Decreased membrane fluidity and altered susceptibility to peroxidation and lipid composition in overweight and obese female erythrocytes

Abstract

The increased generation of reactive oxygen species that occurs in the condition of obesity may be responsible for oxidative injury to erythrocyte membranes, which could lead to a decrease in tissue oxygenation. Therefore, we have looked into the effects of obesity on both indexes of oxidative damage and physical-chemical properties of erythrocyte membranes in 50 overweight or obese [25 < body mass index (BMI) < 33], normotensive, nondiabetic women and 50 age-matched lean healthy women (BMI < 25). In the obese group compared with the lean group, we found that a) the onset of free radical-induced erythrocyte hemolysis and the ratio between reduced and oxidized glutathione were reduced, whereas the rate of free radical-induced damage increased; b) the n-3 fatty acid and the phospholipid contents decreased; c) the ratio between cholesterol and phospholipids increased; and d) the membrane fluidity decreased.These findings suggest an impairment of erythrocyte membrane physical-chemical properties in overweight and obese people as a consequence of oxidative injury that might be part of a pathogenetic mechanism responsible for obesity-related pathologies such as atherosclerosis and hypertension. The increased generation of reactive oxygen species that occurs in the condition of obesity may be responsible for oxidative injury to erythrocyte membranes, which could lead to a decrease in tissue oxygenation. Therefore, we have looked into the effects of obesity on both indexes of oxidative damage and physical-chemical properties of erythrocyte membranes in 50 overweight or obese [25 < body mass index (BMI) < 33], normotensive, nondiabetic women and 50 age-matched lean healthy women (BMI < 25). In the obese group compared with the lean group, we found that a) the onset of free radical-induced erythrocyte hemolysis and the ratio between reduced and oxidized glutathione were reduced, whereas the rate of free radical-induced damage increased; b) the n-3 fatty acid and the phospholipid contents decreased; c) the ratio between cholesterol and phospholipids increased; and d) the membrane fluidity decreased. These findings suggest an impairment of erythrocyte membrane physical-chemical properties in overweight and obese people as a consequence of oxidative injury that might be part of a pathogenetic mechanism responsible for obesity-related pathologies such as atherosclerosis and hypertension. Physiological oxidative metabolism and neutrophil activation occurring in the blood give rise to oxygen-reactive substances and other very active radical species that can irreversibly damage the surrounding macromolecules. In particular, these radicals can attack both the amino and thiol groups of proteins and the double bonds of polyunsaturated fatty acids in lipoproteins. Statistically significant correlations have been found between lipoprotein susceptibility to peroxidation, the degree of obesity, and the risk of developing cardiovascular disease (1Dandona P. Mohanty P. Ghanim H. Aljada A. Browne R. Hamouda W. Prabhala A. Afzal A. Garg R. The suppressive effect of dietary restriction and weight loss in the obese on the generation of reactive oxygen species by leukocytes, lipid peroxidation, and protein carbonylation.J. Clin. Endocrinol. Metab. 2001; 86: 355-362Crossref PubMed Scopus (340) Google Scholar). Because any increase in the rate of lipoprotein peroxidation not only diminishes their levels of polyunsaturated fatty acids but also consumes and reduces their antioxidant contents (vitamin E, β-carotene, coenzyme Q, etc.), the consequence of these biochemical events in overweight and obese people is also a probable reduction of the “exchange rate” of both polyunsaturated fatty acids and lipophilic antioxidants that are normally transferred in the blood from the donor lipoproteins to the erythrocyte acceptor membranes. A decrease in both the degree of polyunsaturation of lipids and the antioxidant levels of the erythrocyte membrane could thus be expected, together with a decrease of both membrane fluidity and the activity of its membrane-bound enzymes. Because the erythrocyte membrane serves as a variable barrier to oxygen transport, the changes in its composition can induce cellular hypoxia in the tissue bed. Furthermore, because the size, shape, and diffusion capacity of a red blood cell depend on the structure of its membrane, alterations in membrane structure could lead to a decrease in tissue oxygenation (2Buchwald H. Menchaca H.J. Michalek V.N. Rohde T.D. Hunninghake D.B. O'Dea T.J. Plasma cholesterol: an influencing factor in red blood cell oxygen