy subjects supplemented for 2 weeks with (study group) or without (controls) sulfurous mineral water both before (T0) and after (T1) treatment. RESULTS: At T1, a significant decrease (P<0.05) in both lipid and protein oxidation products, namely malondialdehyde, carbonyls and AOPP, was found in plasma samples from subjects drinking sulfurous water with respect to controls. Concomitantly, a significant increment (P<0.05) of the total antioxidant capacity of plasma as well as of total plasmatic thiol levels was evidenced. Tocopherols, carotenoids and retinol remained almost unchanged before and after treatment in both groups. CONCLUSIONS: The improved body redox status in healthy volunteers undergoing a cycle of hydropinic therapy suggests major benefits from sulfurous water consumption in reducing biomolecule oxidation, possibly furnishing valid protection against oxidative damage commonly associated with aging and age-related degenerative diseases.

Potential role of levocarnitine supplementation for the treatment of chemotherapy-induced fatigue in non-anaemic cancer patients.

Ifosfamide and cisplatin cause urinary loss of carnitine, which is a fundamental molecule for energy production in mammalian cells. We investigated whether restoration of the carnitine pool might improve chemotherapy-induced fatigue in non-anaemic cancer patients. Consecutive patients with low plasma carnitine levels who experienced fatigue during chemotherapy were considered eligible for study entry. Patients were excluded if they had anaemia or other conditions thought to be causing asthenia. Fatigue was assessed by the Functional Assessment of Cancer Therapy-Fatigue quality of life questionnaire. Treatment consisted of oral levocarnitine 4 g daily, for 7 days. Fifty patients were enrolled; chemotherapy was cisplatin-based in 44 patients and ifosfamide-based in six patients. In the whole group, baseline mean Functional Assessment of Cancer Therapy-Fatigue score was 19.7 (+/-6.4; standard deviation) and the mean plasma carnitine value was 20.9 microM (+/-6.8; standard deviation). After 1 week, fatigue ameliorated in 45 patients and the mean Functional Assessment of Cancer Therapy-Fatigue score was 34.9 (+/-5.4; standard deviation) (P<.001). All patients achieved normal plasma carnitine levels. Patients maintained the improved Functional Assessment of Cancer Therapy-Fatigue score until the next cycle of chemotherapy. In selected patients, levocarnitine supplementation may be effective in alleviating chemotherapy-induced fatigue. This compound deserves further investigations in a randomised, placebo-controlled study.

Protein thiols and glutathione influence the nitric oxide-dependent regulation of the red blood cell metabolism.

Nitric oxide (NO) can modulate red blood cell (RBC) glycolysis by translocation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) (EC 1.2.1.12) from the cytosolic domain of the membrane protein band 3 (cdb3) in the cytosol. In this study we have investigated which NO-reactive thiols might be influencing GAPD translocation and the specific role of glutathione. Two highly reactive Cys residues were identified by transnitrosylation with nitrosoglutathione (GSNO) of cdb3 and GAPD (K(2) = 73.7 and 101.5 M(-1) s(-1), respectively). The Cys 149 located in the catalytic site of GAPD is exclusively involved in the GSNO-induced nitrosylation. Reassociation experiments carried out at equilibrium with preparations of RBC membranes and GAPD revealed that different NO donors may form -SNO on, and decrease the affinity between, GAPD and cdb3. In intact RBC, the NO donors 3-morpholinosydnonimine (SIN-1) and peroxynitrite (ONOO(-)) significantly increased GAPD activity in the cytosol, glycolysis measured as lactate production, and energy charge levels. Our data suggest that ONOO(-) is the main NO derivative able to cross the RBC membrane, leading to GAPD translocation and -SNO formation. In cell-free experiments and intact RBC, diamide (a thiol oxidant able to inhibit GAPD activity) was observed to reverse the effect of SIN-1 on GAPD translocation. The results demonstrate that cdb3 and GAPD contain reactive thiols that can be transnitrosylated mainly by means of GSNO; these can ultimately influence GAPD translocation/activity and the glycolytic flux.

Polymeric protein-polyamine conjugates: a new class of uremic toxins affecting erythropoiesis.

