Biochemical assessments of oxidative stress, erythrocyte membrane fluidity and antioxidant status in professional soccer players and sedentary controls.

BACKGROUND: Physical exercise is characterized by an increase in oxygen consumption by the whole body. This leads to a decrease in antioxidant levels that could promote both an increase in the markers of lipoprotein peroxidation and damage to the erythrocyte membrane with consequent modification of membrane fluidity. MATERIALS AND METHODS: Different markers of oxidative stress, erythrocyte membrane fluidity and antioxidant status were determined in 20 professional soccer players and 20 sedentary controls. Plasma lipoperoxides and kinetics of Cu-stimulated plasma peroxidation were measured together with hydrosoluble (albumin, uric acid and vitamin C), liposoluble (vitamin E and bilirubin) and enzymatic (superoxide dismutase and glutathione peroxidase) serum antioxidants. Erythrocyte membrane rigidity was determined by measuring fluorescence anisotropy (rs) of the fluorescent probe 1, 3, 5 diphenylexatriene. RESULTS: The sportsmen showed higher levels of the following plasmatic antioxidants: ascorbic acid (P<0.0001), uric acid (P<0.0001), alpha-tocopherol (P=0.03) and superoxide dismutase activity (P=0.0001). According to this evidence, the lipoperoxide levels (P=0.0158), the duration of the latency phase of plasma peroxidation (P=0.0123) and erythrocytes membrane fluidity (P=0.0152) were found to be significantly higher in the soccer players. DISCUSSION: Athletes undergoing regular and adequate training show improved antioxidant status together with a more fluid membrane status, which could contribute to improving both peripheral resistance to insulin and all the functional metabolic interchanges in the cellular membrane.

Oxidative stress, vitamin A and vitamin E behaviour in patients submitted to conservative surgery for complicated Crohn's disease.

AIMS: To assess whether plasma peroxidation and plasma levels of antioxidant compounds are correlated with clinical and biochemical activity in complicated Crohn's disease patients, and to evaluate whether the relief of obstructive complication by conservative surgery has any effect on the oxidative stress. PATIENTS AND METHODS: From May 1998 to May 2000, 20 Crohn's disease patients were studied. Basal peroxidative state (basal thiobarbituric acid reactive substances), peroxidative state after stimulation with copper sulfate (stimulated thiobarbituric acid reactive substances], lag time of plasma peroxidation susceptibility, plasma levels of vitamin E and A, C reactive protein, erythrocyte sedimentation rate and Crohn's disease activity index, were determined, before surgery, then 2 months and 1 year after surgery. A group of 134 healthy volunteers were used as controls. All patients were treated by conservative surgical procedures (i.e., strictureplasty and/or minimal resections). Student t test for paired and unpaired data and Spearman R correlation coefficient were calculated. RESULTS: Peroxidative plasma levels, as well as inflammatory indices, are significantly reduced 2 months and 1 year after surgery (p < 0.005), but basal levels of peroxidation and antioxidant scavengers seem to be disregulated in Crohn's disease patients compared to those in controls (p < 0.005). A correlation was found between basal thiobarbituric acid reactive substances, lag-time and erythrocyte sedimentation rate (R:0.51; p < 0.05. R:0.56; p < 0.05) and C reactive protein (R:0. 6; p < 0.005. R:0. 65; p < 0.005). CONCLUSIONS: An imbalance between pro- and antioxidant mechanisms, due to chronic gut inflammation, is present in complicated Crohn's disease, and an excess of lipid peroxidation is probably an important pathogenetic factor Conservative surgery can reduce the oxidative stress avoiding repeated or extended resections that could lead to intestinal malabsorption and short bowel syndrome.

A fluorescence method for the determination of plasma susceptibility to lipid peroxidation.

OBJECTIVE: We propose a fluorescence kinetics method for monitoring plasma susceptibility to peroxidation. DESIGN AND METHOD: Plasmatic peroxidation was induced by CuSO4 (500 microM), and fluorescence was measured every 30 min. Kinetics were represented by a sigmoidal curve from which it was possible to calculate the latency time (lag-time) and the propagation velocity (slope) of plasma peroxidation. RESULTS: The lag-time monitored by the fluorescence kinetics method corresponded to the formation of thiobarbituric acid reactive substances, and to progressive depletion of polyunsaturated fatty acids and alpha-tocopherol. The mechanism of reaction appeared to be dependent upon plasmatic hydroperoxides, and independent of oxygen radicals. Plasma storage is possible for at least two months at -80 degrees C, and reproducibility of the method is very good. CONCLUSIONS: Fluorescence kinetics provide a highly comprehensive picture of plasma susceptibility to peroxidation in comparison with the conventional measurements of anti- and pro-oxidant ratios.