release and cellular oxygen availability.J. Am. Coll. Surg. 2000; 191: 490-497Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Such modifications of oxygen available in cardiovascular cells might be part of a pathogenetic mechanism responsible for obesity and obesity-related pathologies such as atherosclerosis and hypertension. To our knowledge, the physical-chemical properties of the erythrocyte membrane in overweight and obese people have not been thoroughly investigated. With the aim of providing more detailed information on these properties, we performed the following study in which we measured the susceptibility to peroxidation, the lipofuscin levels, the fluidity, the activity of ATPase and acetylcholinesterase, and the cholesterol and fatty acid compositions of the erythrocyte membrane in two adult female groups: overweight and obese patients and lean healthy subjects. The correlations between all of these indices of erythrocyte antioxidant status, the lipid profile, the activity of ATPase and acetylcholinesterase, membrane fluidity, and the most significant anthropometrics and chemical-clinical parameters were also calculated, and on this basis, their possible contributions to the etiopathogenesis of both obesity and obesity-related cardiovascular pathologies are discussed. The patients were 50 overweight [25 < body mass index (BMI) < 29] or obese (first level, moderate obesity: 30 < BMI < 33), normotensive, nondiabetic women. Other pathologies were excluded by clinical history and physical examination. Clinical data, alcohol intake, smoking habits, and physical activity were recorded. Thyroid-stimulating hormone, free thyroxine, free triiodothyronine, and free urinary cortisol levels were in the normal range in all patients. No subject was on a restricted diet. Fifty age-matched, lean healthy women (BMI < 25) were studied as a control group. The “Istituto Santa Margherita” Institutional Review Board approved the study protocol, and each subject signed a consent form that stated the purpose of the study and the sampling to be done. Blood samples were collected at rest and after a 12 h fasting period. Whole blood (EDTA as an anticoagulant) was used for hematological procedures and packed erythrocyte preparation. In this last case, blood was immediately centrifuged (3,000 g for 5 min at 4°C), plasma and buffy coat were removed by careful suction, and the cells were resuspended in a 154 mM NaCl, 50 mM EDTA solution. After mixing by inversion, the samples were centrifuged again at 1,500 g for 5 min at 4°C. This washing procedure was repeated twice. Packed erythrocytes were used for pro-oxidant and antioxidant status measurements and membrane isolation. White blood cell and red blood cell counts, total cholesterol, HDL cholesterol, triacylglycerol, glucose, protein, hemoglobin, and hematocrit levels were quantified by standard laboratory procedures. Free radical-induced hemolysis of erythrocytes was measured on packed erythrocytes resuspended in PBS at 10% hematocrit. Hemolysis was induced by incubating erythrocytes for 6 h at 37°C with 25 mM 2,2′-azobis-(amidinopropane)dihydrochloride (AAPH; Wako Pure Chemical Co.), and the time curve of hemoglobin release was determined by measuring the absorbance at 410 nm as previously described (3Zou C.G. Agar N.S. Jones G.L. Oxidative insult to human red blood cells induced by free radical initiator AAPH and its inhibition by commercial antioxidant mixture.Life Sci. 2001; 69: 75-86Crossref PubMed Scopus (110) Google Scholar). The kinetic profile of each erythrocyte sample, divided into latency, propagation, and steady-state phases, allows the evaluation of two important kinetic parameters: the lag time, expressed in minutes and calculated as the intercept of the linear regression of the propagation phase with that of the latency phase; and the rate of hemolysis in the propagation phase, calculated from the slope of the curve in this phase (Fig. 1). Reduced and oxidized glutathione levels were measured fluorometrically according to Hissin and Hilf (4Hissin P.J. Hilf R. A fluorimetric method for the determination of oxidized and reduced glutathione in tissues.Anal. Biochem. 1976; 74: 214-226Crossref PubMed Scopus (3703) Google Scholar). The erythrocyte membranes (ghosts) were prepared according to Raccah et al. (5Raccah D. Dadoun F. Coste T. Vague P. Decreased Na/K ATPase ouabain binding sites in red blood cells of patients with insulin-dependent diabetes and healthy north African control subjects: relationship with insult and diabetic neuropathy.Horm. Metab. Res. 1996; 28: 128-132Crossref PubMed Scopus (11) Google Scholar). Membrane proteins were determined according to Lowry et al. (6Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), and multiple fractions of 0.2 ml were immediately frozen in liquid nitrogen and stored at −80°C until use. Erythrocyte membranes partially depleted of their cholesterol content were prepared by ghost incubation with β-cyclodextrins according to the method by Ohtani et al. (7Othani Y. Irie T. Ubkama K. Fukunaga K. Pitha J. Differential effects of α-, β- and γ-cyclodextrins on human erythrocytes.Eur. J. Biochem. 