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It has been suggested that amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder resulting in motor neuron death, is associated with oxidative damage induced by free radicals. Our study aimed to get an assessment of the blood oxidative stress status in a population of 167 ALS patients (aged 59+/-13 years), treated or not with riluzole, compared with 62 age-matched healthy control subjects (aged 60+/-11 years) simultaneously included in the study. We determined the level of plasma lipid peroxidation (thiobarbituric acid-reactive substances, TBARS); the status of the major lipophilic plasma antioxidant defenses (vitamin E, vitamin A and beta-carotene); the activities of erythrocyte Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and of plasma and erythrocyte glutathione peroxidase (GSH-Px). Plasma selenium was also determined as a trace element essential to the activity of the GSH-Px. In comparison with controls, we observed in ALS patients (mean+/-S.D.) significantly higher TBARS values (ALS=1.34+/-0.28 micromol/l; controls=1.11+/-0. 20 micromol/l) and a significant enhancement of the erythrocyte SOD activity (ALS=710+/-114 U/g Hb; controls=667+/-93 U/g Hb). No differences were observed for selenium level, GSH-Px activity, plasma vitamin E, beta-carotene and vitamin A concentrations. These data confirm the presence of an oxidative stress in blood of ALS patients. The elevated plasma TBARS, without any deficiency in plasma lipophilic antioxidants such as vitamin E, vitamin A and beta-carotene, suggest an enhancement in the production of free radicals. No correlation was found in our study between the level of any of the blood oxidative stress markers and the disease duration. Comparison between patients treated or not with riluzole did not display any modification of the plasma TBARS concentration, but we observed a slight decrease of erythrocyte SOD activity in treated patients (treated=705+/-113 U/g Hb; not treated=725+/-118 U/g Hb), suggesting a possible activity of riluzole on the oxygenated free radical production.

Susceptibility of lipoproteins to oxidation is partly determined by their content in endogenous antioxidants, but also by the polyunsaturated fatty acids (PUFA)/monounsaturated fatty acids (MUFA) ratio. The aim of our study was to enrich human high-density lipoproteins (HDLs) with dioleoylphosphatidylcholine (DOPC) in order to modify the PUFA/MUFA ratio while maintainig the alpha-tocopherol/PUFA ratio constant and to appreciate the consequences of this enrichment before and after copper-induced oxidation. The enrichment of HDLs with DOPC was obtained by incubation of these lipoproteins with DOPC liposomes and further reisolation of HDLs. The consequent 40% HDL enrichment in MUFA was concomitant with a 35% loss in PUFA (MUFA/PUFA ratio = 1.43). The enrichment of HDLs with DOPC led to a 40% decrease in alpha-tocopherol content, which kept a constant alpha-tocopherol/PUFA ratio. The DOPC-HDLs exhibited a lower oxidizability by copper than the nonenriched HDLs (NE-HDLs), as shown by their twofold longer lag phase and the threefold lower propagation rate. Moreover, DOPC-HDLs led to a six- to sevenfold lower production of hydroperoxide molecular species from phosphatidylcholine and cholesteryl esters than NE-HDLs after 24 h copper oxidation. With regard to the cholesterol effluxing capacity, copper oxidation of HDLs led to a decrease of this property. However, our results clearly showed that DOPC enrichment of HDLs allowed us to keep a better effluxing capacity than in NE-HDLs after 24 h oxidation (22.3% vs 17.4%, respectively). Since apo A-I was degraded as well in DOPC-HDLs as in NE-HDLs, the better effluxing capacity of DOPC-HDLs could not come from a preserved integrity of apo A-I. It could be partly related to the improved fluidity of oxidized DOPC-HDLs compared to oxidized NE-HDLs, as shown by electron spin resonance data (correlation-relaxation time at 24 degreesC = 2.20 ns vs 3.00 ns after 24 h oxidation, in DOPC-HDLs and in NE-HDLs, respectively). Besides, it could also be hypothesized that the sevenfold lower content of phosphatidylcholine hydroperoxides in DOPC-HDLs than in NE-HDLs after 24 h copper oxidation could be involved in the better ability of oxidized DOPC-HDLs to mobilize cellular cholesterol.

