Effect of non-enzymatic glycation on aluminium-induced lipid peroxidation of human high density lipoproteins (HDL).

BACKGROUND AND AIM: Several studies have shown that non-enzymatic glycation and oxidative damage play an important role in the pathogenesis of neurological diseases. Increased levels of advanced glycation end-products (AGEs) and of lipid peroxidation products have been observed in the brain, in the cerebrospinal fluid (CSF) and in the plasma of subjects affected by Alzheimer's disease (AD). The aim of this study was to investigate the effect of non-enzymatic glycation on aluminium-induced lipid peroxidation and on the stimulatory effect exerted by aluminium on iron-triggered oxidation of high density lipoproteins (HDL) isolated from human plasma. METHODS AND RESULTS: Aluminium (10-200 microM) and iron (20 microM) induced a significant increase in lipid hydroperoxides in HDL compared to untreated HDL. Therefore, our results confirm that aluminum and iron exert an oxidant effect on HDL. Moreover, aluminium exerted a stimulatory effect on iron-induced lipid peroxidation of HDL, in agreement with our previous studies. The aluminum/iron-induced increase in lipid hydroperoxides was significantly higher in HDL incubated for different time periods (24-72 hours) in the presence of 50 mM glucose (Gly-HDL) compared to HDL incubated alone. These results demonstrate that Gly-HDL is more susceptible to aluminium and iron-oxidative treatment with respect to control HDL. CONCLUSION: We suggest that aluminium and iron-induced oxidative damage on HDL could be involved in the development of neurological diseases and that glycation of HDL could represent an additional risk factor for these human diseases.

Effect of aluminium on lipid peroxidation of human high density lipoproteins.

We investigated the effect of aluminium (Al3+) on lipid peroxidation and physico-chemical properties of high density lipoproteins (HDL) isolated from human plasma. Our results demonstrated that Al3+ enhances lipid peroxidation of human HDL as shown by the significant increase in lipid hydroperoxides in Al-treated HDL with respect to control HDL. The oxidative effect was higher at acid pH (pH 5.5) with respect to pH 7.4. Moreover, a stimulating effect of Al3+ on iron-induced lipid peroxidation of HDL was demonstrated. The study of the effect of Al3+ on the physico-chemical properties of HDL, using the fluorescence polarization (Pf) of the probes TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide) and DPH (1,6-diphenyl-1,3,5-hexatriene), showed a significant decrease of Pf in Al-treated HDL with respect to control. These results suggest that Al3+ induces a decrease of molecular order at the lipoprotein surface. Moreover, the study of tryptophan (Trp) fluorescence demonstrated that aluminium induces structural modifications of HDL apoproteins and on HDL physico-chemical properties. The effect of Al3+ on lipid peroxidation of HDL was observed at aluminium concentrations similar to those observed in the brain of patients affected by neurological diseases. Aluminium-induced oxidative damage of HDL could be involved in the development of neurological diseases.

Increased susceptibility to peroxidation of VLDL from non-insulin-dependent diabetic patients: a possible correlation with fatty acid composition.

Recent studies suggested that both oxidized very low density lipoproteins (VLDL) and oxidized high density lipoproteins (HDL) might play a role in the pathogenesis of atherosclerosis. The aim of the present work was to analyse the susceptibility to in vitro peroxidation of VLDL and HDL from apparently normolipidemic subjects affected by insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) in good metabolic control and to examine the possible relations between oxidisability and lipoprotein fatty acid composition. VLDL and HDL were isolated from 13 IDDM patients, 12 NIDDM patients and 18 healthy subjects. The degree of lipoprotein oxidation was determined by the measurement of hydroperoxide levels and thiobarbituric acid-reactive substances (TBARS) before and after in vitro peroxidative stress with CuSO4. Fatty acid analysis was performed by gas chromatography. VLDL and HDL from NIDDM patients showed a decrease in the saturated fatty acid content with a concomitant increase in unsaturated fatty acids and higher basal peroxide levels compared with healthy subjects. Oxidisability of VLDL from NIDDM subjects was higher than in controls and was significantly related with the unsaturated fatty acid content. The present work suggests that alterations in the composition and functions of both VLDL and HDL able to produce more atherogenic lipoproteins are present in NIDDM.

