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High density lipoproteins (HDL) are susceptible to structural modifications mediated by various mechanisms including oxidation, glycation, homocysteinylation or enzymatic degradation. Structural alterations of HDL may affect their functional and atheroprotective properties. Oxidants, such as hypochlorous acid, peroxyl radicals, metal ions, peroxynitrite, lipoxygenases and smoke extracts, can alter both surface and core components of HDL. The formation of lipid peroxidation derivatives, such as thiobarbituric acid reactive substances, conjugated dienes, lipid hydroperoxides and aldehydes, is associated with changes of physical properties (fluidity, molecular order) and of apoprotein conformation. Non-enzymatic glycation, generally associated with lipoxidation, leads to form irreversible complexes called advanced glycation end products. These HDL modifications are accompanied with altered biological activities of HDL and associated enzymes, including paraoxonase, CETP and LCAT. Homocysteine-induced modification of HDL is mediated by homocysteine-thiolactone, and can be prevented by a calcium-dependent thiolactonase/paraoxonase. Tyrosylation of HDL induces the formation of dimers and trimers of apo AI, and alters cholesterol efflux. Phospholipases and proteolytic enzymes can also modify HDL lipid and apoprotein structure. HDL modification induces generally the loss of their anti-inflammatory and cytoprotective properties. This could play a role in the pathogenesis of atherosclerosis and neurodegenerative diseases such as Alzheimer's disease.

Background Anorexia nervosa (AN), a psychosomatic disorder, has serious negative effects on multiple organs and systems of the human body. Anorexia nervosa usually runs a chronic course and is associated with significant morbidity and mortality. In order to elucidate the role played by lipids in AN, in the present study we compared the plasma lipid profile and the chemical-physical properties of lipoproteins obtained from subjects affected by AN. Materials and methods The study was performed on lipoproteins of AN subjects and of age-matched healthy subjects used as controls. We tested the susceptibility to oxidative stress in vitro, the fatty acid content, the fluidity using 2-dimethylamino-(6-lauroyl)-naphthalene (Laurdan) and 1,6-difenil-1,3,5-esatriene (DPH) probes. Results Present results indicate that AN patients present a deep alteration of the composition and of chemical-physical properties in circulating lipoproteins, even in the absence of significant modifications to clinical metabolic parameters. A significantly decreased body mass index (BMI) was found in AN patients in comparison with controls. Anorexia nervosa patients showed a significant modification of phospholipids to protein ratio and a significantly increased percentage of unsaturated fatty acids compared with control subjects as well as a decreased fluidity, a significantly increased percentage of liquid-crystalline phase in VLDL, and a significantly reduced susceptibility to oxidative stress, more pronounced in LDL. Conclusions These results confirm the hypothesis that anorexia is accompanied by changes of lipid metabolism in the central nervous system (CNS).

High-density lipoproteins (HDL) plays a key role in the protection against oxidative damage of lipoprotein and biological membranes. The aim of the present study was to investigate the relationship between the antioxidant role of HDL and the HDL-paraoxonase (PON) activity in healthy subjects and in type 1 diabetic patients. Moreover, the ability of HDL of controls and diabetic patients to protect and/or repair biological membranes from oxidative damage was studied. HDL were isolated from 31 type 1 diabetic patients and 31 sex- and age-matched healthy subjects and immediately used to evaluate lipid hydroperoxides and HDL-PON activity. Erythrocyte membranes obtained from healthy subjects were oxidized with 2,2-azo-bis(2-aminidinopropane)dihydrochloride and then incubated in the presence of HDL isolated from healthy or type 1 diabetic subjects, with measurements of membrane lipid hydroperoxides before and after the incubation. HDL from type 1 diabetic patients showed higher levels of lipid hydroperoxides and a lower activity of HDL-PON than healthy subjects. Moreover, HDL of type 1 diabetic patients protected less efficiently erythrocyte membranes against oxidative damage compared with HDL from healthy subjects. A negative correlation was found between HDL-PON activity and the levels of hydroperoxides of HDL, confirming the relationship between PON and lipid peroxidation and suggesting that subjects with low PON activity are more exposed to oxidative damage than subjects with high PON activity. The ability of HDL to protect erythrocyte membranes was positively correlated with HDL-PON activity and negatively correlated with the levels of lipid hydroperoxides of HDL of healthy subjects. These results confirm a linkage between PON activity and lipid peroxidation of lipoproteins and suggest that the ability of HDL to protect erythrocyte membranes might be related to the PON activity. It might be hypothesized that the decrease of PON activity in diabetic patients and the lower HDL protective action against membrane peroxidation could contribute to acceleration of arteriosclerosis in type 1 diabetes mellitus.

Homocysteine-thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL). Structural and functional alterations of Hcy-LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy-LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10–100 μM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico-chemical properties were observed in Hcy-LDL with respect to control LDL (c-LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy-LDL incubated at 37°C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy-LDL with respect to cell incubated with c-LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy-LDL. The relationship between the levels of –SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy-LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC. © 2004 Wiley-Liss, Inc.