BACKGROUND: Preliminary evidence on the accumulation of polyamine-protein conjugates (PPCs) was obtained in uremic patients. The presence of these substances in the plasma of hemodialysis (HD) patients was evaluated, and their possible contribution to uremic anemia was investigated by testing the effect of PPC synthesized in vitro on erythroid cell proliferation. METHODS: Plasma PPC was measured by high-performance liquid chromatography. The in vitro synthesis of PPC from human plasma was carried out by means of the enzyme transglutaminase in the presence of either [3H]-labeled or unlabeled spermidine (SPD). After gel filtration chromatography and detection of the fractions containing [3H]SPD, the latter were tested for their effect on mononuclear bone marrow cell proliferation. RESULTS: In three out of four patients examined, mainly SPD-protein conjugates (SPD-PC) were observed to accumulate during HD. The levels ranged from 0.17 to 4.93 pmol/mg proteins before dialysis, and these values increased at 30 minutes and at the end of the dialysis up to levels 11.90 pmol/mg. SPD-PC levels in healthy controls were 1.46 +/- 0.82. SPD-PCs synthesized in vitro were recovered in two main fractions showing a molecular weight of> 100 kD (peak 1) and of approximately 30 to 50 kD (peak 3), respectively. The SPD-PC contained in peak 1 showed the greatest inhibitory effect on colony-forming units-erythroid (CFU-E) proliferation without any appreciable effect on burst-forming units-erythroid (BFU-E). CONCLUSION: We demonstrate that SPD-PC can accumulate in HD patients. These substances, which affect CFU-E proliferation, can be considered as an at yet unrevealed class of uremic toxins contributing to the onset of the uremic anemia.

Vitamin E, lipid profile, and peroxidation in hemodialysis patients.

BACKGROUND: Hypertriglyceridemia, lipid peroxidation, and abnormalities of the plasma fatty acid (PUFA) profile may be important risk factors for the atherosclerotic cardiovascular disease in hemodialysis (HD) patients. METHODS: We investigated how these factors are affected by vitamin E supplementation carried out by oral administration (clinical study 1) and dialysis with vitamin E-modified dialyzers (clinical study 2). RESULTS: In the HD patients, conditions of relative vitamin E deficiency were observed [lowered vitamin E/triglyceride (TG) ratio] in the presence of high levels of thiobarbituric acid reactants (TBARs) and decreased levels of the polyunsaturated fraction of PUFAs paired with an increased amount of monounsaturated ones (MUFA). In both studies, vitamin E supplementation significantly increased the levels of vitamin E in the plasma without affecting TG levels and provided a partial correction of TBAR levels. Of note was the relative increase in the PUFA fraction, which gave solid proof of an anti(per)oxidant effect of vitamin E supplementation in HD patients. Vitamin E supplementation was also observed to increase plasma levels of reduced glutathione and NOx (NO2 + NO3). CONCLUSION: The results suggest that vitamin E supplementation may be an effective accessory therapy to combat oxidative stress-lowering lipid peroxidation in HD patients.

Overexpression of erythrocyte glutathione S-transferase in uremia and dialysis.

BACKGROUND: Overexpression of glutathione S-transferase (GST; EC 2.5. 1.18) has been documented in the erythrocytes of patients with chronic renal failure, and this event may well be of relevance from a clinical standpoint. In fact, it could serve as a marker of uremic toxicity overall, which can contribute to impair the function and survival of the erythrocytes. However, the biochemical details of this phenomenon are poorly understood. METHODS: In this study, we characterized the expression of GST in erythrocytes of 118 uremic patients under different clinical conditions. The mechanisms responsible for the regulation of protein expression and enzyme activity were investigated in light of different dialysis approaches, oxidative stress, uremic toxins, erythrocyte age, and erythropoietin (EPO) supplementation. RESULTS: Mean GST activity in uremic patients was highly overexpressed with respect to controls, and this phenomenon was exclusively attributable to an increased expression of GST. Overexpression of GST did not appear to be dependent on oxidative stress and was not influenced by vitamin E supplementation. In the same manner, both erythrocyte age and EPO supplementation apparently did not interfere with the GST concentrations, which were the same in controls and patients. Preliminary experiments suggested that high-molecular weight or protein-bound toxins could play some role in the overexpression of GST. CONCLUSIONS: GST expression may be a useful marker for the individual accumulation of uremic toxins as well as of the efficiency of new dialysis strategies in removing them.

Biological effects of oxidant stress in haemodialysis: the possible roles of vitamin E.