Influence of low density lipoprotein from insulin-dependent diabetic patients on platelet functions.

In the present work we studied in vitro the action of low density lipoproteins (LDL) isolated from normolipemic insulin-dependent diabetic (IDDM) patients on transmembrane cation transport, nitric oxide synthase (NOS) activity, and aggregating response to stimuli of platelets from healthy subjects to elucidate whether the modified interaction between circulating lipoproteins and cells might be one of the pathogenetic mechanisms of the increased platelet activation in IDDM. LDL were obtained by discontinuous gradient ultracentrifugation from 15 IDDM out-patients and 15 sex- and age-matched healthy subjects and used for incubation experiments with control platelets. Lipid composition and hydroperoxide concentrations were studied in LDL. Platelet aggregation responses to ADP, NOS activity, cytosolic Ca2+ concentrations, and platelet membrane Na+/K+-adenosine triphosphatase (Na+/K+-ATPase) and Ca2+-ATPase activities were measured after incubation. IDDM LDL showed an increased lysophosphatidylcholine content compared with that of control LDL. IDDM LDL significantly increased the platelet aggregating response to ADP, cytosolic Ca2+ concentrations, and plasma membrane Ca2+-ATPase activity and significantly reduced NOS activity and platelet membrane Na+/K+-ATPase activity compared with those of platelets incubated in buffer or cells incubated with control LDL. The effects exerted by IDDM LDL on platelet suspensions from healthy subjects mimic the alterations observed in platelets from diabetic subjects in basal conditions. Both the decreased activity of NOS and the higher cytoplasmic concentrations of Ca2+ might cause increased platelet activation, as observed in IDDM. In conclusion, the present study suggests a new mechanism with a potential role in the early development of atherosclerosis in diabetic patients, i.e. an altered interaction between circulating lipoproteins and platelets.

Study of fluidity of low density lipoproteins from liver cirrhotic patients.

BACKGROUND AND AIM: Liver disease is accompanied by major quantitative and qualitative modifications in plasma lipoprotein metabolism. Alterations in plasma lipoprotein composition and a lower susceptibility to in vitro peroxidation of low density lipoprotein (LDL) and erythrocyte membranes have been observed in liver cirrhosis. The main objective of the present work was to investigate LDL chemical composition and fluidity in liver cirrhosis using the fluorescence polarization (Pf) of the 1,6-diphenyl-1,3,5-hexatriene (DPH) probe. METHODS AND RESULTS: The chemical composition of LDL was studied in 12 cirrhotic patients and 22 controls by conventional methods and its fatty acid composition by gas chromatography. LDL fluidity was determined by measuring the DPH Pf values. A decrease in molecular order was demonstrated by the significant (p < 0.05) decrease in Pf values in the cirrhotics. Modifications in LDL fluidity are correlated with its composition. A significant increase in triglyceride content (p < 0.05), and significant increases in triglyceride/protein and triglyceride/phospholipid ratios were observed in the cirrhotics. CONCLUSIONS: Our results demonstrate that the higher LDL fluidity of cirrhotic patients may be due to an increased triglyceride content.

Binding steps of apolipoprotein A-I with phospholipid monolayers: adsorption and penetration.

In whole HDL particles, the arrangement of apoA-I relative to phospholipids is of crucial interest with respect to the physiological formations of HDL. We report here new data concerning the nature of the interaction of apoA-I with condensed phospholipid (PL) monolayers (phosphatidylcholine and phosphatidylserine). The use of alternative current polarography allowed the detection for the first time of different binding steps which are dependent on apoA-I concentration. At low concentration (below 10 micrograms/mL), apoA-I adsorbs on PL polar headgroups, through electrostatic interactions. Above this threshold concentration, apoA-I penetrates within the monolayer (i.e., part of apoA-I crosses the PL polar headgroup/hydrocarbon chain interface). The process of penetration described here brings experimental evidence supporting Segrest's "snorkel" model. Penetrated helices are lying at the interface, their apolar face in contact with PL hydrocarbon chains and their polar face in contact with PL polar headgroups. In the absence of cholesterol, a second level of penetration was detected at higher apoA-I concentrations. It was facilitated in the presence of phosphatidylserine in comparison to phosphatidylcholine and disappeared in the presence of cholesterol. It is proposed that the C-terminal domain is involved in the first binding steps and that hinged domains may also be implicated. Furthermore, we propose that the apoA-I binding states stabilize the protein/phospholipid layer complex. These different binding states are discussed with respect to their roles in HDL metabolism.