We investigated the effect of homocysteine (Hcy)-thiolactone on the activity of the enzyme paraoxonase (PON) associated with human high-density lipoprotein (HDL-PON). HDL were isolated from plasma of normolipidemic subjects. The increase in the levels of sulfhydryl groups (−SH) in HDL incubated with Hcy-thiolactone demonstrates that homocysteinylation of HDL occurs. The increase of −SH groups correlated with the basal values of HDL-PON activity (r = −0.73, P

The interaction between low density lipoproteins (LDL) and platelets might play a central role in the development of atherosclerosis in diabetes. The aim of the present study was to investigate whether the glycation of LDL is associated with modifications of their physico-chemical and functional properties and to study the action of glycated LDL (glycLDL) on platelets. LDL and platelets were isolated from 15 healthy subjects. The content of thiobarbituric acid-reactive substances and the generalized polarization of the fluorescent probe Laurdan were determined in LDL glycated in vitro. Platelets were incubated with native LDL, GlycLDL, and minimally oxidized LDL, and the following parameters were evaluated: platelet aggregation, nitric oxide production, intracellular Ca(2+) concentrations, Na(+)/K(+)-adenosine triphosphatase (Na(+)/K(+)-ATPase), and Ca(2+)-ATPase activities. GlycLDL showed increased thiobarbituric acid-reactive substance levels, a red shift of the Laurdan emission maximum, and a decrease in generalized polarization, indicating a higher polarity and a reduced molecular order compared with native LDL. GlycLDL caused a significant increase in platelet nitric oxide production, intracellular Ca(2+) concentration, and aggregating response to ADP; an inhibition of the platelet membrane Na(+)/K(+)-ATPase activity; and a stimulation of Ca(2+)-ATPase activity. Minimally oxidized LDL did not cause statistically significant changes in the parameters studied. The present work demonstrates that glycation induces compositional and structural changes in LDL and suggests that an altered interaction between glycLDL and platelets might play a role in the vascular complications of diabetes.

The behavior of the 2 sialidase forms present in the erythrocyte membrane was investigated in 117 subjects with type 2 diabetes mellitus versus 95 healthy controls. A significant increase of the acidic form of sialidase, which is anchored to the membrane by a glycosylphosphatidylinositol bridge, was observed in erythrocyte resealed membranes. On the contrary, the neutral form of the enzyme, the only one capable of removing lipid- and protein-bound sialic acid from endogenous and exogenous sialoderivatives, was significantly reduced with a consequent increase of erythrocyte membrane total sialic acid content. Disease duration, therapy, glycemia, parameters of metabolic control, and presence of complications, except nephropathies, had no influence on the tested enzyme activities. Diabetic subjects showed a different erythrocyte age distribution, with an almost double proportion of young red cells and only one quarter of senescent ones compared with controls. In young erythrocytes, diabetic and control subjects had the same distribution of the 2 enzymes, while in senescent cells the acidic enzyme was increased 3.5-fold and the neutral form was reduced by half in the diabetic subjects. The increase of both acidic sialidase and total membrane-bound sialic acid, together with an overpresence of young red cells in diabetics, suggests that in this pathological condition there might be an altered aging process with a diminished expression of the neutral form of the enzyme and an increase of bound sialic acid. It has been suggested that the expression of the neutral enzyme requires some activation mechanism that is impaired in diabetes.

Recent studies have demonstrated that Apo AIV exerts a protective effect against atherosclerosis. Moreover, Qin et al. (Am. J. Physiol. 274 (1998) H1836) have demonstrated that Apo AIV, isolated from rat plasma, exerts an inhibitory effect against Cu2+-induced lipid peroxidation of intestinal lymph and LDL. The aim of the study was to investigate whether human Apo AIV exerts a protective effect against Cu2+-induced lipid peroxidation. Our results demonstrated that human Apo AIV exerted an inhibitory effect against Cu2+ and AAPH induced lipid peroxidation of VLDL, as shown by the lower increase in the levels of TBARS and conjugated dienes in lipoproteins preincubated with Apo AIV. In addition, the tryptophan (Trp) and probe 2-(dimethylamino)-6-lauroylnaphthalene (Laurdan) fluorescence studies demonstrated that the modifications of spectral properties in both lipoproteins preincubated with Apo AIV were lower with respect to ox-lipoproteins, suggesting that Apo AIV prevents the modification of physico-chemical properties due to peroxidation.

We investigated the effect of incubation of high density lipoprotein (HDL) under hyperglycaemic conditions on lipid composition, physicochemical properties and activity of paraoxonase (PON), a calcium-dependent enzyme associated with HDL that contributes to the antiatherogenicity of this lipoprotein. HDL incubated for three days with various glucose concentrations (0-100 mM) had significant increases in thiobarbituric acid-reactive substances (TBARS) and conjugated dienes with respect to control HDL. These results suggest that lipid peroxidation accompanies HDL glycation in vitro. The susceptibility to lipid peroxidation was higher in HDL isolated from subjects with low HDL-paraxonase activity with respect to subjects with higher HDL-PON activity. The lipid compositional changes were associated with modifications of apoprotein conformation as shown by the red-shifted position of the maximum emission of tryptophan in treated HDL. The decrease in the Gp (generalized polarization) value and the red-shifted position of the maximum emission of Laurdan incorporated in treated HDL demonstrate modifications of order and polarity with respect to control HDL. The negative correlation established between the Gp value and TBARS demonstrates that the modifications in molecular order are likely related to the increase in lipid peroxidation products. The activity of paraoxonase was significantly decreased in HDL incubated at 37 degrees C; a greater decrease occurred in the presence of 50 mM and 100 mM glucose. This study demonstrates modifications of lipid composition, apoprotein conformation and physicochemical properties of HDL incubated in the presence of glucose. These modifications affect the activity of HDL-associated paraoxonase. The physicochemical properties of lipoproteins play a regulatory role in lipoprotein function. The modification of order and polarity of glycated HDL and the alterations in paraoxonase activity could potentially contribute to the accelerated atherosclerosis in diabetic patients.

