Active Forces of Myosin Motors May Control Endovesiculation of Red Blood Cells.

By using Monte Carlo (MC) simulations, we have shown that the active forces generated by (NMIIA) motor domains bound to F-actin may partially control the endovesiculation of the red blood cell (RBC) membrane. The myosin generated active forces favor pancake-like (torocyte) RBC endovesicles with a large flat central membrane region and a bulby periphery. We suggest that the myosin generated active forces acting on the RBC membrane in the direction perpendicular to the membrane surface towards the interior of the RBC may influence also other RBC shape transformations and the stability of different types of RBC shapes and should be therefore considered in the future theoretical studies of the RBC vesiculation and shape transformations.

Fatty acids derived from a food frequency questionnaire and measured in the erythrocyte membrane in relation to adiponectin and leptin concentrations.

BACKGROUND/OBJECTIVES: Evidence on the association between fatty acids and adiponectin and leptin concentrations is scarce and inconsistent, which may in part be due to limitations of dietary reporting methods. We aimed to estimate the association of fatty acids, derived from a food frequency questionnaire (FFQ) and measured in the erythrocyte membrane, with adiponectin and leptin concentrations. SUBJECTS/METHODS: We studied 330 non-institutionalized inhabitants of Porto (52.4% women; age range: 26-64 years) evaluated in 2010-2011, as part of the EPIPorto cohort study. Fatty acids were derived from a validated semiquantitative FFQ and measured in the erythrocyte membrane by gas chromatography. Serum concentrations of adiponectin and leptin were determined through radioimmunoassay. Regression coefficients (ß) and 95% confidence intervals (95% CI) were obtained from linear regression models, after controlling for gender, age, education, leisure time physical activity and total body fat percentage (obtained from dual energy X-ray absorptiometry). RESULTS: Fatty acids measured by FFQ showed no significant associations with both adipokines. Lauric and linoleic acids, measured in the erythrocyte membrane, were significantly and positively associated with adiponectin (ß=0.292, 95% CI: 0.168-0.416; ß=0.150, 95% CI: 0.020-0.280) and leptin (ß=0.071, 95% CI: 0.003-0.138; ß=0.071, 95% CI: 0.002-0.140), whereas total n-3, eicosapentaenoic and docosahexaenoic acids were significantly but negatively associated with adiponectin (ß=-0.289, 95% CI: -0.420 to -0.159; ß=-0.174, 95% CI -0.307 to -0.040; ß=-0.253, 95% CI -0.383 to -0.124) and leptin (ß=-0.151, 95% CI: -0.220 to -0.083; ß=-0.080, 95% CI: -0.151 to -0.009; ß=-0.146, 95% CI: -0.214 to -0.078). Positive significant associations of palmitic and trans-fatty acids with adiponectin were also observed. CONCLUSIONS: A positive association of lauric and linoleic acids and a negative association of total n-3 fatty acids with both adipokines were observed only with fatty acids measured in the erythrocyte membrane.

Association of marine-origin n-3 polyunsaturated fatty acids consumption and functional mobility in the community-dwelling oldest old.

OBJECTIVE: To examine whether habitual dietary intake of marine-origin n-3 polyunsaturated fatty acids (MOPUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are associated with functional mobility in the community-dwelling oldest old, 85 years or older, who are at high risk for physical disability. DESIGN: A cross-sectional study. SETTING: A community-based survey conducted at university research center or home-based. PARTICIPANTS: Four hundred seventeen (189 men, 228 women) out of 542 participants in the baseline examination of the Tokyo Oldest Old Survey on Total Health, a community-based ongoing longitudinal study among the oldest old living in the center of Japan. MEASUREMENTS: Habitual dietary intake of MOPUFA was assessed by the brief-type self-administered diet history questionnaire (BDHQ), and functional mobility was assessed by the timed up and go test. Plasma inflammatory biomarkers (C-reactive protein, interleukin-6 and tumor necrosis factor-α) were measured. We evaluated the cross-sectional association between habitual intake of MOPUFA and functional mobility using multivariate logistic regression analysis. Prior to the analysis, validation of BDHQ in this study was confirmed among 190 participants (96 men, 94 women) based on the EPA and DHA concentrations in the erythrocyte membrane phospholipids as reference. RESULTS: Moderate correlation between estimated dietary intake of EPA/DHA and concentration of EPA/DHA in the erythrocyte membrane phospholipids was obtained (Spearman's r=0.29-0.58, p<0.01). Multivariate logistic regression analysis revealed that a lower habitual intake of EPA+DHA was significantly associated with poor functional mobility in men but not in women (OR (95% CI) per 1 SD increase of EPA+DHA intake; 0.55 (0.33-0.91), 0.88 (0.59-1.32), men and women respectively). CONCLUSIONS: Habitual intake of MOPUFA was associated with functional mobility in community-dwelling oldest old men.