Effect of nicotine and cotinine on the susceptibility to in vitro oxidation of LDL in healthy non smokers and smokers.

Cigarette smoke of which the major component is nicotine plays an important role in the development of cardiovascular diseases. To study the effect of in vitro incubation of LDL with nicotine and its metabolite, cotinine on a copper-induced peroxidation, we monitored the formation of conjugated dienes, hydroperoxides and thiobarbituric acid-reactive substances production. The LDL studied were taken from six non-smokers (aged 41.5 years) and six smokers who consumed at least ten cigarettes per day (40.7 years). LDL oxidation with CuSO4 showed that cigarette smoking promotes LDL susceptibility to peroxidative modification. During the peroxidation of LDL with nicotine (O to 5 mmol/1) and CuSO4 (5 micromol/l), the formation of hydroperoxides decreased when nicotine concentrations increased and the production of TBARS increased in a concomitant manner. The results showed that the presence of nicotine destabilized the production of hydroperoxides in LDL and increased the formation of secondary oxidation products. On the other hand, cotinine had no effect on LDL oxidative susceptibility in smokers and non-smokers.

Physico-chemical properties of copper-oxidized very low density lipoproteins.

The aim of our study was to investigate the effect of Cu2+ catalyzed oxidation on VLDL physico-chemical properties and secondary structure of apo B-100. Incubation of very low density lipoproteins with copper ions resulted in a decrease in tryptophan and lysine residues parallel to lipid peroxidation products, conjugated dienes and TBARS. Fluorescence polarization showed an increase in the molecular order at the lipoprotein surface of VLDL, as demonstrated by the increase in Pf values of DPH. The secondary structure of apo B-100 was investigated by infrared spectroscopy. Increased order and structural changes, as observed after oxidative stress on VLDL, could be of relevance in the abnormal interactions between lipoproteins and cell membranes.

Effect of aluminium ions on liposomal membranes as detected by Laurdan fluorescence.

We report here an investigation of the influence of aluminium on iron-induced peroxidation in brain model membranes. Laurdan fluorescence emission spectra and generalised polarisation measurements have been used to investigate how ferrous and aluminium ions can affect the phase components of phospholipid membranes. An increase in the generalised polarisation of oxidised liposomes with respect to controls has been observed, which reveals the presence of a less polar environment surrounding the probe that changes the properties of the bilayer. Aluminium has been shown to facilitate iron-mediated oxidation as detected from emission fluorescence spectra. However, no quantitative influence has been calculated relative to general polarisation and derived phase state determinations. The structural influence of aluminium on membranes may therefore be less significantly marked than initially expected.

Prevention of preatheromatous lesions in sand rats by treatment with a nutritional supplement.

Sand rats fed a hypercholesterolaemic diet containing 0.01% of the anti-thyroid agent 2-mercapto-1-imidazole develop preatheromatous lesions similar to those found in humans, in addition to obesity and insulin resistance. The effects of a nutritional supplement rich in essential fatty acids and garlic extract (Arterodiet) on the appearance and evolution of the lesions were studied. Treatment with this nutritional supplement significantly decreased circulating triglycerides and low-density lipoprotein (LDL)-cholesterol levels but did not alter plasma insulin or glucose levels. Intra-arterial cholesterol levels were also decreased by the treatment which resulted in a normalisation of the atherosclerotic lesions in these animals.

Influence of the environment in space on the biochemical characteristics of human low density lipoproteins.

The purpose of this experiment was to study the efficiency of protective substances on the effects of cosmic radiation in space on low density lipoproteins. This environment induced modifications in LDL consisting of an increase of lipid peroxidation markers (hydroperoxides, thiobarbituric acid reactive substances). In contrast, apo B was not affected by cosmic radiation as shown by the stability of the trinitrobenzenesulfonic acid reactivity and the tryptophan content. Furthermore, oxidation of LDL was partially inhibited by the addition of cysteamine or/and probucol before the spaceflight experiment. The hydroperoxide formation was almost completely inhibited by cysteamine. It was concluded that antioxidants can exert a protective effect against peroxidative stress induced by the space environment.