Background Na+,K+-ATPase activity was evaluated in relation to membrane composition and molecular organization in erythrocyte membranes from obese patients by the amphyphylic molecule 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan). Its possible relationship with fat distribution and hyperinsulinaemia was also investigated. Design Subjects were 10 obese men (OM), 12 women with subcutaneous obesity (FSO), 10 women with abdominal obesity (FAO) and 41 healthy lean subjects, 26 women (FC) and 15 men (MC). An oral glucose tolerance test was administered to all subjects to evaluate insulin secretion and glucose tolerance. Results Na+,K+-ATPase activity was increased in all obese patients. Values were higher in FSO and FAO than in FC (with FAO greater than FSO) and in OM than in MC. The erythrocyte membrane cholesterol-to-phospholipid ratio was increased in obese patients and was significantly different in FSO patients compared with FC. The erythrocyte membrane protein-to-phospholipid ratio was also increased in all obese subjects, reaching statistical significance only in FSO vs. FC. The liquid crystalline phase, as tested by Laurdan generalized polarization (GP), was decreased in obese patients, indicating the presence of greater molecular environmental order; all patients groups showed lower GP values than control subjects, but only FAO reached statistical significance compared with FC. There was no evident correlation between membrane Na+,K+-ATPase activity and insulin levels, nor did membrane composition and properties show any evident relationship with insulin levels. Conclusion Both increased Na+,K+-ATPase activity and altered fluidity and lipid composition were observed in the erythrocyte membrane of all obese patients. These findings are in line with previous observations by our group and indicate that the changes in Na+,K+-ATPase activity observed in obese patients could be related to changes in plasma membrane organization and composition.

The aim of this study was to evaluate changes in the concentration of certain components of human unstimulated whole saliva during aging, in order to better understand the role played by aging in oral health. In particular, we studied total protein concentration, alpha-amylase activity, sialic acid content and calcium and phosphorus concentrations in 100 healthy subjects of both genders, aged between 10 and 80 years, who were subdivided into four groups according to their age: 10-25 years, 26-40 years, 41-65 years, and 66-80 years. Other than sialic acid, the concentrations of the components studied were not affected by age. There was a significant negative correlation between sialic acid content and age. Our data indicate the presence of a decreased submandibular/sublingual function with aging, thus suggesting the possibility of a concomitant reduction in the modulating action of unstimulated whole saliva on the oral flora.

The fluorescence emission spectrum of N-dansyl-S-nitrosohomocysteine was enhanced approximately 8-fold upon removal of the NO group either by photolysis or by transnitrosation with free thiols like glutathione. The fluorescence enhancement was reversible in that it could be quenched in the presence of excess S-nitrosoglutathione. Attempts were then made to utilize N-dansyl-S-nitrosohomocysteine as an intracellular probe of thiols/S-nitrosothiols. Fluorescence microscopy of fibroblasts in culture indicated that intracellular N-dansyl-S-nitrosohomocysteine levels reached a maximum within 5 min. N-Dansyl-S-nitrosohomocysteine fluorescence was directly proportional to intracellular GSH levels, directly determined with HPLC. N-Dansyl-S-nitrosohomocysteine preloaded cells were also sensitive to S-nitrosoglutathione uptake as the intracellular fluorescence decreased as a function of time upon exposure to extracellular S-nitrosoglutathione.

Phase-modulation fluorescence lifetime measurements were used to study the single Trp residue of the Ca(2+)-binding protein S-100a both in the absence and in the presence of Ca2+ and/or Mg2+. Trp fluorescence decay for the protein was satisfactorily described by Lorentzian lifetime distributions centered around two components (approximately 4 ns and 0.5 ns). Lifetime values were unchanged by 2 mM Ca2+, but the fractional intensity associated with longer lifetime increased up to 75%. In the presence of Mg2+, the Ca2+ induced increase of the fractional intensity associated with longer lifetime was only 57%. For the protein in buffer, about the 85% of the recovered anisotropy was associated to a rotational correlation time of 6.7 ns. After the addition of Ca2+, this value was increased to 16.08 ns. In the presence of Mg2+, Ca+2 increased the rotational correlation time to 33.75 ns. Similar studies were performed with S-100a interacting with egg phosphatidylcholine vesicles (SUV). Our data suggest that the conformation of the protein may be influenced by structural features of the lipidic membrane. Moreover, data obtained in the presence of Mg2+ indicate some interaction between lipids and S-100, likely mediated by this ion.

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.

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.

The changes in plasma membrane polarity of polymorphonuclear leukocytes (PMN) during the activation of the respiratory burst were investigated by measuring the steady-state fluorescence emission spectra of 2-dimethylamino(6-lauroyl) naphthalene (Laurdan), which is known to be incorporated at the hydrophobic-hydrophilic interface of the bilayer, displaying spectral sensitivity to the polarity of its surroundings. Laurdan shows a marked steady-state emission blue shift in nonpolar solvents, with respect to polar solvents. Our results show a blue shift of the fluorescence emission spectra of Laurdan during activation of PMN with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. These results suggest that the activation of the respiratory burst of PMN is accompanied by a decrease in polarity in the hydrophobic-hydrophilic interface of the plasma membrane.

The interaction between PAF and human platelet membranes was investigated by measuring the steadystate fluorescence anisotropy and fluorescence decay of 1 (4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated in platelet plasma membranes. PAF induced a time-limited and significant increase of the lipid order in the exterior part of the membrane and a decrease in membrane heterogeneity. These changes were blocked in the presence of the PAF antagonists, L-659,989 and 1-O-hexadecyl-2-acetyl-sn-glycero-3-phospho(N,N,N-trimethyl)hexanolamine.H2O. These results indicate that the observed changes in the physico-chemical properties of the membrane are attributed to the PAF–receptor interaction and signal transduction.