Variability in alpha-tocopherol antioxidant activity in the core and surface layers of low- and high-density lipoproteins.

The effect of alpha-tocopherol enrichment of low- and high-density lipoproteins on Cu(2+)-catalyzed lipid peroxidation in the hydrophobic core and in the hydrophilic envelope of lipoproteins was investigated by using two pyrene derivatives, namely, cholesteryl pyrenyl hexanoate (P6Chol) and pyrene dodecanoyl sulfatide (P12CS). The progressive decrease in fluorescence of P6Chol was used to monitor lipid peroxidation in the core of LDL and HDL, whereas that of P12CS was used to follow lipid peroxidation in the envelope of both lipoproteins. alpha-Tocopherol enrichment of LDL and HDL was obtained by incubating blood plasma at 37 degrees C with different concentrations of the vitamin (25-500 microM) before lipoprotein separation. The incorporation of alpha-tocopherol in LDL and HDL presents a progressive, time-dependent increase up to 200 microM alpha-tocopherol, then a plateau up to 500 microM. In the envelopes, the added tocopherol causes a great decrease in the rate of peroxidation and a dramatic increase in the latency phase in both lipoproteins. In the cores the lengthening of latency phase resulting from alpha-tocopherol enrichment was by far greater in LDL than in HDL, and the decrease in the rate of peroxidation in both lipoproteins was less than in the envelopes.

Studies on the antioxidant activity of milk caseins.

The antioxidant properties of milk casein subunits (alpha-casein, beta-casein and kappa-casein) were evaluated in liposomal models. All the subunits of casein are able to inhibit Fe-induced peroxidation of arachidonic acid inserted into multilamellar liposomes of dipalmitoylphosphatidylcholine (0.2 mM and 0.8 mM, respectively). The peroxidation was monitored as thiobarbituric acid reactive substances, and the strongest inhibitory effect occurred when 500 micrograms of alpha-casein were added to 0.5 ml of liposomal suspension. At this concentration, peroxidation was completely inhibited in our experimental conditions (incubation for 2 h at room temperature, with a mixture of ferrous sulfate and ascorbate, 50 and 500 microM final concentration, respectively). The mechanisms of antioxidant action are complex, but the strongest effect is achieved by modifying the Fe2+/Fe3+ equilibrium; in fact, caseins seem to favour the autoxidation of iron, and thus inhibit lipid peroxidation.

Red wine effects on peroxidation indexes of rat plasma and erythrocytes.

Moderate wine intake has been associated with low risk for cardiovascular disease, possibly due to its polyphenol content. We investigated the influence of these compounds on peroxidative indices of blood. Forty Sprague-Dawley rats were divided into four groups given the same AIN-76 diet but different types of isocaloric beverage: red wine, alcohol and dealcoholated wine and water with added sucrose. Rats consumed about 35 ml day(-1) of beverage and 17 g day(-1) of diet for a total of about 95 kcal day(-1). Plasma alpha-tocopherol was higher in the alcohol group and the peroxidation kinetic slope was higher in the control and dealcoholated wine groups. Ascorbic acid in whole blood was higher in the alcohol group than the others. Erythrocytic glutathione peroxidase activity was lower in the red wine group. The other variables studied, were not significantly different in the four groups. Despite the significant differences observed, the oxidative status of blood does not appear seriously altered by heavy ingestion of wine and thus of polyphenols. Probably the balanced diet and adequate intake of micronutrients were sufficient to counteract any oxidative damage.

Pyrene lipids as markers of peroxidative processes in different regions of low and high density lipoproteins.

Three different pyrene derivatives, pyrene decanoyl phosphatidylcholine (P10PC), pyrene dodecanoyl sulfatide (P12CS) and cholesteryl pyrenyl hexanoate (P6Chol), were used to follow lipid peroxidation in low and high density lipoproteins. Probe-labelled lipoproteins were subjected to Cu2+ catalyzed peroxidation. In all cases the fluorescence of the probes progressively decreased due to the involvement of pyrene in the peroxidative reaction. Thus, we used the fluorescence decrease of P6Chol to monitor the lipid peroxidation in the hydrophobic core of LDL and HDL, and that of the amphipatic probes, P10PC and P12CS, to follow lipid peroxidation in the envelope of both lipoproteins. The possibility of following lipid peroxidation in individual lipoprotein regions could lead to more detailed information on the oxidative modifications that play an important role in the altered cholesterol homeostasis involved in the formation of atherosclerotic lesions. No differences were observed in the peroxidation kinetics of the hydrophobic core of HDL and LDL monitored with P6Chol. On the contrary kinetics obtained with P10PC and P12 CS demonstrated the HDL envelope to be more susceptible to Cu2+ -dependent lipid peroxidation than that of the LDL. This could be due to a greater radical generating capacity of the HDL envelope and can be explained on the basis of low vitamin E levels and large amounts of polyunsaturated fatty acids esterified on phospholipids determined in HDL, and on literature evidence that indicates HDL as the principal vehicle of circulating plasma lipids peroxides.