ESA's Biopan 1--"Vitamin" experiment preliminary results.

The purpose of "Vitamin" experiment is to study the efficiency of protective substances on three biological acellular systems aqueous solutions exposed to cosmic radiation in space. The first system "LDL" is a low density lipoprotein. The second is "E2-TeBG complexe" in which estradiol (E2) is bound to its plasmatic carrier protein, testosterone-estradiol binding globulin (TeBG). The third is "pBR 322", a plasmid. "Vitamin" experiment was accommodated in the Biopan which had been mounted on the outer surface of a Foton retrievable satellite. The experiment was exposed to space environment during 15 days. A stable temperature of about 2O degrees C was maintained throughout the flight. "Vitamin" experiment preliminary results are presented and discussed.

Increased susceptibility to lipid oxidation of low-density lipoproteins and erythrocyte membranes from diabetic patients.

The aim of the present study was to determine if low-density lipoproteins (LDLs) and red blood cell (RBC) membranes from diabetic patients present an increased susceptibility to lipoperoxidation, which might be related to the increased incidence of atherosclerosis in diabetes. LDLs and RBC membranes were isolated from 11 insulin-dependent (IDDM) and 18 non-insulin-dependent diabetic (NIDDM) patients and exposed to a peroxidative stress by incubation with phenylhydrazine. The susceptibility to peroxidation was determined by measuring the production of thiobarbituric acid-reactive substances (TBARS) after the incubation. The following parameters were also evaluated: plasma glucose, triglycerides (TG), phospholipids (PL), total and high-density lipoprotein (HDL) cholesterol, apolipoprotein (apo) A-I, apo B, hemoglobin A1c (HbA1c), LDL PL and cholesterol, LDL fatty acid composition, and RBC membrane PL and cholesterol. Although they were apparently normolipidemic, diabetic patients showed an increased susceptibility to peroxidation in LDLs and erythrocyte membranes as compared with control subjects. The amount of arachidonic acid in LDLs and the PL concentration of RBC membranes from diabetic patients were significantly higher than in normal subjects. The increased lipoperoxidability of both RBC membranes and LDLs might play a central role in the pathogenesis of the vascular complications of diabetes mellitus.

Reduced Na(+)-K(+)-ATPase activity and plasma lysophosphatidylcholine concentrations in diabetic patients.

A fraction from normal human plasma inhibiting Na(+)-K(+)-ATPase has been recently identified as lysophosphatidylcholine (LPC). The aim of this study was to investigate the existence of a relationship between the activity of the cellular membrane Na(+)-K(+)-ATPase and plasma LPC in human diabetes. We studied 10 patients with insulin-dependent-diabetes mellitus (IDDM), 14 patients with non-insulin-dependent diabetes mellitus (NIDDM), and 10 sex- and age-matched control subjects. Plasma LPC concentrations were increased in both IDDM and NIDDM patients compared with control subjects. Na(+)-K(+)-ATPase activity was reduced in both groups of patients in erythrocyte and platelet membranes. There was a significant correlation between the concentrations of plasma LPC and Na(+)-K(+)-ATPase activity in both erythrocyte and platelet membranes (P < 0.01). To investigate the effect of LPC on the enzyme, Na(+)-K(+)-ATPase activity was determined in erythrocyte membranes obtained from six healthy subjects after in vitro incubation with increasing concentrations of LPC (1-10 microM). Enzymatic activity was significantly reduced by in vitro LPC at a concentration of 2.5 microM, with a further decrease at 5 microM. These data suggest that the decrease in Na(+)-K(+)-ATPase activity in diabetes might be due to increased LPC concentrations.

Susceptibility to in vitro lipid peroxidation of low density lipoproteins and erythrocyte membranes from liver cirrhotic patients.