We have studied plasma lipid patterns and erythrocyte membrane fluidity in 60 obese children and 20 normal children. Plasma levels of total cholesterol and associated lowdensity lipoproteins were significantly increased in 20 obese patients with respect to controls. A significant decrease in membrane fluidity, measured as an increase in the fluorescence polarization value of the probe 1,6-diphenyl-1,3,5-hexatriene, associated with an increase in the cholesterol/protein ratio has been shown in obese patients. The study of the correlation between erythrocyte membrane fluidity and plasma cholesterol has indicated that significant changes in fluidity and membrane lipid composition also occur in erythrocytes of obese patients with normal plasma lipid levels. These findings confirm that the erythrocyte membrane responds very early to modifications of plasma lipoproteins and suggest that in childhood obesity a modified transfer of cholesterol from plasma to erythrocyte membrane may take place.

The effect of atrazine on Ca 2+ induced fusion of cardiolipin(CL) and phosphatidylserine (PS) vesicles is studied by Tb 3+ /dipicolinic acid fluorescence and turbidity measurements. The interaction of herbicide with CL and PS membranes is studied by DPH fluorescence polarization. At low concentrations the pesticide partially inhibits fusion, especially in CL vesicles. Higher concentrations of atrazine decrease inhibition of fusion in CL, while fusion is slightly increased in PS. The Ca 2+ -induced increase of turbidity is not affected by atrazine in both PS and CL aggregation experiments. DPH polarization measurements show a perturbation only of the membrane hydrophobic core of PS, in presence of Ca 2+ . It is hypothesized that this biphasic effect shown by low and high atrazine concentrations on Ca 2+ -induced fusion of vesicles is due to a different localization of the pesticide in the membrane.

Atrazin (2-chloro-4 ethylamino-6-(isopropylamino)-s-triazine) is one of the most widely used herbicides. Fourier transform infrared spectroscopy, differential scanning calorimetry and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of its derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) were used to study the interaction of atrazine with dipalmitoyl phosphatidylcholine liposomes used as a model for biological membranes. The results show that atrazine does not perturb the hydrophobic core of the lipid bilayer and suggest that the herbicide localizes near the glycerol backbone of the lipid.

The effect of diacetylrhein on lymphocyte membrane fluidity in osteoarthritis patients before and after 10 and 30 days of treatment was studied using the 1,6-diphenyl-1,3,5-hexatriene fluorescence polarization. Moreover we studied the in vitro effect of rhein, the active metabolite of diacetylrhein, on lymphocyte membrane fluidity of controls using the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and its cationic derivative 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene. Our results showed that the patients with active osteoarthritis have higher fluorescence polarization values than those in the other osteoarthritis patients and in the controls. Moreover after 10 days of diacetylrhein treatment, we observed a significant decrease of fluorescence polarization values only in lymphocytes of the patients who showed active osteoarthritis before therapy. However after 30 days of treatment, significant changes of fluorescence polarization values were observed also in those patients who did not show painful osteoarthritis before diacetylrhein treatment. We observed that in vitro rhein also induces an increase in lymphocyte membrane fluidity, more evident with 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5-hexatriene with respect to 1,6-diphenyl-1,3,5-hexatriene.

Several studies have demonstrated that the isoflavone genistein exerts a protective effect against lipid peroxidation of low density lipoproteins (LDL). Aim of our study was to investigate whether genistein protects high density lipoproteins (HDL), isolated from normolipemic subjects, against Cu ++ -induced lipid peroxidation. Our results demonstrated that genistein exerts an inhibitory effect against Cu ++ -induced lipid peroxidation of HDL, as shown by the lower increase in the levels of conjugated dienes in lipoproteins oxidized after preincubation with different concentrations of genistein (0.5–2.5μM). Moreover the analysis of fluorescence emission spectra of tryptophan (Trp) and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene) demonstrated that genistein prevents the alterations of apoprotein structure and physico-chemical properties, associated with Cu ++ -triggered lipid peroxidation of lipoproteins. The protective effect exerted by genistein against oxidative damage of lipoproteins was realized at concentrations similar to those observed in plasma of human subjects consuming a traditional soy diet or receiving a soy supplement. Therefore, we suggested that antioxidant activity exerted by genistein against lipid peroxidation of HDL in vitro could be of physiological relevance.

In the present study, we confirmed that copper ions induce oxidative damage in human astrocytes in culture, as demonstrated by the significant increase in the levels of hydroperoxides and in the fluorescence intensity of the fluorescent probe dichloro-dihydrofluorescein diacetate (H2DCFDA). The compositional changes were associated with a significant decrease in cell viability in astrocytes treated with 10 μM Cu++ with respect to control cells. Astrocytes incubated with copper ions in the presence of high density lipoproteins (HDL) isolated from plasma of normolipemic subjects showed lower levels of hydroperoxides and a higher cell viability with respect to cells oxidized alone. Moreover, a significant decrease in the levels of hydroperoxides was observed in oxidized astrocytes treated with HDL. These results demonstrate that HDL exert a protective role against lipid peroxidation. The protective effect could be related to the ability of HDL to bind metal ions at the lipoprotein surface and/or to a stimulation of the efflux of lipid hydroperoxides from cell membranes as demonstrated in other cell types. Oxidative damage of astrocytes was induced at a copper concentration similar to that observed in cerebrospinal fluid (CSF) of patients affected by neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's diseases (PD). Lipoprotein particles similar for density and chemical composition to plasma HDL were recently isolated in human CSF, therefore, the protective role exerted by HDL against Cu++-induced oxidative damage of astrocytes could be of physiological relevance.