This study was performed to elucidate the mechanism by which adenosine triphosphate (ATP) encapsulated into liposomes was able to protect against experimental brain ischemia in the rat. After intracarotidal administration of liposomally entrapped ATP, the ATP blood level increased dramatically whereas no change was observed after administration of free ATP. This suggested that liposomes may protect ATP from its degradation by endothelial ectonucleotidases. On the other hand, it was observed that after administration of liposomally entrapped carboxyfluorescein (CF) to ischemic rats, the distribution of the brain fluorescence under the form of numerous punctiform structures was completely different from the diffuse fluorescence obtained with free CF injections. These data suggest that under certain hypoxic conditions the blood-brain barrier is open allowing the liposomes to reach the cerebral parenchyma. The mechanism of brain uptake is, however, still unclear: endothelial tight junctions opening or endothelial transcytosis.

Background: Oxidative modification of low-density lipoprotein (LDL) plays a key role in the pathophysiology of atherosclerosis. LDL-apheresis, which involves direct removal of plasma LDL from circulating blood, is an efficient treatment of homozygous familial hypercholes terolemia (FH). Methods: We evaluated impact of long-term LDL apheresis treatment on the atherogenicity of the major LDL subclasses (light, LDL1, and LDL2, density [d] 1.018-1.030 g/mL; inter mediate, LDL3, d 1.030-1.040 g/mL, and dense LDL, LDL4 and LDL5, d 1.040-1.065 g/mL) separated by density gradient ultracentrifugation in severe FH patients. Therefore, we compared the oxidative resistance as well as the chemical and physical properties of each LDL subpopulation in the FH group with those in the corresponding LDL subfractions from normocholesterolemic control subjects. Results: Both intermediate and dense LDL subfractions were significantly more resistant to copper-mediated oxidation in FH patients treated regularly by LDL-apheresis than their counterpart controls. The lag phases for LDL3, LDL4, and LDL5: 63.9 ± 1 1.6, 55.8 ± 1.2, and 47.2 ± 6.5 min. in FH patients were significantly longer than those of the corresponding subfractions in normocholesterolemic controls (P < .01 for LDL3 and LDL5, P < .005 for LDL4). This protective effect was reflected in the delayed formation of biologically active lipid oxidation products such as oxysterols, lipid hydroperoxides, dienes, and dienals in the intermediate and dense LDL subfractions of FH patients. These findings may result from lower "seed" contents of lipid hydroperoxide (LOOH) detected as dienes in plasma LDL from apheresis-treated FH patients; indeed, baseline LOOH/diene contents in all 5 LDL subclasses from FH patients were significantly lower than those of the corresponding subclasses in normolipidemic subjects (P < .0005). On the other hand, the enhanced oxidative resistance of both intermediate (LDL3) and dense (LDL4 and LDL5) LDL subpopulations in FH patients could not be accounted for by any consistent modification in chemical composition or in lipophilic antioxidant content, although minor differences were observed between patients and controls in unsaturated fatty acid profile. In contrast, sphingomyelin content was enriched in FH LDL subclasses, potentially resulting in reduced penetration of the hydrophilic surface layer of LDL by oxygen radicals. Conclusion: We conclude that low concentrations of preformed lipid hydroperoxides and dienes, together with surface sphingomyelin enrichment, can account for the enhanced oxi dative resistance of intermediate (LDL3) and atherogenic dense LDL (LDL4, LDL5) induced by long-term LDL apheresis in severe FH patients.

Energy charge and capacity for adenosine triphosphate (ATP) synthesis have been demonstrated to play a major role in the maintenance of organ function after liver preservation for transplantation. The aim of this study was to evaluate whether a supply of liposomally-entrapped ATP during preservation could improve the energy state and metabolism of cold-stored rat liver.In the first set of experiments, the uptake of ATP-containing liposomes and their effects on hepatic viability were determined in isolated perfused unstored rat liver. In the second set of experiments, rat livers were preserved for 18 h at 4 degrees C in UW solution in the presence of these liposomes, and effects on energy state, cell volume and metabolism were evaluated. In each part, data were compared with adequate control, unloaded liposome-treated, and free ATP-treated groups (n=6 in each group).In non-stored livers, ATP-containing liposomes were taken up by the liver; they did not alter hepatic viability and induced a decrease in energy substrate consumption (glucose and amino acids), and an improvement in intrahepatic ATP content (+23% vs. Control). Addition of liposomally-entrapped ATP during cold storage produced a significant attenuation of the decrease in hepatic ATP content (Lip ATP 2: 524+/-45 vs. Control 2: 364+/-106 nmol/g; p0.05), and induced, during reperfusion, a decrease in proteolysis associated with an increase in cell volume compared with the other groups (Lip ATP 2: 633+/-63 vs. Control 2: 532+/-38, Unloaded Lip 2: 483+/-55 and Free ATP 2: 500+/-29 microl/g; p0.01).These data indicate that liposomally-entrapped ATP represents an effective means to improve liver graft energy state and function. The decrease in protein degradation may be related to the modification of cell volume.