The aim of the present study was to investigate the susceptibility of low density lipoprotein (LDL) and red blood cell membranes (RCM) from liver cirrhotic patients and control subjects, to the peroxidative stress, induced in vitro by phenylhydrazine. The susceptibility to peroxidation was determined by measuring the formation of the thiobarbituric acid reactive substances (TBARS) after incubation with phenylhydrazine. Moreover the levels of cholesterol (C) and phospholipid (PL) were evaluated in plasma and isolated LDL and RCM. A significant decrease in plasma C, LDL-C and RCM-C content (p < 0.001, p < 0.01 and p < 0.05 respectively) and RCM-PL content (p < 0.01) were found in cirrhotic patients compared to controls. The lower susceptibility to in vitro peroxidation was shown by the TBARS levels significantly lower in LDL and RCM from cirrhotic patients with respect to controls (p < 0.0001 for both of them). Moreover significant positive correlations were found between TBARS-LDL and LDL-C or LDL-PL (r = +0.72, p < 0.0001; r = +0.58, p < 0.001) and between TBARS-RCM and RCM-C (r = +0.35, p < 0.05) or RCM-PL (r = 0.37, p < 0.05) from combined patients and controls. Our data seem to ascribe the lower in vitro peroxidability to the lower levels of plasma lipids.

Effect of desialylation on low density lipoproteins: comparative study before and after oxidative stress.

Aim of our study was to investigate the effect of the desialylation induced by neuraminidase treatment on low density lipoprotein susceptibility to peroxidative stress induced by incubation with copper ions. Our results show that peroxidative stress induces the formation of aggregates that was not observed in desialylated low density lipoproteins. An increase in thiobarbituric reactive substances and a decrease in polyunsaturated fatty acids content have been shown in oxidized LDL. These modifications were less pronounced in oxidized low density lipoproteins previously treated by neuraminidase. The present data suggest a lower susceptibility to peroxidative stress in previously desialylated low density lipoproteins.

Lipoprotein peroxidation in adult psoriatic patients.

Psoriasis, a chronic inflammatory skin disease characterized by an aberration of lipid metabolism, has been associated with an increased risk for atherosclerosis. Since oxidatively modified lipoproteins are involved in the pathogenesis of atherosclerosis, we investigated the lipid composition and in vitro induced peroxidation of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) from psoriatic patients. 11 male adult psoriatics and 16 male age-matched healthy subjects were studied. Lipid peroxidation of VLDL and LDL was performed by incubation with CuSO4 for 24 h at 37 degrees C. The compositional analysis showed a significant increase in triglycerides and phospholipids, both in VLDL (p < 0.05) and in LDL (p < 0.001) from psoriatic patients, compared with controls. Moreover a significant increase in total cholesterol (TC) (p < 0.01) and apoprotein (P) (p < 0.05) was found in LDL from psoriatics. The levels of thiobarbituric acid reactive substances (TBARS), as a measurement of lipid peroxidation, were significantly higher in Ox-VLDL and in Ox-LDL from psoriatics (p < 0.01) than the corresponding values in controls. Moreover, basal values of TBARS were significantly higher in VLDL and LDL from psoriatic patients than those from controls. In conclusion, the lipoprotein compositional changes associated with the modifications of TBARS before and after Cu2+ treatment of lipoproteins may suggest an increased risk for atherosclerosis in adult psoriatic patients.

Physico-chemical properties of copper-oxidized high density lipoprotein: a fluorescence study.

The modifications of the physico-chemical properties of the high density lipoprotein (HDL) before and after in vitro induced oxidation by copper ions have been studied using the fluorescence polarization (Pf) of the phosphatidylcholine derivative of 1,6-diphenyl-1,3,5-hexatriene (DPH-PC) and of the cationic derivative (TMA-DPH). We have observed that HDL oxidation is associated with a decrease of the molecular order at the lipoprotein surface as demonstrated by the increase in Pf with respect to untreated HDL. Moreover in oxidized HDL the polarity-sensitive probe laurdan has shown a decrease of the polarity in its microenvironment. It has been suggested that a decrease in HDL fluidity would inhibit cholesterol reverse transport from peripheral tissues in form of HDL core cholesteryl esters. Peroxidation of HDL, if occurring in vivo, could contribute to the progress of atherogenesis by decreasing cholesterol efflux from peripheral tissues.