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Several factors are known to limit cardiac transplantation, such as number of donors, quality of cardiac graft preservation, and ischemia-reperfusion injury. Some mechanisms of reperfusion injury are now recognized; they include oxygen free radical (OFR), white blood cells activation, changes in calcium influx, alteration of microvascular blood flow, and sympathetic activation. The goal of this study was to assess the effects of two types of cardioplegia with long-term storage, either static or continuous perfusion, in 30 isolated sheep hearts as a model for heart transplantation. We examined myocardial function, histology, ischemic damage, and markers of oxidative stress. Two types of cardioplegia and storage conditions using a Langendorff reperfusion were studied in a combined approach: crystalloid (CP) [groups I and III] or cold oxygenated autologous blood (BC) [groups II and IV], immediate storage during 8h in profound hypothermia (groups I and II), or reperfused with crystalloid (group III), or blood cardioplegia (group IV). All perfusate samples were drawn from the coronary sinus. Lactate levels increased progressively in groups I, II, and IV, but not in group III, as no significant elevation was shown [90 min: 13.6 ± 1.7 versus 5.2 ± 1.0 mmol/L (P

We investigated whether the increase of urokinase-type plasminogen activator (uPA) monocyte expression and chemokine releases induced by oxidised low density lipoproteins (LDL), which participate to vascular tissue remodeling and to atherosclerotic plaque rupture, involved proinflammatory phospholipid products having platelet-activating factor (PAF)-like activity via the PAF-receptor pathway.uPA monocyte expression was stimulated by either copper ions-oxidised or O2*-/HO* free radical-oxidised LDL. The effects of PAF and oxidised LDL on the production of monocyte chemoattractant protein-1 and interleukin-8 were also examined.Synthetic PAF significantly enhanced chemokine releases (P0.001) without modifying uPA expression. Copper-oxidised LDL, which exhibit a higher content in lysophosphatidylcholines than free radical-oxidised LDL, induced a significantly higher enhancement in uPA expression (P0.05). By contrast, free radical-oxidised LDL were more efficient than copper-oxidised LDL to increase chemokine releases (P0.01). Oxidised LDL-enhanced uPA expressions were not altered by the PAF-receptor antagonist SR27417, whereas increases in chemokine releases induced by oxidised LDL and by PAF were abolished. PAF-acetylhydrolase activity was rapidly and largely inhibited in free radical-oxidised LDL when compared to copper-oxidised LDL, suggesting that free radical-oxidised LDL would contain a higher content in PAF-like products than copper-oxidised LDL.Our results indicated that PAF-like oxidation products are responsible for the monocyte chemokine releases, but did not contribute to the enhanced monocyte uPA expression by oxidised LDL.

This study was designed to evaluate the effect of high concentrations of melatonin on the peroxidation of human low density lipoproteins (LDLs) initiated by O(2)(*-) and ethanol-derived peroxyl radicals (RO(2)(*)) from water gamma radiolysis in the presence of ethanol. LDL (3 g/l; total LDL concentration) was oxidized in the absence of melatonin or in its presence at three concentrations (50 x 10(-6), 100 x 10(-6) or 250 x 10(-6) mol/l) in ethanol. Radiolytic yields (i.e. number of mole consumed or produced per Joule) of the markers of lipid peroxidation were determined (i.e. decrease in the endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances [TBARS]). Melatonin decreased the yields of lipid peroxidation products and delayed the onset of the propagation phase for conjugated dienes and TBARS in a concentration-dependent manner. Nevertheless, melatonin did not protect endogenous alpha-tocopherol against peroxyl-induced oxidation (probably due to a lower scavenging capacity than that of alpha-tocopherol towards peroxyl radicals), but delayed the consumption of LDL endogenous beta-carotene and decreased its rate of disappearance. The effect of melatonin seemed to be the highest for a melatonin concentration of 250 x 10(-6) mol/l.