Several studies support the role of copper-mediated oxidative injury in the development of neurodegenerative diseases. In this study we demonstrated that copper exerts an oxidant effect on cultured astrocytes as shown by the significant increase in the levels of hydroperoxides in astrocytes oxidized with 10 micromol/L Cu2+ for 4 h with respect to control cells. Using the fluorescent probe 2,7-dichloro-dihydrofluorescein diacetate (H2DCFDA) that represents an useful approach for measuring the production of reactive oxygen species (ROS), a significant increase in fluorescence intensity was observed in Cu(2+)-oxidized cells. The increase in the levels of lipid peroxidation products was associated with a significant decrease in cell viability in Cu(2+)-treated cells with respect to untreated cells. Many evidences suggest that human high-density lipoproteins (HDL) play a key role against lipid peroxidation of plasma low-density lipoproteins (LDL) and peripheral cells. We investigated whether HDL isolated from human plasma exert a protective role against copper-induced lipid peroxidation on astrocytes in culture. The increase in the levels of hydroperoxides and in the fluorescent intensity of H2DCFDA was significantly lower in astrocytes oxidized after preincubation for 20 h in the presence of HDL (50-200 microg/mL) with respect to cells preincubated without HDL. The decrease in viability in Cu(2+)-treated astrocytes preincubated with HDL was significantly lower with respect to cells preincubated without. These results demonstrate that preincubation of astrocytes with HDL for 20 h makes cells more resistant to the Cu2+ oxidant effect. The protective effect exerted by HDL against copper-induced oxidative damage on astrocytes was concentration dependent. These data suggest that copper, at concentrations similar to those observed in cerebro-spinal fluid of patients affected by neurodegenerative diseases, induces oxidative damage to astrocytes and confirm that HDL exert a protective effect against oxidative damage induced by copper ions.

Human whole saliva contains a number of antimicrobial agents, and lysozyme, lactoferrin, secretory IgA and peroxidase are among the best known. Peroxidase catalyzes a reaction involved in the inhibition of bacterial growth and metabolism, and the prevention of hydrogen peroxide accumulation, thus protecting proteins from the action of oxygen and reactive oxygen species (ROS). To better understand the role played by the oxidative stress in the aging process, we studied the relationship between total protein content, peroxidase activity, malondialdehyde (MDA) and nitric oxide (NO) content of human unstimulated whole saliva in 169 healthy subjects subdivided into groups according to age. Our results show a significant decrease in peroxidase activity with age. Moreover, the increase in saliva lipid peroxide levels indicates an enhanced free radical production that may contribute to tissue damage. On the other hand, findings concerning human unstimulated whole saliva NO content showed a significant increase in elderly subjects, suggesting that an enhanced NO production might depend on a stimulation of leukocyte-inducible NO synthase (i-NOS) activity. Our results suggest that during aging the oral tissues may become more susceptible to environmental factors due to a modification in the balance between different antimicrobial agents.

A modified platelet response to aggregating stimuli is supposed to play a role in the pathogenesis of diabetic macroangiopathy. We studied the fluidity and microheterogeneity of the external surface of the platelet membrane and the activities of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase in 21 men with type 1 diabetes and in 20 control subjects before and after in vitro thrombin addition. In the resting state, platelets from type 1 diabetic patients showed an increased fluidity and microheterogeneity of the platelet membrane, a higher Ca2+-ATPase activity, and a reduced Na+-K+-ATPase activity in comparison with platelets from healthy subjects. The fatty acid composition was also modified, with increased C 16:1 and decreased C 18:0 content. Control cells incubated with thrombin showed a modification of the membrane parameters opposite to the response observed in type 1 cells after the stimulation. The incubation of control platelets in the resting state with high concentrations of glucose modified the fluidity of the plasma membrane Na+-K+-ATPase and Ca2+-ATPase activities in an opposite way in comparison with the alterations observed in type 1 platelets. This study suggests that in type 1 diabetic patients, the platelet membrane responds to activation with a molecular remodeling different from the response of healthy subjects. The abnormal organization of the membrane might contribute to the altered platelet functions in type 1 diabetic patients, but acute exposure to high glucose levels does not seem able to modify the platelet membrane in the way observed in type 1 diabetes.

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.

The secondary structure of apolipoprotein B-100 in low-density lipoprotein (LDL) subfractions was analysed by Fourier-transform IR spectroscopy. LDLs were isolated in three density ranges by gradient centrifugation of human plasma from healthy volunteers. The spectra revealed differences in the lipid content and composition of the three LDL fractions. The secondary structure of apolipoprotein B-100 was the same in the two fractions corresponding to the large less-dense LDL particles, whereas a lower content of β-strands was found in the third fraction corresponding to the smaller denser LDL particles. Analysis of the spectroscopic data suggests that, in the same set of LDL subfractions, the particle size is probably the cause of the observed differences in apolipoprotein B-100 secondary structure.