Oxidative stress has been proposed to play a role in many disease states, including cardiovascular and infectious diseases, cancer, diabetes and neurodegenerative pathologies. The fact that these diseases have an increased incidence in uremia, and particularly in dialysis patients, suggests an increased exposure to oxidative stress in this condition. In haemodialysis (HD), the absence of a complete correction of the uremic toxicity together with the untoward effects of the dialysis, malnutrition and the progressive worsening of the clinical condition, can lead to a high susceptibility to oxidative stress by an abnormal production of oxidants - including reactive oxygen species (ROS) and uremic toxins with prooxidant function - and defective antioxidant protection. One of the most investigated biological effects of the oxidative stress in the HD patients is lipid peroxidation in plasma and blood cell membranes. Moreover, we have recently described how abnormal apoptosis in peripheral blood leukocytes is associated with cell oxidative stress (intracellular thiol depletion). Vitamin E, in both in vitro and in vivo conditions, has been proposed to partially correct these effects. In this review we evaluated some features of two new dialysis strategies using an antioxidant approach to the protection against the oxidant stress in HD. Their rationale is based on the emerging role of vitamin E in counteracting some biological effects associated with oxidant stress namely lipid peroxidation and apoptosis. These techniques use: 1) the recirculation of the dialysate through a suspension of vitamin E-enriched liposomes combined with the supplementation by the dialysate with ascorbic acid, this method has been called hemolipodialysis; 2) the coating of the dialysis membrane with vitamin E (vitamin E- modified dialysis membranes). These unconventional approaches to the antioxidant therapy in HD open a widely unexplored and promising field in the evolution of the biomaterials and dialysis quality.

S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase decreases the enzyme affinity to the erythrocyte membrane.

The effects of nitric oxide (NO) or related molecules on the binding of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to the red blood cell (RBC) membrane were investigated. It was demonstrated that submillimolar concentrations of the NO donor sodium nitroprusside (SNP) not only strongly inactivated GAPDH by S-nitrosylation of the enzyme thiols but also decreased the binding affinity of GAPDH for the RBC membrane. In fact, the incubation with SNP for 60 min at 30 degrees C and at a concentration > 50 microM induced the dissociation of the native GAPDH from the white unsealed membranes (standard ghosts) in a concentration-dependent manner with a partial recovery of the enzyme activity and thiols when SNP concentrations higher of 1 mM were used. Binding experiments under saturating conditions indicate a Ka value for the nitrosylated GAPDH of 3.5 +/- 0.8 x 10(6) M-1, which was more than 50% less than the Ka value of 7.6 +/- 0.6 x 10(6) M-1 observed for the native enzyme. These data were also confirmed in reassociation experiments under nonsaturating conditions. Dithiothreitol (DTT), which at concentrations of less than 1 mM catalyzed the S-nitrosylation of GAPDH and the consequent modification of the binding properties described above, the concentrations higher than 5 mM restored both the enzyme activity and the binding properties. Furthermore, the enzyme-membrane association induced before the incubation step afforded at least partial protection from the loss of titrable thiols and from the inactivation induced either spontaneously or by SNP. Taken together, these data not only confirm the key role of the active site cysteine residues in the catalytic function of GAPDH but also suggest that they may be involved in the NO-dependent regulation of GAPDH binding to the RBC membrane.

Bioreactivity and biocompatibility of a vitamin E-modified multi-layer hemodialysis filter.

BACKGROUND: The present study was designed to test the biocompatibility of a new vitamin E-modified multi-layer membrane (CL-E filter), as well as its ability to protect against oxygen free radicals during hemodialysis (HD). METHODS: We investigated, both in vitro and in vivo, the bioreactivity of the filter with respect to the blood antioxidants and its ability to prevent lipoperoxidation. The effects on the leukocyte respiratory burst were also studied. Cuprammonium rayon was used as a comparison material (CL-S filter). RESULTS: The in vitro results demonstrated that, under controlled conditions, CL-E is able to preserve blood antioxidants, and particularly vitamin E, from the spontaneous consumption observed in the incubation with CL-S filters and in control incubations. In accordance with this observation, the rate of the oxidative demolition of lipids either in plasma and red blood cells (RBC) or from rat brain homogenate decreased after the exposure to CL-E filters in comparison with the CL-S filter. Moreover, in the absence of any significant cytotoxic effects due to both the types of material studied, the production of oxygen free radicals and nitric oxide (NO) by leukocytes was higher after their in vitro exposure to CL-S, but was quite similar to that of the control leukocytes after exposure to CL-E. In vivo, a one-month treatment with the CL-E filter increased plasma vitamin E by 84.3% with respect to treatment with CL-S; this gain slightly decreased to 68.9% when CL-E treatment was prolonged to three months. In the RBC, vitamin E was found to have increased by 76.7% and 113.4% at one and three months, respectively. Plasma glutathione (GSH) levels determined at three months were significantly increased from 0.10 +/- 0.02 to 0.33 +/- 0.12 mumol/ml, while the erythrocyte GSH was only slightly increased. The leukocyte function estimated as responsiveness to soluble chemical stimuli in CL-S-treated patients was significantly improved both qualitatively and quantitatively after CL-E treatment. The presence of an increased number of mononuclear cells undergoing programmed cell death (apoptosis) in CL-S-treated patients (18.8 +/- 1.7% vs. a control value of 6.5 +/- 2.3%) as well as the apoptogenic effect of their plasma in vitro on U937 cells was significantly corrected after CL-E treatment (mean decrease in apoptotic mononuclear cells at 24 hours of culture, 25.5% and 27.1% at 1 and 3 months, respectively). The anti-apoptogenic effect of CL-E treatment showed a close dependence on the increase in vitamin E in the blood cell compartment. CONCLUSIONS: This study suggests that this vitamin E-modified membrane can be considered a highly biocompatible material, the antioxidant properties of which can exert a site-specific and timely scavenging function against oxygen free radicals in synergy with a hypostimulatory action on the PMN respiratory burst.