Resume L'endothelium vasculaire joue un role primordial notamment dans la regulation du tonus vasculaire, en reponse a differents stimuli tels que la prostacycline, l’endotheline et surtout le monoxyde d'azote (NO • ). Le stress oxydant, caracterise par une augmentation relative des especes reactives de l'oxygene (ERO), peut diminuer la biodisponibilite du NO • , ce qui conduit a un dysfonctionnement vasculaire. En outre, les ERO (produites notamment par la NADPH oxydase) participent soit directement, soit par le biais des lipoproteines de basse densite oxydees, a la transduction du signal dans les cellules vasculaires. Elles sont ainsi impliquees dans les phenomenes d’apoptose, la proliferation des cellules musculaires lisses, l'adhesion des monocytes aux cellules endotheliales et l'agregation plaquettaire. Des perspectives therapeutiques visant par exemple a moduler la production radicalaire au niveau des cellules endotheliales pourraient etre envisagees pour restaurer la fonction endotheliale.

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 (RO(2)(*))] 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-beta-carboline (GWC20) which is a pinoline derivative. Their effects were compared with those of melatonin at the same concentration (100 micromol/L). None of the three tested compounds protected endogenous LDL alpha-tocopherol from oxidation by RO(2)(*)/O(2)(*)- free radicals. By contrast, they all protected beta-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.

This study was designed to evaluate the effect of ethanol on the peroxidation of human low-density lipoprotein (LDL) initiated by oxygen free radicals (O(2)(.-) and (.)OH in the absence of ethanol; O(2)(.-) and ethanol-derived peroxyl radicals, RO(2)(.), in the presence of ethanol) generated by gamma radiolysis. Initial radiolytic yields as determined by several markers of lipid peroxidation [i.e. decrease in endogenous antioxidants alpha-tocopherol and beta-carotene, formation of conjugated dienes and of thiobarbituric acid-reactive substances (TBARS)] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence of ethanol or its presence at six different concentrations (0.42-17 x 10(-2) mol liter(-1)). Ethanol acted as an antioxidant by decreasing the rate of consumption of LDL endogenous antioxidants and the yields of formation of lipid peroxidation products, and by delaying the onset of the propagation phase for conjugated dienes and TBARS. With regard to the different markers studied, except for alpha-tocopherol and beta-carotene consumption, the effect of ethanol did not appear to be dependent on its concentration. Indeed, (.)OH were scavenged by ethanol at the lowest ethanol concentration (0.42 x 10(-2) mol liter(-1)), leading to RO(2)(.). These RO(2)(.) resulted in lower radiation-induced yields related to endogenous antioxidant consumption or to formation of lipid peroxidation products (for example, approximately 10% of RO(2)(.) oxidized LDLs from TBARS). Thus, under our in vitro conditions, ethanol behaved as an antioxidant when added to the LDL solutions. This should be taken into account in the reported antioxidant activity of wine. This is also of interest when lipophilic compounds have to be added as ethanolic solutions to LDLs to evaluate in vitro their antioxidant activity toward LDL peroxidation.

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.

This study was designed to evaluate the antioxidant effect of aminoguanidine toward human low-density lipoproteins (LDLs) initiated by oxygenated free radicals (*OH/O(2)*-) generated by gamma radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous alpha-tocopherol and beta-carotene, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined in 3 g liter(-1) LDLs (expressed as total LDL concentration) in the absence and presence of 10 different concentrations of aminoguanidine (from 0.04 to 5 mmol liter(-1)). Fluorescence and relative electrophoretic mobility of oxidized LDLs were also studied as markers that indirectly reflect the attack of the protein moiety of LDLs (namely apolipoprotein B). Our data clearly showed the inhibitory effect of aminoguanidine on lipid peroxidation induced in LDLs by *OH/O(2)*- in a concentration-dependent manner. This effect probably resulted from a scavenging activity of aminoguanidine toward *OH. In contrast, aminoguanidine did not appear to react significantly with O(2)*-, which resulted in a poor residual lipid peroxidation. Our data led us to determine an optimum [aminoguanidine]/[LDL] ratio ranging from 250 to 500 to obtain the best in vitro protection of LDLs under our experimental conditions. It is also of great interest that aminoguanidine was able to protect endogenous alpha-tocopherol and beta-carotene of LDLs upon *OH/O(2)*(-)-induced oxidation.