Apolipoprotein E (apo E) is postulated to be a major lipid carrier protein in the brain involved in brain development and repair. Multiple sclerosis (MS) is a major demyelinating disease characterized by destruction of myelin and marked alteration of myelin cholesterol and lipid metabolism. We have determined serum and cerebrospinal fluid (CSF) apo E concentrations using an original time-resolved immunofluorometric assay and calculated intrathecal apo E concentration. Apo E concentrations were determined in 13 control subjects and 129 neurological patients: 34 definite MS patients, 25 with Guillain-Barre syndrome (GBS), 32 with amyotrophic lateral sclerosis (ALS) and 38 with other neurological diseases. Seven clinical parameters (sex, age, age at MS onset, duration of the disease, course, clinical status and disability score) were considered in MS patients. Significant (P < 0.01) decrease in CSF apo E was observed in MS, linked to a decrease in intrathecal apo E. The decreased CSF apo E concentration in MS patients occur independent of the apo E genotype. Apo E is considered as a neurotrophic factor in the brain. Any decrease in intrathecal apo E synthesis would thus contribute to progression of neurological diseases, such as MS.

In clinical research, the definition of the upper limit of normal (ULN) alanine transaminase (ALT) is never detailed. However, such a definition can vary and may have practical consequences. Our aim was to assess factors associated with serum ALT activity in apparently healthy subjects and then to apply seven different definitions of ULN in three different populations so as to assess the prevalence of subjects with normal ALT among blood donors and among hepatitis C patients before (normal ALT hepatitis C patients) and after treatment (interferon [IFN] responders). ALT measurements were performed in the same laboratory using the same technique; 1,033 donors were prospectively investigated, 186 patients with hepatitis C never treated and 40 patients treated with 3 MU three times per week of IFN-alpha for at least 6 months. The seven definitions (D) of ULN were: D1: 95th percentile of ALT; D2: 95th percentile after separating males and females; D3: males and females separately, ULN=10 (mean of log10 ALT + 1.96 SD); D4: ULN=45 IU/L given by the manufacturer; D5: mean + 1 SD after exclusion of the 5% extreme values; D6: 95th percentile after separating subjects with body mass index (BMI) under or equal to the median (23); and D7: 95th percentile after separating subjects according to BMI and sex. BMI and male sex were independently associated (P.0001; logistic regression) with ALT, without an association with alcohol. The range of ULN varied from 26 IU/L in females (D5) to 66 IU/L in males with BMI23 (D7). Depending on the definition, the prevalence of blood donors with normal ALT varied from 82% to 96%, i.e., a range of 14%; that of hepatitis C patients with normal ALT varied from 16% to 27%, i.e., a range of 11%; the prevalence of IFN responders varied from 25% to 42%, i.e., a range of 17%. Definitions of normal ALT values should be adjusted for sex and BMI to reduce artificial heterogeneity in blood donor selection and in hepatitis C clinical studies.

patients, evidenced by a decrease in plasma seleniumBackground. An oxidative stress has been reported in levels and in P-GSH-Px activity. However, plasmapatients with chronic renal failure (CRF) treated by TBARS were not higher in CAPD patients than inhaemodialysis. To our knowledge, only scant informa- age-matched non-CRF control subjects, probably ontion is available concerning CRF patients treated by account on the patients’ ages.continuous ambulatory peritoneal dialysis (CAPD)with regard to their redox and nutritional status. Key words: Antioxidants; chronic renal failure; continu-Methods. The oxidative stress and the biological nutri- ous ambulatory peritoneal dialysis; denutrition; retinoltional status were evaluated in 20 elderly CRF patients binding proteintreated by CAPD, compared with a control group of30 elderly non-CRF patients. Plasma peroxidationproducts were assayed as thiobarbituric acid-reactivesubstances (TBARS), and two enzymatic antioxidant Introduction systems were determined: erythrocyte superoxide dis-mutase (SOD), glutathione peroxidase activity inplasma (P-GSH-Px) and in erythrocytes (E-GSH-Px). Oxidative stress, which occurs when there is excessiveSelenium, vitamin E, b-carotene and vitamin A were free radical production or low antioxidant levels, hasevaluated as plasma non-enzymatic antioxidants. been reported in patients with chronic renal failureNutritional status and iron status were assessed by (CRF) treated by haemodialysis [1,2]. In addition, thedetermining serum albumin, prealbumin, iron, ferritin incidence of atherosclerosis is increased in suchpatientsand transferrin concentrations. [3]. The oxidative stress could be involved in theseResults. Plasma TBARS concentration was high in cardiovascular complications, which include athero-sclerosis, accelerated ageing, cataract, b2both groups (CAPD: 1.37±0.06 mmol/l versus non- -microglobulinCRF: 1.41±0.06 mmol/l; P=NS), compared with arthropathy, impaired erythrocyte deformability,usual values (0.60 to 1.20 mmol/l), on account of the increased haemolysis and platelet dysfunction.patients’ ages. SOD and E-GSH-Px activities were Numerous studies in haemodialysed patients investi-normal in both groups. A significant lowering in gated markers of oxidative stress, such as malon-P-GSH-Px activity was observed only in CAPD dialdehyde (MDA) or thiobarbituric acid reactivepatients (211±14 U/l, usual values: 480 to 650 U/l). substances (TBARS), or antioxidant defence systemsPlasma selenium concentration, decreased in both such as erythrocyte superoxide dismutase (SOD),groups, was significantly lower in CAPD than in non- glutathione peroxidase activity in erythrocytesCRF patients (P