Oxidative damage during hemodialysis using a vitamin-E-modified dialysis membrane: a preliminary characterization.

A comparison of the oxyradical exposure during hemodialysis (HD) carried out with vitamin-E-modified cellulose (CL-E) or conventional membranes, studying red blood cell (RBC) and plasma lipoperoxidation and RBC glutathione metabolism, was done. In this preliminary characterization of a new and original approach to the prevention of free radical damage in HD, the results obtained indicate that lipoperoxidation in plasma and RBC is decreased and therefore oxidative damage can be significantly decreased using CL-E dialysis membranes instead of conventional membranes.

Bicarbonate versus lactate buffer in peritoneal dialysis solutions: the beneficial effect on RBC metabolism.

OBJECTIVE: Using the erythrocyte as a model for other kinds of cells not directly exposed to peritoneal dialysis (PD) solutions, we investigated the tolerance of the cell metabolism to lactate and bicarbonate buffers. DESIGN: We studied, in vivo (in two groups of 5 PD patients each) and in vitro, the Embden-Meyerhof pathway (EMP) because it represents a potential target for the unphysiological effects of lactate or bicarbonate buffers. The EMP is the main glucose-utilizing route in the red blood cell (RBC), producing energy and reducing power. METHODS: The enzymatic activities of the key steps in the glycolytic pathway and the energy charge (EC), determined by the levels of phosphorylated adenine nucleotides, were investigated spectrophotometrically and by high performance liquid chromatography (HPLC) in two groups of patients undergoing lactate (L-group) and bicarbonate (B-group) PD, respectively. The in vitro effects of both bicarbonate and lactate buffers on some EMP enzyme activities and energy production were determined. Cellular pH (pHi) was also investigated. RESULTS: The B-group showed an EC value near the control levels, while in the L-group a significantly lower EC value was observed (t-test: p < 0.05 vs both B-group and controls). The key enzymes in the EMP, and in particular hexokinase, were higher in the L-versus B-group (p < 0.03 for the comparison of the Hk mean values). As demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the bound form of glyceraldehyde-3-phosphate dehydrogenase (G-3-PD), an inactive form of this EMP enzyme, was significantly higher in the L-group with respect to the B-group (p < 0.004). In the in vitro experiments, high lactate concentrations acutely inhibited the key enzymatic steps of glycolysis, producing a significant decrease in glucose consumption and adenosine triphosphate production. These effects were not observed when bicarbonate was used in the incubations. Both in vivo and in vitro lactate, but not bicarbonate, induce a significant drop in pHi (p < 0.05). Decreased levels of pHi like those observed in the lactate-incubated RBC were demonstrated to be able to inhibit G-3-PD activity (25 +/- 2%) here used as an indicator of the actual decrease in pH. CONCLUSION: This study provides evidence for a damaging action of lactate with respect to bicarbonate buffer on the RBC metabolism. This condition was demonstrated observing a cell energy depletion, which coincides in vitro with an acute EMP impairment; the lactate accumulation together with the consequent lowering of pHi seem to be responsible for this effect, which was not observed when bicarbonate was used instead of lactate.

Morphological alterations and increased resistance to hemolysis in t-butyl hydroperoxide incubated RBC from elderly subjects.