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.

ln a preliminary report (Skiba-Lahiani, M. et al.), we showed that dilauroylphosphatidylcholine (PC12) liposomes entrapping ATP sustain sperm motion over time and in some experiments a concomittant increase in the percentage of human acrosome-reacted spermatozoa. With the present study, we confirmed an interaction between PC12 liposomes and human spermatozoa vizualizing the morphologic aspects when we used several ultrastructural techniques. Investigations using on the one hand liposomes made from fluorescently labeled phosphatidylcholines analogue, acyl- N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-phosphatidylcholine (Acyl-NBD-PC), and on the other hand liposomes encapsulating a fluorochrome (calceine), allowed to approach the mode of this interaction. Electron-microscopic examination has shown on the one hand the transfer of Acyl-NBD-PC from vesicles to cells, and on the other hand the transport of encapsulated fluorochrome (calceine) into the cell, supporting the suggestion of membrane perturbation as a cause of an increase of its permeability to trigger the acrosomal exocytosis. Under conditions which stimulate the acrosome reaction by liposomes entrapping ATP (L ATP ) and blank liposomes (L B ) treatments, we have not shown an increase in intracellular calcium when we used the fluorescent probe [1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane N , N , N ′, N ′-tetraacetic acid] (Fura-2) suggesting that the interaction PC12 liposomes-human spermatozoa induced an acrosome reaction different from the physiologic acrosome reaction.

In the aim to study the effect of an in vitro enrichment of high-density lipoprotein (HDL) with α -tocopherol in alcoholic solution on a copper-induced peroxidation, we monitored several markers of lipid peroxidation ( α -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 α -tocopherol molecules per HDL particle were enriched whith α -tocopherol in alcoholic solution to obtain an average of 3.7 and 21 α -tocopherol molecules per HDL particle. HDL oxidation with 5 μ M CuSO 4 at 37°C resulted in the total disappearance of endogenous α -tocopherol after 2 h, but after 24 h about 19% of α -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 α -tocopherol concentration, whereas TBARS production decreased. These results showed that α -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 CuSO 4 . Copyright © 1996 Elsevier Science Inc.

Efficient transfer of genes maintaining a correct hormonal control in transfected cells is the prerequisite for gene regulation studies and for gene therapy. Differentiated cells, like adipocytes or hepatocytes, are difficult to transfect. In an attempt to improve gene transfer, we first transiently transfected cultured 3T3-F442A adipocytes with a construct containing the simian virus 40 (SV40) promoter fused to the chloramphenicol acetyltransferase (CAT) gene (pSV2-CAT), using various cationic liposomes. Among these, only lipofectAMINE was five times more efficient than the standard calcium phosphate procedure. To further augment efficiency, we transfected 3T3-F442A adipocytes and FAO hepatoma cells with the lipofectAMINE/pSV2-CAT complex in the presence of replication-deficient recombinant type-5 adenovirus at 200 pfu/cell. CAT activity of transiently transfected cells was increased about 50-fold when compared to the calcium phosphate procedure. To determine whether this methodology would be useful for obtaining stable transfectants and would not interfere with correct gene regulation, we used a construct containing -2100 to +69 bp of the phosphoenolpyruvate carboxykinase gene fused to the CAT gene (pPL1-CAT). This construct was shown previously to be cAMP-responsive after calcium-phosphate-mediated transfection of adipocytes and hepatoma cells. 3T3-F442A or FAO cells in which pPL1-CAT was either transiently or stably transferred by lipofectAMINE and adenovirus responded to isoproterenol or cAMP, respectively, with a 2-3-fold increase in CAT activity. Therefore the association of liposomes and adenovirus is an efficient method for transient or stable transfer of regulated genes in adipocytes and hepatoma cells.