1989; 186: 17-22Crossref PubMed Scopus (545) Google Scholar). Free radical-induced ghost peroxidation was promoted by incubating erythrocyte membranes for different times at 37°C with 25 mM AAPH. Erythrocyte membrane fatty acid composition was determined on lipids extracted in 2:1 chloroform-methanol (Merck, Darmstadt, Germany) containing 0.2% butylated hydroxytoluene (Merck) according to the method of Folch, Lees, and Sloane Stanley (8Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The lipid extracts were methylated with methanolic HCl (Merck) at 90°C for 2 h. Fatty acid methyl esters were analyzed using capillary gas chromatography (GC). The GC apparatus (Hewlett Packard 6890 series equipped with autoinjector) was used in split mode (ratio 50:1) with hydrogen as the carrier gas. The analytical column was a fused silica capillary column (BPX70; length 50 m, internal diameter 0.25 mm, film thickness 0.25 μm; SGE Europe Ltd.). The GC autoinjector temperature was 250°C. The GC temperature program was as follows: initial temperature of 150°C for 3 min, increase of 5°C per min to 210°C, and a run time of 60 min. The fatty acid methyl esters were identified according to their retention times in comparison with known standards (Sigma-Aldrich, Dorset, UK) and were quantified by using heptadecanoic acid (Sigma-Aldrich) as the internal standard. The amount of each considered fatty acid was calculated as nanomoles per milligram of protein and expressed as a percentage of the total fatty acid concentration. The degree of unsaturation of erythrocyte membrane (unsaturation index) was calculated as the sum of each unsaturated fatty acid concentration multiplied by its double bond number and divided by the total unsaturated fatty acid concentration. Vitamin E levels were determined by measuring the ghost α-tocopherol content by HPLC (9Viani P. Cazzola R. Cervato G. Gatti P. Cestaro B. Pyrene lipids as markers of peroxidative processes in different regions of low and high density lipoproteins.Biochim. Biophys. Acta. 1996; 1315: 78-86Crossref PubMed Scopus (20) Google Scholar). Total phospholipids and cholesterol of native and peroxidized ghosts were measured according to the method of Alcindor et al. (10Alcindor L.G. Kadra M. Antebi H. Guerrito M.J. Piot M.C. Heraud G. A rapid assay for cholesterol and choline phospholipids in erythrocyte membranes.in: Siest G. Perspective Biology. J. Libbey Eurotext, Montrouge, France1989: 181-184Google Scholar). Lipofuscins, a cross-linked complex of proteins and lipid peroxidation products, were measured fluorometrically on native and peroxidized ghosts as previously described (11Dillard C.J. Tappel A.L. Fluorescent damage products of lipid peroxidation.Methods Enzymol. 1984; 105: 337-341Crossref PubMed Scopus (146) Google Scholar). The Na,K ATPase activity was measured as the ouabain-inhibitable inorganic phosphorus released after incubation of membranes with ATP (5Raccah D. Dadoun F. Coste T. Vague P. Decreased Na/K ATPase ouabain binding sites in red blood cells of patients with insulin-dependent diabetes and healthy north African control subjects: relationship with insult and diabetic neuropathy.Horm. Metab. Res. 1996; 28: 128-132Crossref PubMed Scopus (11) Google Scholar). The acetylcholinesterase activity was determined according to Vander Jagt et al. (12Vander Jagt D.L. Intress C. Heidrich J.E. Mrema J.E. Rieckmann K.H. Heidrich H.G. Marker enzymes of Plasmodium falciparum and human erythrocytes as indicator of parasite purity.J. Parasitol. 1982; 68: 1068-1071Crossref PubMed Scopus (38) Google Scholar). Membrane fluidity was evaluated by measuring the steady-state anisotropy of diphenylhexatriene (Molecular Probes Europe BV, Leiden, The Netherlands) as previously described (13Cazzola R. Cervato G. Cestaro B. of oxidative erythrocyte membrane fluidity and antioxidant status in and J. Clin. PubMed Scopus Google Scholar). The diphenylhexatriene was at a of and the was at were with and the of and was was calculated with the and thickness of were measured between and after an weight was measured on a and was measured using a BMI was calculated by weight by were measured with a at 5 min at each using a standards of weight and body composition by and for of status of and the J. Clin. 1984; PubMed Scopus Google Scholar). measurements