Sialic acid (SA) content and Na+/K+-ATPase activity of red blood cell (RBC) membranes were studied in 26 normoalbuminuric patients with insulin-dependent diabetes mellitus (IDDM), 25 normoalbuminuric patients with non-insulin-dependent diabetes mellitus (NIDDM), and 40 healthy nondiabetic subjects with a negative family history for diabetes. A decrease in RBC membrane SA content and Na+/K+-ATPase activity was observed in older control subjects compared with younger controls. A significant correlation between age, Na+/K+-ATPase activity, and SA content was also found. No difference was observed in RBC membrane SA content between IDDM and NIDDM subjects, but Na+/K+-ATPase activity was significantly lower in IDDM patients. SA content was increased in NIDDM subjects compared with healthy subjects of similar age, whereas Na+/K+-ATPase activity was significantly lower in both IDDM and NIDDM subjects compared with controls. In NIDDM, Na+/K+-ATPase activity was significantly correlated with age, whereas both Na+/K+-ATPase activity and SA content were significantly correlated in IDDM and NIDDM patients. Hemoglobin A1c, (HbA1c) levels did not show any significant correlation either with Na+/K+-ATPase or with SA content in diabetic patients. The modified SA content and Na+/K+-ATPase activity in elderly subjects described in the present study indicate a similar behavior of the erythrocyte membrane during both RBC senescence and aging of subjects.

The aim of this study was to investigate plasma membrane fluidity in human keratinocytes using fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its cationic derivative 1-[4-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Keratinocytes from normal or psoriatic skin were isolated using trypsin-EDTA or dispase. In keratinocytes isolated from normal skin, TMA-DPH anisotropy values were higher than those observed using DPH; the difference must be related to the different localization of the two probes. In fact, DPH in whole cells localizes in plasma as well as intracellular membranes, yielding an average value of fluidity, while the cationic derivative TMA-DPH resides in the plasma membrane of the whole cells for a sufficient time for anisotropy measurements. Moreover, it has to be considered that plasma membrane is more ordered than intracellular membranes. The kinetics of incorporation of TMA-DPH was similar in keratinocytes isolated using trypsin-EDTA and those isolated using; dispase, however, the fluorescence anisotropy values were lower in keratinocytes isolated with dispase (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.029). This difference is probably related to modifications of lipid-protein interactions after trypsin treatment. Since no damage to plasma membrane after incubation with dispase seems to have been reported, we decided to use this separation procedure to study plasma membrane fluidity in psoriasis, a human pathological condition characterized by excessive cell proliferation and incomplete differentiation. Lower anisotropy values (0.260 +/- 0.01 vs 0.270 +/- 0.01, p = 0.001), indicating an increase in fluidity, were observed in keratinocytes isolated from skin of psoriatic patients than in epidermal cells isolated from normal human skin. We suggest that the measurement of fluorescence anisotropy in living cells is a convenient and useful tool to study membrane fluidity in human keratinocytes isolated from normal and diseased skin. Its application represents a technical advance because plasma membrane fluidity can be measured using very limited amounts of tissue, as obtained from biopsies.

Erythrocytes of Salmo irideus trout were separated in the range from 45 to 65% Percoll, yielding three well-separated different fractions. Steady-state fluorescence of probes embedded in erythrocyte membranes and/or in liposomes from extracted lipids was used to characterize their physicochemical properties. Furthermore, the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH), embedded in the same liposomes, was measured by a frequency decay fluorometer. DPH decay was analyzed on the assumption of continuous distribution of lifetimes, for evaluating modifications of membrane microheterogeneity. Significant differences were observed in the parameters measured for the three erythrocyte fractions, possibly connected with the specific lipid composition of the samples.

Publisher Summary Low-density lipoproteins (LDL) can be oxidized by transition metals, by cells including endothelial cells, smooth muscle cells, monocytes, macrophages, and fibroblasts, and also by irradiation. Low-density lipoproteins oxidatively modified by metal ions are characterized by alterations in biochemical composition because of the hydrolysis of phospholipids, a decrease in unsaturated fatty acids, and the generation of aldehydes with a concomitant increase in thiobarbituric acid-reactive substances. The compositional changes observed in ox-LDL and the role of antioxidants in the process is also presented in the chapter. The compositional changes of ox-LDL are associated with modifications in properties, for example, density, electrophoretic mobility, and fluidity, and in functions. Ultraviolet radiation also induces a strong peroxidation of the lipid content of LDL without changing the apolipoprotein moiety. Beside the biochemical modifications, ox-LDL are toxic for cultured cells. The abnormal receptor-mediated interactions between ox-LDL and cells are mainly related to the modifications of apoB structure, as the apoprotein plays a key role in receptor recognition; ox-LDL are cleared through the scavenger receptor pathway in macrophages and can induce the formation of foam cells involved in the atherogenesis pathway.

Psoriasis has been associated with an abnormal plasma lipid metabolism, and changes of erythrocyte membrane lipid composition and fluidity have been shown in adult patients. To investigate whether the alterations of plasma lipids appear also in pediatric patients, we have studied plasma lipids and lipoproteins in 15 prepubertal children affected by mild-to-moderate psoriasis with respect to healthy controls. The patients showed higher levels of plasma total cholesterol (4.44 +/- 0.78 versus 4.03 +/- 0.58 mmol/L), a significant increase of cholesterol associated with HDL (1.39 +/- 0.26 versus 1.13 +/- 0.28 mmol/L, p = 0.02), and a significant decrease of the ratio LDL cholesterol to HDL cholesterol (1.73 +/- 0.6 versus 2.46 +/- 0.8, p = 0.02). By using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, we have shown a significant increase in fluidity in erythrocyte membrane of psoriatic children that was associated with a slight, but not significant, decrease in the cholesterol to protein ratio (422 +/- 127 versus 503 +/- 117 nmol/mg). No significant changes of phospholipid fatty acid composition have been shown, in disagreement with previous studies in adult patients. Our results support the relation between childhood psoriasis and plasma lipid changes, which are likely related to the slight compositional changes in erythrocytes. However, the observed abnormalities are expressed differently in children than in adults.