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.

Xenotransplantation (XT) reveals a growing interest for the treatment of cardiomyopathy. The major barrier is an acute vascular rejection due to an acute humoral rejection. This pathogenesis is a difficult issue and in order to elaborate means for its prevention, we analysed the implication of oxidative stress (OS) on hearts from mini-pigs followed by reperfusion with either autologous or human blood in an attempt to simulate xenotransplantation. About 14 hearts were studied after a Langendorff blood reperfusion: allografts with autologous blood (n = 7) or xenografts with human blood (n = 7). Blood samples were drawn from the coronary sinus to assess ischemia and OS. In xenografts, arrhythmias occurred more frequently (p0.01, left ventricular systolic pressure decreased more significantly (p0.05), thiobarbituric acid-reactive substances concentrations increased at 30 min (0.7 +/- 0.1 vs. 2.4 +/- 0.3 mmol/l; p0.05) while vitamin A levels decreased (p0.05). XT was associated with a significant increase in ischemic injury and OS production. OS might play an eminent role in hyperacute humoral rejection.

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 (RO(2)(*))] 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-beta-carboline (GWC20) which is a pinoline derivative. Their effects were compared with those of melatonin at the same concentration (100 micromol/L). None of the three tested compounds protected endogenous LDL alpha-tocopherol from oxidation by RO(2)(*)/O(2)(*)- free radicals. By contrast, they all protected beta-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.

The aim of our study was to evaluate the carbonylation and the carbonylated fragmentation of apolipoprotein B upon low-density lipoprotein (LDL) oxidation induced in vitro by copper and OH / O 2 − free radicals generated by γ-radiolysis. Therefore, we developed a very sensitive Western blot immunoassay using 2,4-dinitrophenylhydrazine which allows the revelation of the apolipoprotein B carbonylation and its carbonylated fragmentation. The main results of this study show that (i) apolipoprotein B carboylation is present during the lag phase of LDL oxidation in the two oxidative processes and (ii) apolipoprotein B carbonylated fragmentation was not detected during the lag phase of copper-oxidized LDL but was detected during the propagation phase. By contrast, apolipoprotein B carbonylated fragmentation was detected in the lag phase of OH / O 2 − oxidized LDL.

We investigated the effects of low density-lipoproteins (LDL) and lipoprotein(a) [Lp(a)] oxidized by O 2 • −/HO • free radicals generated by gamma radiolysis of water, on the release of tissue Plasminogen Activator (tPA) and of its main inhibitor Plaminogen Activator Inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC). These effects were compared to those of lipoproteins issued from the same preparations but oxidized by the classical copper ions procedure. The results showed that O 2 • −/HO • free radical oxidized LDL and Lp(a) led to a dramatic decrease of PAI-1 release but did not affect tPA release, whereas copper oxidation of lipoproteins resulted in an increase in PAI-1 release and a decrease in tPA release. Chemical analysis revealed that O 2 • −/HO • free radical oxidized lipoproteins exhibited very much lower levels of phosphatidylcholine hydroperoxides, lysophosphatidylcholine and oxysterols (7-ketocholesterol, 7β-hydroxycholesterol, 5,6β-epoxycholesterol) than copper oxidized LDL. Thus, the discordant effects of O 2 • −/HO • oxidized and copper oxidized LDL and Lp(a) on the endothelial releases of PAI-1 and tPA appeared to be due to qualitatively and/or quantitatively different formation of oxidized components by the two oxidation processes.

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.