Effect of red wine consumption on rat liver peroxidation.

To evaluate the role of wine polyphenols and that of alcohol on lipid peroxidation indexes and membrane composition in the liver, 40 Sprague-Dawley rats were fed for 28 days with a commercial AIN-76 diet to which was added one of four different beverages: red wine, alcohol solution, dealcoholated wine, or water. The beverage provided 26% of the caloric intake. Peroxidation indexes and antioxidative enzymes were determined: no significant differences were detected in catalase and glutathione peroxidase whereas superoxide dismutase was significantly lower in the wine-treated animals (220.3 +/- 15.4 vs. 342.2 +/- 43.0 U/mg protein of controls). The following significant differences in hepatic variables were observed: increased alpha-tocopherol concentration in the alcohol group (0.17 +/- 0.02 vs. 0.11 +/- 0.01 microgram/mg protein of controls); increased concentration of cytochrome P450 in the rats given wine (0.75 +/- 0.06 vs. 0.51 +/- 0.08 nmol/mg protein of the alcohol group); increased concentration of cytochrome b5 in wine and dealcoholated wine treatment groups (0.30 +/- 0.01 vs. 0.23 +/- 0.02 nmol/mg protein of controls). The liver membrane fatty acid composition of the wine and dealcoholated wine groups was similar and showed an increase in the saturated fatty acid percentage and a decrease in the polyunsaturated one. The data presented indicate that the main action of polyphenols seems to be an induction of cytochrome activity and that the modality of red wine administration adopted combined with an adequate diet does not provoke any apparent physiological effect on the animals.

Effect of endothelin-1 induced ischemia on peroxidative damage and membrane properties in rat striatum synaptosomes.

Synaptosomes obtained from rat striata lesioned by central injection of endothelin-1 (ET-1) were analyzed for the levels of lipid peroxidation products, the susceptibility to lipid peroxidation, the phospholipid and free fatty acid composition and the activity of Na+,K(+)-ATPase one hour after ET-1 treatment. The intrastriatal injection of ET-1 promoted an increase of endogenous thiobarbituric reactive substances (TBARS), as index of free radical mediated lipid damage, and a greater susceptibility to iron/ascorbate-induced lipid peroxidation. The pattern of free fatty acids showed a significant decrease of arachidonic and docosahexaenoic acid consequent to ET-1 treatment. The analysis of lipid composition showed a significant loss of phospholipids: among phospholipid species, sphingomyelin and phosphatidylethanolamine plasmalogen were particularly reduced by ET-1 treatment. The activity of membrane-bound Na+,K(+)-ATPase was also significantly reduced in synaptosomes obtained from ET-1 lesioned striata. Taken together these results indicate a significant modification of synaptosomal membrane of ET-1 treated rat striata, possibly due to a free radical mediated damage.

Fluorescence-determined kinetics of plasma high oxidability in diabetic patients.

The peroxidative potential of whole plasma was studied in thirteen control subjects and twenty-three diabetic patients divided into two groups on the basis of their metabolic control, defined as good (n = 12) or poor (n = 11) according to the fasting blood glucose, glycated hemoglobin and fructosamine levels. The amounts of thiobarbituric reactive substances (TBARS) were determined before and after an exhaustive peroxidation induced by incubation with cupric sulfate; the kinetics of Cu-dependent plasma peroxidation, determined by the time course of endogenous fluorescence emission at 430 nm, were also evaluated. Our data indicate that in diabetic patients, and particularly in those with a long-lasting poor metabolic control, a significant increase in plasma susceptibility to peroxidative stress is evident only when we consider the lag-time of fluorescence emission in the course of Cu-induced plasma peroxidation. A good inverse correlation was observed between the lag-time and the blood glucose concentration, the glycated hemoglobin and the fructosamine blood levels. Furthermore a multivariate analysis, performed by Principal Component Analysis, strongly supports the relation between the extent and duration of the diabetic pathology, and the decrease in the lag-time of oxidation.

Further studies on the antioxidant role of pyrophosphate in model membranes.

