Jacques Delattre

Blood oxidative stress in amyotrophic lateral sclerosis

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.

High Density Lipoproteins (HDL) and the Oxidative Hypothesis of Atherosclerosis

Abstract The oxidative hypothesis of atherosclerosis classically implies a central role for low density lipoprotein (LDL) oxidation. However, new antiatherogenic properties have been recognized for high density lipoproteins (HDL), apart from their ability to reverse cholesterol transport. Indeed, native HDL could protect LDL from oxidation, thereby minimizing the deleterious consequences of this process. Several mechanisms have been suggested to explain this protective role. Two HDL-associated enzymes, paraoxonase and PAF-acetylhydrolase, detoxify oxidized phospholipids produced by lipid peroxidation. In addition, HDL could reduce hydroperoxides to their corresponding hydroxides. It has also been suggested that HDL could inhibit oxidized LDL-induced transduction signals. However, in vivo HDL oxidation in the subendothelial space would favor the atherosclerotic process. Indeed, atherogenic properties of these oxidized HDL partly result from some loss of their cholesterol effluxing capacity and from an inactivation of the lecithin-cholesterol acyltransferase, which is a HDL-associated enzyme involved in reverse cholesterol transport. Finally, oxidized HDL could induce cholesterol accumulation in macrophages. Further in-depth investigation is needed to assess these antagonistic effects and their consequences for the atherosclerotic process.

Copper and cell-oxidized low-density lipoprotein induces activator protein 1 in fibroblasts, endothelial and smooth muscle cells

The effect of cupric ion‐ or endothelial cell‐oxidized low‐density lipoproteins (LDL) on transcription factor AP1 activation was investigated by electrophoretic mobility shift assay. Both oxidized LDL induced AP1 activation in fibroblasts, endothelial and smooth muscle cells. This phenomenon was also observed in the presence of cycloheximide. α‐Tocopherol, a lipophilic free radical scavenger, and N‐acetylcysteine, an hydrophilic antioxidant, partially inhibited the stimulatory effect of Cu2+‐oxidized LDL. LDL modified by the mixture of the oxygen radicals OH· and O2·−, which generated lipid peroxidation products, also initiated AP1 activation, whereas LDL modified by OH· alone, which did not lead to marked LDL lipid peroxidation, was ineffective. Thus, lipid peroxidation products seem at least partially involved in the activation mechanism. Since AP1 activity is essential for the regulation of genes involved in cell growth and differentiation, our study suggests that the oxidative stress induced by oxidized LDL might be related to the fibroproliferative response observed in the atherosclerotic plaque.

Protection of insulin from enzymatic degradation by its association to liposomes

Abstract The degradative action of digestive enzymes (pepsin, α-chymotrypsin and trypsin) was investigated against insulin associated or entrapped in positively charged liposomes. Under the chosen experimental conditions, both insulin preparations were protected against enzymatic degradation. Furthermore, the nature of the interaction between external phospholipidic bilayer and insulin was found to result from an ionic association. Finally, the protective role of liposomes appeared highly dependent upon the molar proportion phospholipid/hormone.

α-Tocopherol Enrichment of High-Density Lipoproteins: Stabilization of Hydroperoxides Produced During Copper Oxidation

In the aim to study the effect of an in vitro enrichment of high-density lipoprotein (HDL) with alpha-tocopherol in alcoholic solution on a copper-induced peroxidation, we monitored several markers of lipid peroxidation (alpha-tocopherol consumption, formation of conjugated dienes and of fatty acid hydroperoxides, production of thiobarbituric acid-reactive substances) and the integrity of apolipoprotein A-I. High-density lipoproteins (1.063 < d < 1.21) with a mean of 0.58 alpha-tocopherol molecules per HDL particle were enriched with alpha-tocopherol in alcoholic solution to obtain an average of 3.7 and 21 alpha-tocopherol molecules per HDL particle. HDL oxidation with 5 microM CuSO4 at 37 degrees C resulted in the total disappearance of endogenous alpha-tocopherol after 2 h, but after 24 h about 19% of alpha-tocopherol remained in the most enriched HDL. In agreement with the tocopherol-mediated peroxidation, the formation of conjugated dienes and of fatty acid hydroperoxides was very fast and increased with alpha-tocopherol concentration, whereas TBARS production decreased. These results showed that alpha-tocopherol enrichment stabilized the production of hydroperoxides in HDL and decreased the formation of secondary oxidation products. These latter products are known for deleterious effects towards apolipoproteins. This could explain why we observed that the apolipoprotein A-I of the most enriched HDL was only slightly altered after incubation with CuSO4.

Liposomally-entrapped ATP: improved efficiency against experimental brain ischemia in the rat.

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 was administered intracerebroventricularly to rats submitted to brain ischemic episodes by both carotid artery 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.

Efficiency of liposomal ATP in cerebral ischemia : bioavailability features

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.

Melatonin related compounds inhibit lipid peroxidation during copper or free radical‐induced LDL oxidation

Abstract:  This study was designed to evaluate the protective effect of two melatonin related compounds towards low density lipoproteins (LDL) oxidation initiated in vitro either by defined free radicals [i.e. superoxide anion (O2·–) and ethanol‐derived peroxyl radicals (RO2·)] produced by gamma radiolysis or by copper ions. The compounds studied were N‐[2‐(5‐methoxy‐1H‐indol‐3‐yl)ethyl]‐3,5‐di‐tert‐butyl‐4‐hydroxybenzamide (DTBHB) and (R,S)‐1‐(3‐methoxyphenyl)‐2‐propyl‐1,2,3,4‐tetrahydro‐β‐carboline (GWC20) which is a pinoline derivative. Their effects were compared with those of melatonin at the same concentration (100 μmol/L). None of the three tested compounds protected endogenous LDL α‐tocopherol from oxidation by RO2·/O2·– free radicals. By contrast, they all protected β‐carotene from the attack of these free radicals with GWC20 being the strongest protector. Moreover, melatonin and DTBHB partially inhibited the formation of products derived from lipid peroxidation (conjugated dienes and thiobarbituric acid‐reactive substances or TBARS) while GWC20 totally abolished this production. As previously shown, melatonin (at the concentration used) inhibited copper‐induced LDL oxidation by increasing 1.60‐fold the lag phase duration of conjugated diene formation over the 8 hr of the experimental procedure, however, DTBHB and GWC20 were much more effective, because they totally prevented the initiation of the propagation phase of LDL oxidation. It would be interesting to test in vivo if DTBHB and GWC20 which exhibit a strong capacity to inhibit in vitro LDL oxidation would reduce or not atherosclerosis in animals susceptible to this pathology.

Intracarotidal administration of liposomally-entrapped ATP: improved efficiency against experimental brain ischemia.

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.

Liposomally entrapped adenosine triphosphate: improved efficiency against experimental brain ischaemia in the rat

Liposomally entrapped adenosine triphosphate (ATP) was administered intracerebroventricularly and intracarotidally to rats subjected to brain ischaemic episodes by clamping of the carotid arteries and lowering of the systemic blood pressure. It was observed that, when entrapped in liposomes, ATP greatly increased the number of ischaemic episodes before brain electrical silence and death. The results open new perspectives in brain ATP supply, which will potentially be useful in human resuscitation from deep brain hypoergic states.