To the Editor: We read with interest the editorial by Tracy1 on inflammation in cardiovascular disease (CVD). Serum concentrations of C-reactive protein (CRP) were demonstrated to be related to increased risk of CVD, which underlines the potential inflammatory nature of human atherosclerosis.2 3 Interestingly, an unexpected association between CRP and body mass index (BMI) was found in several population studies2 3 4 without any explanation.1 The production of CRP is regulated by cytokines, …

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.

Desensitization therapy for type I allergy is now current practice. Liposomes have been proposed as a support for allergens to improve safety and effectiveness. The aim of this work was to optimize liposomal formulations of three different standardized allergen extracts and to test their allergenicity in vitro and in a preclinical trial. Allergen extracts (Dactylis glomerata, Dermatophagoides pteronyssinus, and cat hair and dander) were associated with multilamellar liposomes of varying compositions at different pH. Liposome-bound allergens were quantified by RAST inhibition after ultracentrifugation, and analyzed qualitatively by SDS-PAGE followed by immunoblotting. Their allergenicity was assessed by basophil degranulation in vitro, as compared with the allergenicity of an aqueous extract, and by skin tests in allergic subjects. The best association, about 50% of the added allergen, was obtained with negatively charged liposomes, when the pH of the allergen solution was adjusted so as to impart a net positive charge to the proteins. One-third of the liposome-associated allergens was located on the surface of the liposomes and was free to interact with antibody, as shown by RAST inhibition assays and the basophil degranulation test; the remaining two-thirds was encapsulated within the liposomes. All the major immunoreactive proteins in the extract were included. These liposomes could be readily freeze-dried and reconstituted without changing their properties. This study reveals the allergenic characteristics of liposomes and suggests their potential use in the treatment of allergic patients.

Adenosine triphosphate (ATP) was proposed in various medical applications, as a possible bioenergetic substrate. Unfortunately, ATP is very difficult to use at a therapeutic level because of its high sensitivity to enzymatic hydrolysis making this molecule unstable in biological fluids. ATP is also a highly hydrophilic molecule that is unable to cross biological membranes. To try to develop a system able to protect ATP against degradation and to efficiently deliver this bioenergetic substrate, its liposomal encapsulation in multilamellar vesicles was carried out. One of the studies described in this paper deals with the efficiency of liposomal ATP in the treatment of cerebral ischemia. Our results show that encapsulation was able to protect ATP from its degradation by ectonucleotidases and that liposomal ATP was active against experimental brain ischemia. The other study deals with the effect of ATP on the motility and the acrosomal reaction of human spermatozoa. The results show that co-incubating ATP-loaded liposomes with sperm cells was able to induce the process of capacitation in vitro and might therefore be a useful tool in the procedure of in vitro fertilization.

The fate of a drug after administration in vivo is determined by a combination of several processes: distribution and elimination when given intravenously and absorption, distribution and elimination when an extravascular route is used. Regardless of the mechanisms involved, each of these processes depends mainly on the physico-chemical properties of the drug and therefore, for the most part, on its chemical structure.

This study was designed to evaluate the antioxidant effect of probucol on peroxidation of low-density lipoproteins (LDLs) initiated by oxygenated free radicals ( O2· -) and ethanol-derived peroxyl radicals ( ${\rm RO}_{2}{}^{\cdot}$ ) generated by ? radiolysis. Initial radiolytic yields related to the markers of lipid peroxidation [i.e. decrease in endogenous ?-tocopherol, formation of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes] were determined as a function of LDL concentration (1.5 and $3\ {\rm g}\ {\rm l}^{-1}$ , expressed as total LDL) and in the absence or the presence of probucol at different concentrations (2.3× 10-6, $3.5\times 10^{-6}$ , 9× 10-6 and $20.5\times 10^{-6}\ {\rm mol}\ {\rm l}^{-1}$ ). Our results showed that probucol was able to decrease not only the yields of TBARS and conjugated dienes but also the levels of these peroxidation products obtained at high doses (2500 Gy) compared ...