The response of human red blood cells (RBC) to oxidative stress has been studied with the aim to evaluate any difference in the behavior of cells from young and old subjects. Thus, RBC from 5 young (27 +/- 2 years) and 5 old (80 +/- 5 years) individuals have been treated with the organic peroxide t-butyl hydroperoxide (TBHP). The two groups behaved differently: after 4 hrs of incubation in 0.5 mM TBHP, RBC from young donors showed a higher level of hemolysis; instead, RBC from old individuals showed abnormal morphologies, being absent in unstressed RBC, with constriction and budding, which could be identified as poikilocytosis. The same abnormal forms are found in patients with spectrin mutation, leading us to hypothesize that TBHP causes damage to the cytoskeletal spectrin. This suggests that poikilocytosis might be an early stage of red blood cell hemolysis because their presence is associated to a lower level of hemolysis.

Redox state, antioxidative activity and lipid peroxidation in erythrocytes and plasma of chronic ambulatory peritoneal dialysis patients.

Red blood cells and plasma reduced and oxidized glutathione levels, glutathione peroxidase (GSH-Px) activity, thiobarbituric acid reactants (TBAR) of both chronic ambulatory peritoneal dialysis (CAPD) patients and a matched control group were investigated in this study. Oxidized and reduced pyridinic nucleotides in red blood cells (RBC), in which NADPH is a direct expression of hexose monophosphate shunt function, were also studied. The results obtained indicate that RBC and plasma are exposed to oxidative stress in CAPD. This condition is characterized by a decreased GSH/GSSG ratio, particularly evident in RBC as a consequence of the GSSG accumulation. Lipid peroxidation is increased, as indicated by raised TBAR levels, and reduced pyridinic nucleotides are decreased. Increased GSH-Px levels and unmodified or slightly increased GSH content were observed in the RBC but not in plasma, which showed decreased GSH and unmodified peroxidase activity. Peroxidase correlated positively with TBAR levels in the RBC lysates. In a subgroup of patients treated with erythropoietin (vs. untreated patients and controls) no differences were observed in the glutathione-related parameters studied. These data suggest that a mechanism for adaptation to oxidative conditions may be present in CAPD and its effects on RBC integrity are discussed in comparison with the hemodialysis conditions previously studied.

Erythrocyte Na+,K(+)-ATPase properties and adenylate energy charge in normotensives and in essential hypertensives.

The activity and some kinetic properties of RBC Na+,K(+)-ATPase (EC 3.6.1.37) were investigated in essential hypertensives (EH; 40 subjects) and normotensives (NT; 20 subjects). A decrease in ouabain-sensitive 86Rb uptake as well as ouabain-sensitive ATPase activity was found in EH. [Na+]i and [K+]i of EH did not show any statistical difference from NT. Na+,K(+)-ATPase showed a reduced Mg2+ activation and the apparent Km value for Mg2+ was 2-fold increased in the EH group. The influence of temperature on the Na+,K(+)-ATPase showed a reduced modulation and a minor activity peak at 37 degrees C in the patients, consequently the calculated activation energy of the enzyme was increased at temperatures lower than 40 degrees C. Increased RBC adenylate energy charge (EC) was observed in EH when compared with NT. A negative correlation between EC and total Na+,K(+)-ATPase activity was found when all subjects were compared and also in both groups, showing a possible pump involvement in the regulation of the RBC metabolic flux in EH. These data provide evidence about some modifications in active Na+,K+ transport and in EC in RBC which allows a further characterization of membrane cation fluxes in EH.

Erythrocyte redox state in uremic anemia: effects of hemodialysis and relevance of glutathione metabolism.

Reduced and oxidized glutathione and pyridine coenzymes, glutathione-related enzymes and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were investigated in the RBC of patients with chronic renal failure (CRF) and in age- and sex-matched controls. The effects of hemodialysis (HD) were also studied. A defective RBC redox state was shown in the CRF group based on a decreased GSH/GSSG ratio and NADPH levels. Increased activities of glutathione transferase (GSH-S-T) and Cu,Zn-SOD were observed before HD. Dialysis apparently restores the levels of antioxidant enzymes and at the same time strongly affects the redox state. Thus we can speculate that HD can generate severe redox impairment inducing damage in RBC and plasma antioxidant enzymes. Increased erythrocyte GSSG and GSM-S-T levels coupled with a reduced hexose monophosphate shunt (HMPS) function may be useful indexes of oxidative stress in uremic anemia.