Our previous studies (G. Cervato et al., Chem. Phys. Lipids 56 (1990), 91-99) demonstrated that 100 microM pyrophosphate (PPi) inhibits Fe2+/ascorbate-induced peroxidation of arachidonic acid (AA) inserted in unilamellar liposomes (SUVs) of dipalmitoylphosphatidylcholine (DPPC), by chelating ferrous ions in the aqueous phase. In this work we demonstrate that the kinetics of AA peroxidation in DPPC SUVs are strongly affected by PPi, also at very low concentration (1 microM). In fact at low PPi concentration there is a longer lag-phase, while the maximum of thiobarbituric acid reactive substances (TBARS) is similar in both the presence and absence of 1 microM PPi. The lag-phase of peroxidation of AA in lysophosphatidylcholine (palmitoyl) (LysoPC) micelles is also prolonged. The AA peroxidation in membrane models without choline as the polar headgroup (dipalmitoylphosphatidylethanolamine (DPPE) SUVs, or Triton X-100 micelles), or in which AA is not free but esterified with glycerol (stearoyl-arachidonyl-phosphatidylcholine (SAPC)), is unaffected by the presence of 1 microM PPi.

Effects of alcohol abuse: studies on human erythrocyte susceptibility to lipid peroxidation.

The susceptibility to lipid peroxidation induced by an iron-ascorbate system was studied in human erythrocyte membranes (ghost), and measured as thiobarbituric acid reactive substances (TBARS) formed. The male and female subjects were 13 chronic alcoholic patients aged 34-54 who assumed 200-250 g/die of ethanol, and 12 healthy controls aged 23-46. In alcoholics ghost sensitivity to lipid peroxidation was significantly decreased compared with that of the controls. Peroxidation on lipids extracted from the ghosts of both control and alcoholic subjects confirmed an increased resistance to free radical damage of the erythrocyte membranes obtained from alcoholic patients, thus indicating that in alcoholic patients the lipid component is involved in the resistance to free radical damage of erythrocyte membranes. Lipid analysis revealed no significant difference in cholesterol and phospholipid content, nor in the phospholipid classes, of the two groups considered. Nevertheless the fatty acid composition of ghosts from the alcoholic subjects was significantly lower in docosahexaenoic acid content than in the controls, and it is with this in mind we propose the explanation of the results we obtained.

Plasma dependent pH sensitivity of liposomes containing sulfatide.

In this study we investigated the possibility to define relatively plasma-stable liposomal preparations in which the sensitivity to moderate drops of pH (i.e., from 7.4 to 6.8) would be induced by the presence of plasma itself. The liposome stability was monitored by determining the release of entrapped 5,6-carboxyfluorescein (CF). Using small unilamellar vesicles composed of egg phosphatidylcholine (EPC) and bovine brain sulfatide (CS) (4:1, molar ratio), the amount of CF released at pH 6.8 in the presence of 50% plasma was 3-fold that at pH 7.4, whereas no significant differences in the amount of CF released were observed when the same liposomes were incubated in buffer at pH 7.4 and 6.8, respectively. The increase in plasma induced leakage as a consequence of a drop in the pH medium, seems to specifically depend on the presence of sulfatide molecule in the bilayer since neither the acidic cholesterol 3-sulfate nor galactocerebroside, are able to induce pH sensitivity in EPC liposomes. Of all the plasma components considered (VLDL, LDL, HDL, protein fraction), VLDL seemed preferentially involved in the pH sensitivity induced by CS since they promoted an almost complete release of CF from EPC/CS small unilamellar vesicles. Thus, these liposomes are potentially a useful tool for a specific drug delivery to those pathological tissues such as tumors, inflammation sites and ischemic areas in which it is known that a lowering of the pH can occur.

Calcitonin-induced changes in the organization of sulfatide-containing membranes.

The interactions of salmon calcitonin with glycosphingolipid sulfatide are studied by right angle light scattering from the lipid suspension, by the excimer to monomer ratio (E/M) of the fluorescence intensity of pyrene phosphatidylcholine and pyrene sulfatide and by the leakage of carboxyfluorescein. It was found that calcitonin strongly modified the structure of the sulfatide aggregate, as indicated by the light scattering determinations. At a lipid peptide ratio 100:1 (molar ratio) light scattering from the suspension was negligible, indicating the formation of peptide-sulfatide complexes with a structure different from that of the lipid aggregate. The interactions of calcitonin with sulfatide when the latter is a component of a bilayer were also evaluated. A specific calcitonin-membrane sulfatide interaction was demonstrated by determining the temperature-dependent E/M of pyrene phosphatidylcholine and pyrene sulfatide in dipalmitoyl phosphatidylcholine/sulfatide (80:20, molar ratio) liposomes. The E/M curves were modified by calcitonin only when the liposomes were labelled with fluorescent sulfatide which probes the sulfatide behavior in the membrane. Furthermore, the addition of calcitonin to the incubation medium of liposomes containing sulfatide promoted the release of vesicle entrapped carboxyfluorescein without disrupting the bilayer structure, the release being correlated with the amount of sulfatide in the bilayer and the calcitonin concentration in the medium.