The aim of this work was to specify the mechanisms involved in the radical oxidation of human high-density lipoprotein (HDL) and to compare these mechanisms with those described previously for the oxidation of low-density lipoprotein (LDL) under the same experimental conditions (Bonnefont-Rousselot et al., Radiat, Res. 134, 271-282, 1993). The oxidation of HDL, initiated by .OH or .OH/O(.-)2 free radicals from gamma radiolysis of water, was evaluated as a function of increasing radiation dose by analyzing quantitatively the decrease of endogenous alpha-tocopherol and the formation of oxidation products (thiobarbituric acid-reactive substances and conjugated dienes). All qualitative conclusions were supported by quantitative data (radiation yields and concentrations of the oxidation markers at high radiation doses) and by the mechanisms of the kinetics, .OH free radicals in the absence of oxygen were less efficient in initiating HDL oxidation than in the presence of oxygen (action of .OH/O(.-)2 free radicals), which was in agreement with the enhancement of the action of .OH free radicals by oxygen. The remaining significant level of vitamin E in HDLs at high radiation doses in the absence of oxygen could be explained by a regeneration of vitamin E by an oxidation product that was able to reduce the alpha-tocopheroxyl radical. The yields related to the decrease in the vitamin E content of HDLs after exposure to radiation with .OH or .OH/O(.-)2 free radicals were slightly higher than those obtained previously in LDLs under similar experimental conditions. Moreover, in the presence of oxygen, .OH free radicals led to a lower formation of thiobarbituric acid-reactive substances in HDLs than in LDLs. Such discrepancies in the behavior of these two lipoprotein fractions could be related to the differences in the chemical composition of HDLs and LDLs.

The aim of this study was to analyze quantitatively the oxidative modification of low-density lipoproteins (LDLs) induced by OH· free radicals produced by gamma radiolysis, in the absence or in the presence of oxygen (action of OH· free radicals, or simultaneous action of OH· and ${\rm O}_{2}^{\overline{\cdot}}$ free radicals, respectively). The effects of increasing radiation doses on aqueous LDL solutions have been monitored by several parameters: a decrease in endogenous vitamin E, the formation of thiobarbituric acidreactive substances (TBARS) and conjugated dienes, the appearance of a differential fluorescence (excitation wavelength = 360 nm), and an increase in the relative electrophoretic mobility. Initial radiation yields (decrease in vitamin E, formation of TBARS) have been determined at pH 7 as a function of LDL concentration (from 0.75 to 9 g liter-1). From the comparison of these yields with those of OH· and ${\rm O}_{2}^{\overline{\cdot}}$ free radicals produced by water radiolysis, we have d...

This study aimed to analyze quantitatively the initiation and the consequences of low-density lipoprotein (LDL) peroxidation by O2·−/HO2· free radicals produced by gamma radiolysis. The action of i...

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.

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.

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.

The water-soluble immunomodulator muramyldipeptide has been encapsulated in oligolamellar liposomes (mean diameter 200 nm) of various lipid compositions. These liposomes were sterilized by Millipore filtration without loss of entrapped solute and are therefore suitable for i.v. administration. The amount of encapsulated MDP increased when the volume of aqueous phase (containing a given MDP concentration) used for hydrating the phospholipid film increased and this did not vary for at least 15 days when the liposomes were kept at 4°C. The presence of cholesterol did not modify the encapsulation results but prevented the leakage of encapsulated water-soluble solute in serum-containing media. The internalization of various types of liposomes by murine peritoneal macrophages has been studied in order to define the best conditions for activating macrophages. For a given lipid composition, sterile oligolamellar vesicles (suitable for i.v. injection) were more efficient than multilamellar vesicles in rendering macrophages capable of inhibiting the growth of a syngeneic tumor, particularly when these vesicles contained muramyldipeptide in their aqueous space and cholesterol in their lipidic phase.

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.