[Assessment of some biophysical parameters of human erythrocyte by FT-IR spectroscopy--preliminary study]. / Evaluarea unor parametri biofizici ai eritrocitului uman prin spectroscopie FT-IR--studiu preliminar.

UNLABELLED: Based on the biochemical composition of erythrocyte membrane and asymmetric distribution of phospholipids, proteins, and cholesterol, FT-IR spectroscopy can monitor the distribution and interaction pattern of membrane constituents. MATERIAL AND METHODS: The study series included 21 clinically healthy subjects aged between 20 and 60 years. Complete blood counts were performed and the serum biochemical compounds (cholesterol, triglycerides, total protein, glucose) were determined. RESULTS AND DISCUSSIONS: The parameters that can be assessed by erythrocyte FT-IR in relation with the biochemical factors that may influence membrane fluidity are: degree of fatty acids unsaturation, saturated fatty acids/unsaturated fatty acids ratio, cholesterol/phospholipids ratio, and phospholipids/protein ratio. Based on the obtained results, in the assessment of membrane status the following vibration modes were selected as spectral parameters: vibration associated valence bond (=CH), asymmetric valence CH2 groups, modes associated with P=O bond and amine bands I and II specific for proteins. Other parameters, such as v(C-O) specific to glucose, may provide additional information on glucose metabolic status. CONCLUSIONS: By correlating biochemical markers with these spectral parameters information on red cell membrane status, status that may reflect different pathological processes, can be obtained.

[Computer assessment of membrane structure in various erythrocyte forms].

The photometric computer image analysis method is described. It is based on the creation of the gallery of the images of various erythrocyte forms (discocytes, ecchinocytes, target cells and degenerative forms). Using the Bio Vision program, the structure of membranes of each type of erythrocytes was studied. It was found that the morpho-functional changes of erythrocytes of various degrees were accompanied by the alterations in the relative content of condensed membrane protein-lipid complexes.

Cytoskeleton mediated effective elastic properties of model red blood cell membranes.

The plasma membrane of human red blood cells consists of a lipid bilayer attached to a regular network of underlying cytoskeletal polymers. We model this system at a dynamic coarse-grained level, treating the bilayer as an elastic sheet and the cytoskeletal network as a series of phantom entropic springs. In contrast to prior simulation efforts, we explicitly account for dynamics of the cytoskeletal network, both via motion of the protein anchors that attach the cytoskeleton to the bilayer and through breaking and reconnection of individual cytoskeletal filaments. Simulation results are explained in the context of a simple mean field percolation model and comparison is made to experimental measurements of red blood cell fluctuation amplitudes.

Assessment of erythrocyte phospholipid fatty acid composition as a biomarker for dietary MUFA, PUFA or saturated fatty acid intake in a controlled cross-over intervention trial.

BACKGROUND: Dietary intervention trials rely on self-reported measures of intake for assessment of energy and macronutrient composition. Dietary fat intake is of particular interest due to strong associations with pathophysiology. In epidemiological trials phospholipid fatty acid composition may reflect composition of habitual diet, although strong correlations have been identified only for essential polyunsaturated fatty acids (PUFAs). Preliminary evidence shows that saturated fatty acids (SFA) C15:0 and C17:0 may be acceptable biomarkers. This study measured changes in erythrocyte membrane fatty acids during a period of strictly controlled fat feeding to investigate their use as a short-term marker of compliance, particularly for intake of SFAs. RESULTS: This was a randomised cross-over trial in which diet was provided and strictly controlled. 20 healthy, male subjects were given a 40 energy % (en%) fat diet, high in saturated (high-SFA, 20 en%) or unsaturated (high-USFA, 24 en%) fatty acids for 2 periods of 3 weeks. Subjects were residential during intervention with all food and beverages provided. Dietary composition was verified by direct chemical analysis. Blood samples were collected on days 1,7,14, 21 and analysed for red blood cell (RBC) membrane fatty acid composition. Pearson correlation showed RBC fatty acid composition to mimic dietary composition by 3 weeks, but the relationships were weak. Of the SFAs only RBC C16:0 decreased in response to decreased dietary content on high-USFA treatment (ANOVA, diet, P < 0.05). Of the USFAs, higher levels of C18:1 MUFA, C20:4 and C22:6 long chain PUFA on high-USFA diet lead to higher C18:1, C20:4 and C22:6 within RBCs (ANOVA, time*diet, P < 0.05). Pearson's correlation was significant between dietary and RBC fatty acids during the 21d dietary manipulation for C18:1, and C20:5, C22:6 only (P < 0.05). CONCLUSION: RBC membrane fatty acids cannot reliably be used as an independent measure of compliance for dietary SFA intake in short-term studies. The MUFA oleic acid and PUFAs EPA and DHA may be more useful as markers of compliance during short term intervention trials.