Pyrene derivatives as markers of transbilayer effect of lipid peroxidation on neuronal membranes.

Two different pyrene derivatives, namely 12-(1-pyrene)dodecanoic acid (P12-FA) and N-(12-(1-pyrene)dodecanoyl)-galactosylsphingosine I3-sulfate (P12-CS) have been used to follow lipid peroxidation both in model and natural membranes. The malondialdehyde (MDA) production in small unilamellar vesicles of dipalmitoylphosphatidylcholine/arachidonic acid (80:20, molar ratio), symmetrically labelled with both probes determined a progressive decrease of pyrene fluorescence due to an involvement of pyrene in the peroxidative reaction. Nervous membranes are particularly sensitive to lipid oxidation which differentially acts on the two layers of the membrane determining a greater rigidity of the exofacial one. Thus, we consider the possibility to asymmetrically introduce the pyrene ring, as P12-FA or P12-CS, in synaptosomes for monitoring lipid peroxidation in each layer of the membrane. The amount of the two probes incorporated in the membrane was 20 +/- 3 and 10 +/- 2 nmol/mg of protein for P12-FA and P12-CS, respectively. P12-FA was symmetrically distributed in the two layers, whereas 95% of P12-CS was incorporated in the exofacial layer of the membrane as determined by TNBS measurements. The decrease in fluorescence of synaptosome associated pyrene was, in the early stages of lipid peroxidation, greater for P12-CS than for P12-FA labelled membranes, indicating a greater susceptibility of the exofacial layer to iron-induced peroxidation.

Age-related differences in synaptosomal peroxidative damage and membrane properties.

Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+, K(+)-ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age-related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age-dependent decrease in membrane fluidity, a reduction of Na+, K(+)-ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K(+)-ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to ageing processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.

Studies on peroxidation processes of model membranes: role of pyrophosphate.

Phosphate-containing compounds of both a lipophilic (dipalmitoylphosphatidic acid, lysophosphatidic acid and dicetylphosphate) and hydrophilic nature (orthophosphate, 3-phosphoglyceric acid, 2,3-diphosphoglyceric acid and pyrophosphate in particular) have been found to inhibit to varying degrees the lipoperoxidation of liposomal arachidonic acid. Lipophilic compounds seem to act exclusively at the lipid component level of the membrane, giving rise to polyanionic complexes with free arachidonic acid or its radical derivatives that could bind the Fe2+ strongly (thereby inhibiting the iron pro-oxidation activity) or to minimize the lateral mobility of the lipid radicals (thereby reducing the propagation of lipid peroxidation). The high antiperoxidative power of hydrophilic compounds, and in particular of pyrophosphate, must, on the contrary, be primarily attributed to their ability to form very stable complexes with the Fe present in the solution surrounding the liposomal membranes. The possible contribution of these physiological compounds to the in vivo defense mechanism against radical-induced damage is discussed.

Studies on peroxidation processes of model membranes and synaptosomes: role of phosphatidic acid.

Dipalmitoylphosphatidic acid (DPPA) was found to exert a strong inhibitory effect on Fe-induced peroxidation of arachidonic acid inserted into liposomal dipalmitoylphosphatidylcholine (DPPC) vesicles. This inhibition was quite effective both below and above the phase transition temperature of the liposomes. Moreover, we demonstrated the antiperoxidative activity of phosphatidic acid (PA) in synaptosomal membranes. PA enriched synaptosomes were prepared by the stimulation of the endogenous phospholipase D activity or by the incubation of the synaptosomes with Streptomyces chromofuscus phospholipase D. The possible contribution of PA to the in vivo defense mechanism against free radical-induced damage is discussed.

Studies on peroxidation of arachidonic acid in different liposomes below and above phase transition temperature.

The mechanism of Fe-induced peroxidation of arachidonic acid (AA) in small unilamellar vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) was studied below and above gel to liquid-crystalline phase transition temperature (Tm). In both liposomes the AA peroxidation resulted higher in the temperature range below Tm, but the extent of malonyldialdehyde (MDA) formation was dramatically lower in DPPE vesicles when compared with corresponding DPPC liposomes. A possible explanation for this is discussed.

N-pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes.

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.