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) has been studied in hepatocytes isolated from rat liver and in isolated plasma membrane subfractions (cLPM, canalicular membranes and bLPM, basolateral membranes) using frequency domain fluorometry. The decay has been analyzed either by using a model of discrete exponential components or a model that assumes a continuous distribution of lifetime values in order to study different aspects of membrane heterogeneity. The results obtained by the two analyses are practically superimposable but the distributional approach allows an evaluation of membrane heterogeneity through the width of the distribution that has shown particularly significant differences when freshly hepatocytes are compared with in vitro aged hepatocytes. Moreover, the comparison of the distributional analysis of the two probes has shown in cLPM a tendency to higher values of the main lifetime component and a narrower distribution width with respect to bLPM. These results indicate changes of membrane domain organization that have been discussed in relation with the specific lipid composition that characterizes the two membrane subfractions. Our results indicate that frequency domain fluorometry may be used to study membrane heterogeneity in intact cells and isolated membranes.

We have used phase-modulation fluorescence lifetime measurements to study the single Trp residue of the Ca 2+ -binding protein S-100a. Trp fluorescence decay was not exponential for the protein irrespective of the absence or presence of Ca 2+ . Fluorescence decay was best described by Lorentzian lifetime distributions centered around two components (approx. 3 and 0.7 ns) for protein in absence of Ca 2+ and one component (approx. 2.9 ns) for the protein in presence of 2 mM Ca 2+ . Similar studies were performed with S-100a interacting with cardiolipin, phosphatidylserine or egg phosphatidylcholine, both in absence and in presence of 2 mM Ca 2+ . Out data suggest that the conformation of the protein and its Ca 2+ -binding properties vary depending on the characteristics of charge and structure of phospholipids.

Plasma membrane fluidity of polymorphonuclear leukocytes was investigated in 28 patients with insulin dependent diabetes mellitus and 30 healthy controls. Membrane fluidity was measured by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into the plasma membrane. The fluorescence anisotropy values in resting (unstimulated) polymorphonuclear leukocytes from diabetic subjects were significantly higher than those of controls (0.318 +/- 0.003 vs 0.287 +/- 0.003, P less than 0.001). The addition of the respiratory burst stimulus phorbol myristate acetate induced a stable increase in fluorescence anisotropy values in both groups. Fluorescence anisotropy values of stimulated polymorphonuclear leukocytes from the diabetic and control groups were not significantly different (P greater than 0.05). These data demonstrate a decrease in plasma membrane fluidity of resting polymorphonuclear leukocytes obtained from diabetic subjects. This finding could be in part explained by an increase in their basal respiratory burst activity.

Steady state fluorescence anisotropy (rs) of TMA-DPH was measured to study the effect of respiratory burst activation with PMA, FMLP, and PAF on the physico-chemical structure of PMNs plasma membrane. Our results show a significant increase in rs during the respiratory burst activation. In the presence of NADPH-oxidase inhibitor DPI, only PAF induces changes in rs values. This suggests a non-specific effect of PAF on plasma membrane. Azide, which induces a supranormal release of H2O2, fails to increase the basal rs value after activation. Moreover, the catalase does not abolish the increase in rrs induced upon activation. This rules out the possibility that changes of rs during the respiratory burst activation are attributed mainly to H2O2 release. We conclude that multiple processes accompanying the respiratory burst activation are responsible for the changes in the physico-chemical properties of PMNs plasma membrane.

The fluorescence decay of 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was used to study micro-heterogeneity of 1,2-dimyristoyl-3-sn-phosphatidylcholine (DMPC) liposomes and to characterize the effect of phosphatidic acid on the correlation between fluorescence microheterogeneity and membrane permeability. The fluorescence decay, measured using multifrequency phase fluorometry, has been analyzed either by using a model of discrete exponential components or a model of continuous distribution of lifetime values. Both analyses have shown that TMA-DPH decay is characterized by two components: a long one of about 9 ns and a short one of about 5 ns. In the gel phase, at variance with previous DPH studies, the short component was associated with a large fractional intensity. The distributional analysis showed changes of lifetime values and width in correspondence to the calorimetric transitions. The presence of egg phosphatidic acid increased both long lifetime values and distributional width. The use of TMA-DPH as a probe to evaluate membrane heterogeneity using the distributional width is discussed. The effect of phosphatidic acid on the membrane surface and in the hydrophobic core has been related to its structural properties and to its role in water penetration.

The role of microtubules on membrane fluidity has been investigated on freshly isolated whole rat hepatocytes prepared by the perfusion method and exposed either to the microtubule-depolymerizing drugs colchicine and vincristine or to beta-lumicolchicine, a colchicine analog deprived of biological activity. Exposure of hepatocytes to 6.3 microM colchicine or to 3.0 microM vincristine led to a significant decrease of membrane fluidity as measured by fluorescence polarization of trimethylammoniodiphenylhexatriene (TMA-DPH). No changes were observed in cells exposed to 10.0 microM beta-lumicolchicine. These observations support the hypothesis that the microtubular system plays a role in the modulations of physico-chemical properties of the plasma membrane.

In a recent letter to the editor by Richard et al,[1][1]the letter authors made some comments about our work published early this year.[2][2] In our study we observed a sharp decrease of neutral sialidase activity on the surface of erythrocytes of diabetic patients, which accounts at the same time

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca 2+ -independently. In the presence of Ca 2+ , S-100b potentiates the Ca 2+ -induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca 2+ -dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the α-helical content of S-100b, analogous to that produced by Ca 2+ and that the effects of CL vesicles and of Ca 2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca 2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca 2+ -binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.