Factors influencing zinc status of apparently healthy indians.

OBJECTIVES: To identify dietary, environmental and socio-economic factors associated with mild zinc deficiency, three zinc status indices; erythrocyte membrane zinc (RBCMZn), plasma zinc and super oxide dismutase (SOD) were assessed in free living and apparently healthy Indian population. METHODS: Dietary patterns of 232 men and 223 women (20-65 yr) from rural, industrial and urban regions of Western India were evaluated by food frequency questionnaire. RBCMZn was estimated using atomic absorption spectrometry, hemoglobin and serum ceruloplasmin by spectrophotometer. On a sub sample (48 men and 51 women) plasma zinc and SOD were also assessed. RESULTS: Mean RBCMZn was 0.5 +/- 0.1 micromols/g protein with 46% individuals showing zinc deficiency. Mean plasma zinc was 0.98 +/- 0.12 microg/mL with 25% men and 2.5% women having values below normal range. Mean SOD was 0.97 +/- 0.1 (u/mL cells). A significant positive correlation was observed between intakes of green leafy vegetables, other vegetables and milk products with RBCMZn status (p < 0.05). But these were not correlated with plasma zinc (p > 0.2). Cereal and legume intakes were negatively correlated with RBCMZn (p < 0.05) but positively correlated with plasma zinc (p < 0.05) and not correlated with SOD (p > 0.2). Fruit and other vegetable intake were positively correlated with SOD (p < 0.05) alone. Logistic regression analyses revealed that RBCMZn was positively associated with intakes of beta-carotene, zinc and environmental conditions and negatively associated with family size (p < 0.05). Plasma zinc indicated positive association with zinc, thiamin and riboflavin intakes (p < 0.05) and SOD showed negative association with iron and family size. CONCLUSION: RBCMZn was a more sensitive indicator of long-term zinc status than plasma zinc and SOD. Prominent determinants of zinc status were intakes of beta-carotene and zinc along with environmental conditions and family size.

Spontaneous transfer of phospholipid and cholesterol hydroperoxides between cell membranes and low-density lipoprotein: assessment of reaction kinetics and prooxidant effects.

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH > 5 alpha-OOH > 6 alpha-OOH > 6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOH > SMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.

In situ assessment of erythrocyte membrane properties during cold storage.

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.

Erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans.

During a controlled zinc depletion-repletion study, fifteen men aged 25.3 (SD 3.3) years were fed on a low-Zn diet with high phytate:Zn and phytate x calcium:Zn molar ratios for 7 weeks, followed by a 2 week repletion period when 30 mg supplemental Zn/d was given. Changes in plasma, urine, and hair Zn concentrations, taste acuity, and cellular immune response confirmed the development of mild Zn deficiency. Zn concentrations in neutrophils, platelets, erythrocytes and erythrocyte membranes, mean platelet volume, and activities of alkaline phosphatase (EC 3.1.3.1) and alpha-D-mannosidase (EC 3.2.1.24) in neutrophils did not respond to changes in Zn status. In contrast, alkaline phosphatase activity in erythrocyte membranes showed a significant decline which was consistent in all subjects (nmol product formed/min per mg protein; baseline v. 7-week Zn depletion, 0.656 (SD 0.279) v. 0.506 (SD 0.230), at 7 weeks; P < 0.05); neutral phosphatase activity remained unchanged. Alkaline phosphatase activity in erythrocyte membranes may be a potential index of Zn status in humans.