were at in and are as between measurements were by a The linear between the were using The of was at in the in and plasma red blood total and total protein levels between the group of obese and overweight women and the group of control were not the BMI and all indices of body and were in the group of overweight and obese and parameters of the study 25 BMI blood cells blood cells cholesterol body mass in a body mass The levels of hemoglobin and hematocrit were whereas the of in the and were in the group of overweight and obese women compared with the control group. The parameters of erythrocyte membrane susceptibility to peroxidation lag time, of the total amount of and the of the rate of free radical-induced membrane together with the lipofuscin levels of the between the different and the ratio between reduced and oxidized glutathione of the status of the erythrocyte are in The slope and the lipofuscin levels a significant to increase in the group of overweight and obese women. the the lag time and the ratio were in this group of pro-oxidant and 25 BMI time 154 glutathione ratio and the levels of were determined on native The lag time and the slope were calculated from the kinetic of 2,2′-azobis-(amidinopropane)dihydrochloride hemolysis as described in and in a The glutathione ratio and the levels of were determined on native The lag time and the slope were calculated from the kinetic of 2,2′-azobis-(amidinopropane)dihydrochloride hemolysis as described in and The lipofuscin levels were with the BMI < and the of and whereas the lag time was with the of the and The total of cholesterol and phospholipids in the native and peroxidized membranes in both obese and control women are in The peroxidation promoted a significant decrease in the of phospholipids < and cholesterol < in both In the native and peroxidized membranes, the ratio was in the obese in the control and cholesterol contents of native and peroxidized erythrocyte 25 BMI membranes were peroxidized by incubation at 37°C with 25 mM AAPH for 3 h. were peroxidized by incubation at 37°C with 25 mM AAPH for 3 h. in a The ratio between cholesterol and phospholipids of the native membrane was with the lipofuscin levels < the BMI < and the thickness of the and and was with the lag time < To the of cholesterol on the of peroxidation, the obese ghosts were with a method for the ghost cholesterol content (7Othani Y. Irie T. Ubkama K. Fukunaga K. Pitha J. Differential effects of α-, β- and γ-cyclodextrins on human erythrocytes.Eur. J. Biochem. 1989; 186: 17-22Crossref PubMed Scopus (545) Google Scholar). The ratio was reduced from to by this The native and ghosts were to peroxidation for 30 min. The lipofuscin content after peroxidation was and protein in the native and the percentage levels of the different fatty acids and together with the E ratio in the native erythrocyte The most significant findings from a detailed of these are as acid of native erythrocyte 25 BMI index calculated as the sum of each unsaturated fatty acid concentration multiplied by its double bond number and divided by the total unsaturated fatty acid fatty acid contents are expressed as index calculated as the sum of each unsaturated fatty acid concentration multiplied by its double bond number and divided by the total unsaturated fatty acid concentration. in a The fatty acid contents are expressed as a) In the group, was a significant decrease in the percentage of fatty acids and fatty on the the fatty acids and acids to be Furthermore, in the obese group, the of and acids were b) The sum of all polyunsaturated fatty acids to be but this decrease is to the decrease of the species with the degree of and acids double bonds per and acid double bonds per The significant reduction of the membrane unsaturation index is to these c) The ratio between the and fatty acids particular, between the most polyunsaturated and acid and was in the group. d) The ratio between the total polyunsaturated fatty acids and E was in the group, but not The index of which is to membrane fluidity, and the activity of Na,K ATPase and acetylcholinesterase are in The were the ATPase a to and the acetylcholinesterase in the anisotropy and of Na,K ATPase and acetylcholinesterase of native erythrocyte 25 BMI ATPase in a The were with the native lipofuscin levels the BMI and the thickness of the and The a susceptibility to peroxidation, together with a ratio between cholesterol and phospholipids and a significant increase in the cholesterol content of the erythrocyte membranes of overweight and obese patients in comparison with the normal weight control group. The levels of cholesterol may be of the possible for the pro-oxidant status in the erythrocyte cholesterol been in to rise to a of