The lipid composition and fluidity of plasma membranes have been studied at different stages of liver regeneration (4, 15 and 24 h after surgery). The phospholipid and fatty acid composition is not modified, whereas the cholesterol/phospholipid ratio is lower with respect to control membranes. The modification of the physical properties of the membranes has been studied directly by EPR analysis and indirectly by temperature dependence and cooperativity of some membrane-bound enzymes (Mg2+-ATPase, (Na+ + K+)-ATPase and 5'nucleotidase). Surgical operation or anaesthesia alone causes an early increase in fluidity; such an effect appears to be markedly reduced at a later stage. There seems to be a marked effect of regeneration on plasma membrane fluidity 15 h after partial hepatectomy when several parameters--surface fluidity, cholesterol/phospholipid ratio, and 5'-nucleotidase activity in the presence of concanavalin A -- are modified and indicate an increase in membrane fluidity. It is suggested that this modification of membrane properties could be related to the proliferative process.

The effect of n -butanol on the mobility of phospholipids in phospholipid vesicles and beef heart mitochondrial membranes has been studied using three stearic acid spin labels having a paramagnetic doxyl group in positions 5,12, and 16, respectively, and the fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS). The mobility of the spin labels in the phospholipid aliphatic chains increases from the polar heads toward the methyl groups both in vesicles and in mitochondrial membranes; however, in the latter there is a higher constriction of rotational mobility observed at all levels in the lipid bilayer. Butanol determines a moderate increase in mobility of phospholipids in lipid vesicles, but the effect is more striking in the mitochondrial membranes, where the protein-induced constraint of mobility of the fatty acyl chains is removed at low concentrations of the alcohol. Butanol also enhances the mobility of tightly bound phospholipids residual in lipid-depleted mitochondrial preparations, although higher concentrations of butanol are required for this effect. The effect of the series of aliphatic n -alcohols is related to their hydrophobicity. Alcohols induce a decrease of the fluorescence of ANS bound to both lipid vesicles and mitochondrial membranes. The fluorescence decrease is not the result of a decreased partition of ANS from the aqueous medium to the bilayer, but depends upon a change in the chromophore environment. Since no shift of the emission maximum is observed after alcohol addition, such a change must be ascribed to increased mobility of the probe, in accord with the spin label data. As for the spin label data, the effect of the series of aliphatic n -alcohols is related to their hydrophobicity; at difference with the electron spin resonance results, however, the effects are maximal for pure phospholipid vesicles. It is calculated that alcohols affect both the long-range interactions between phospholipids and proteins in mitochondrial membranes (as detected by spin labels) and the order of phospholipid bilayers near the glycerol region (as detected by ANS). The differences between the two kinds of probes may be related to their differing localization in the lipid bilayer.

This report documents changes in the physical state of synaptosomal particulate fractions (SYN) upon binding of S-100 protein, as monitored by spin labels. Studies were conducted on SYN labeled with either 5-doxylstearic acid or 16-doxylstearic acid, which probe the polar region and the hydrophobic core of the lipid bilayer, respectively. S-100 perturbs to some extent both the polar surface and the hydrophobic core of SYN in a time- and temperature-dependent manner. Ca 2+ is essential for S-100 to perturb the membranes. K + almost completely inhibits the S-100 perturbing effect if present in the incubation medium, but fails to reverse the S-100-induced changes if added after S-100 has interacted with SYN. At room temperature and below, the overall S-100 effect registered after about 30 min of association of the protein with SYN is an increase in the fluidity of both the surface and the interior of the membranes. Spectra registered at intervals at room temperature indicate that the S-100 perturbing effect on the membrane surface is practically monophasic, consisting of an increase in fluidity, while that on the membrane interior is multiphasic, consisting of a decrease in fluidity during the first 10 min of association, followed by an increase in fluidity during the subsequent 20 min and a return to starting values during the second 30 min of association. Around 37 °C, on the contrary, a decrease in fluidity is registered in both regions. The data suggest that S-100 induces a spatial rearrangement of membrane components (proteins) involved in the specific binding and/or partially penetrates into the lipid bilayer.

Experimental evidence of tryptophan incorporation inside dipalmitoyl-lecithin liposomes is reported. The thermodynamic and structural changes induced in the model membranes by tryptophan were investigated by using electron spin resonance (ESR), X-ray diffraction, calorimetry and fluorescence techniques. The amino acid interacts with the polar region of the membrane at low tryptophan concentrations, and with the hydrophobic interior of the membrane at the higher concentrations. The liposomes-tryptophan reciprocal effects are discussed as an example of perturbation induced by an exogenous molecule that penetrates the lipid bilayer. Moreover, they are relevant in relation to the study of lipid-protein interactions and for the possible use of liposomes as amino acid carriers.

Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene has been used to investigate the physical state of erythrocyte membranes from 36 obese children in absence of endocrine and metabolic disorders involving alterations of blood lipid pattern. A decrease of membrane fluidity, as shown by an increase of fluorescence polarization, was evident in obese subjects in comparison with the controls. The study of membrane composition has revealed an increase of cholesterol content and of cholesterol to phospholipid molar ratio, both being positively correlated with the observed changes of membrane fluidity. Moreover a correlation of membrane fluidity is also observed with body-mass index and plasma cholesterol levels. The results are discussed in the light of the possible use of erythrocyte membrane as a model to detect early alterations in the exchanges between erythrocyte membrane and lipoproteins in obesity.