Oxidation of low density lipoprotein is involved in the pathogenesis of atherosclerosis. Epidemiological studies suggest a negative correlation between the occurrence of cardiovascular diseases and blood concentrations of lipophilic antioxidants such as vitamins A and E and beta-carotene. Trace elements, such as selenium, zinc, and copper, are involved in the activity of the antioxidant enzymes glutathione peroxidase and superoxide dismutase. The aim of this study was to determine the antioxidant and trace element status of patients with severe hypercholesterolemia who had been treated with dextran-sulphate low-density lipoprotein apheresis in comparison with two control populations, normocholesterolemic subjects and untreated hypercholesterolemic patients. Our results showed that, patients treated with LDL apheresis, compared with normocholesteromic subjects, were not deficient in vitamin E, beta-carotene, and copper, but had lower plasma levels of selenium, zinc, and vitamin A. The low selenium and vitamin A levels were due to the LDL-apheresis treatment, and the hypercholesterolemia might have provoked the low plasma levels of zinc. The study pointed out the potential benefits of supplemental selenium, zinc, and vitamin A in patients being treated with LDL apheresis.

Summary ATP entrapped into liposomes was administered intracarotidally to rats submitted to brain ischemics episodes by clamping of the carotid arteries and lowering of the systemic blood pressure. It was observed that when entrapped into liposomes, ATP greatly increased the number of ischemic episodes tolerated before brain electrical silence and death appeared. These results added to very similar previous data obtained by i.c.v. treatment excluding the prominent role of cardiovascular effects, could open new possibilities in brain antihypoxic protection. Here and now it cannot be stated if ATP provides direct energetic supply.

ATP was entrapped inside negatively charged liposomes composed of sulfatide, in order to improve its penetration into the brain and to reduce its degradation into other tissues. These liposomes were prepared according to an original method allowing a satisfying stability of the formulation. Liposomally entrapped ATP were administered intracerebroventricularly to rats submitted to brain ischemic episodes by both carotid arteries clamping and systemic blood pressure lowering (during 3 minutes every 15 minutes). Such treatment importantly increases the number of ischemic episodes before brain silence appeared. So, this paper allows new perspectives in the administration of drugs into the brain. Several authors (1,2) have already proposed administration of ATP for the treatment of severe hypoergic syndromes. However, it is generally accepted that strongly charged anions cannot enter into the cells through the plasma membrane (2,3). For this reason, improvement in the treatment of hemorhagic shock after systemic administration of ATP was mostly attributed to peripheral effects, taking place mainly in the liver and the kidneys(1). However, such results were not confirmed by Schloerb et al. (4) who showed that ATP-MgCl2 i.v. neither increased survival rate, nor enhanced tissue levels of ATP in shocked animals. Furthermore, they showed that the administered ATP was rapidly and extensively degraded in various tissue. These results could be explained by the fact that the central nervous system and especially the neo-cortex is characterized by a very tight blood brain barrier, suggesting that ATP does not enter it. On the other hand, liposomes were proposed for introduction of hydrophilic molecules (i.e. enzymes, cytostatics) into the brain (5,6). Furthermore, liposomes composed of sulfatide, in addition to phospholipids, were found to be the optimal system for brain targeting (7). Owing to these considerations, we looked at the possibility of entrapping ATP inside negatively charged liposomes composed of sulfatide. This paper describes preparation and characterization of liposomes loaded with ATP, as well as their efficiency in experimental brain ischemia in the rat.