and J. M. T. Y. Y. determination of cholesterol in human erythrocyte PubMed Scopus Google Scholar). In the cholesterol contents of leukocytes, and cells have been to and this increase been to the of oxygen free radicals A. plasma and tissue cholesterol levels in for the of cholesterol in Full Text PDF PubMed Scopus Google B. R.J. and of cholesterol Clin. PubMed Scopus Google Scholar). in particular, because of their for free radical generation to their very high oxygen might be a very for cholesterol to its pro-oxidant of changes in PubMed Scopus Google Scholar). the membranes of the obese women were in part depleted of their cholesterol by of a with the their susceptibility to peroxidation was possible for the pro-oxidant status of the erythrocyte membrane in overweight and obese patients is the increased susceptibility of lipoproteins to peroxidation previously in these patients A. M. A. A. susceptibility of to in in patients with insulin-dependent and diabetes Res. Google is an risk factor for plasma lipid peroxidation and of erythrocyte enzymes in J. Metab. PubMed Scopus Google this lipoprotein pro-oxidant an increase of both lipid and radicals are in the of peroxidative and a decrease of lipophilic such as E, β-carotene, coenzyme Q, Because lipid radicals and together with lipophilic are normally transferred from donor lipoproteins to the acceptor erythrocyte membrane, we can in overweight and obese women is that a amount of lipid radicals and a amount of antioxidants could be between plasma lipoproteins and erythrocyte membranes, thus to an increase in erythrocyte susceptibility to A consequence of these pro-oxidant events is the significant decrease that we found both in erythrocyte membrane unsaturation index to a reduction of fatty which are very to the peroxidative and in erythrocyte levels of reduced glutathione is more in the of the peroxidation the peroxidation of polyunsaturated fatty the peroxidation of the polyunsaturated fatty to increase the of as which are known to bonds between membrane proteins and The cross-linked are the probable of the of that we found in the group. is a of that the erythrocyte membrane susceptibility to peroxidation in overweight and obese women with the significant of the anisotropy of a more status of the that we measured in this a) The content in the membrane of the polyunsaturated fatty which to the membrane in a more b) The of all of the parameters of the erythrocyte antioxidant the reduced of lag time, of the reduced levels of lipophilic antioxidant in the membrane, and the decrease of reduced of the status of antioxidant in the that normally to the of peroxidation of more polyunsaturated fatty c) The of which to both the of and the of both membrane proteins and phospholipids in these d) The increase in the ratio between cholesterol and which is known as a to the membrane in a more is important to that the of acid fatty acid normally only in was in the obese group. This to the found in the is in that are a of the membrane H. M. P. is to erythrocyte membrane fluidity changes in obese nondiabetic Clin. Endocrinol. Metab. 1996; Google Scholar). This decrease in erythrocyte membrane fluidity in overweight and obese women could thus to the rate of blood in the and the oxygen diffusion the erythrocyte membrane and its with is known that cholesterol content in the erythrocyte membrane and the decrease of its fluidity both the oxygen release and the of the red blood cells (2Buchwald H. Menchaca H.J. Michalek V.N. Rohde T.D. Hunninghake D.B. O'Dea T.J. Plasma cholesterol: an influencing factor in red blood cell oxygen release and cellular oxygen availability.J. Am. Coll. Surg. 2000; 191: 490-497Abstract Full Text Full Text PDF PubMed Scopus (27) Google D. F. F. Membrane fluidity and oxygen diffusion in erythrocyte Biochem. Biophys. PubMed Scopus Google in erythrocyte for oxygen M.C. to Scopus Google Scholar). The reduced of oxygen could thus to both the metabolism of and fatty acid and the in and the both in and cells to the of all of the cardiovascular pathologies that are with this decrease in membrane fluidity promoted by the pro-oxidant status in erythrocytes could be the of a more other as in particular, a significant between membrane fluidity and been both in diabetes P. T. T. D. M. M. R. membrane and in diabetes PubMed Scopus Google and in obese patients H. M. P. is to erythrocyte membrane fluidity changes in obese nondiabetic Clin. Endocrinol. Metab. 1996; Google Scholar). In the of data, could be of to or not a dietary of fatty acids and antioxidants could significant effects for the of the fluidity of erythrocyte membranes in and all other in and this could be of in the of both obesity and all of the other cardiovascular with