Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients

  Citation: Sot, J.; García-Arribas, A.B.; Abad, B.; Arranz, S.; Portune, K.; Andrade, F.; Martín-Nieto, A.; Velasco, O.; Arana, E.; Tueros, I.; et al. Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients. Int. J. Mol. Sci. 2022,23, 1920. https:// doi.org/10.3390/ijms23031920 Academic Editor: Orfeo Sbaizero Received: 21 January 2022 Accepted: 6 February 2022 Published: 8 February 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Erythrocyte Membrane Nanomechanical Rigidity Is Decreased in Obese Patients † Jesús Sot 1,‡ , Aritz B. García-Arribas 1,‡ , Beatriz Abad 2, Sara Arranz 3, Kevin Portune 3, Fernando Andrade 4, Alicia Martín-Nieto 4, Olaia Velasco 4, Eunate Arana 4, Itziar Tueros 3, Carla Ferreri 5, Sonia Gaztambide 4, Félix M. Goñi 1, Luis Castaño 4and Alicia Alonso 1,* 1Instituto BIOFISIKA (CSIC, UPV/EHU), Departamento de Bioquímica, Universidad del País Vasco, 48940 Leioa, Spain; [email protected] (J.S.); [email protected] (A.B.G.-A.); [email protected] (F.M.G.) 2SGIKER, Servicios Generales de Investigación (SGiker), Universidad del País Vasco, 48940 Leioa, Spain; [email protected] 3AZTI, Food Research, Basque Research and Technology Alliance (BRTA), Parque Tecnológico de Bizkaia, Astondo Bidea, Edificio 609, 48160 Derio, Spain; [email protected] (S.A.); [email protected] (K.P.); [email protected] (I.T.) 4Biocruces Bizkaia, Hospital Universitario Cruces, CIBERDEM, CIBERER, Endo-ERN, UPV-EHU, 48903 Barakaldo, Spain; [email protected] (F.A.); [email protected] (A.M.-N.); [email protected] (O.V.); [email protected] (E.A.); [email protected] (S.G.); [email protected] (L.C.) 5 ISOF, Consiglio Nazionale delle Ricerche, Via Piero Gobetti, 101, 40129 Bologna, Italy; [email protected].it *Correspondence: [email protected] † This research is a partial result of the consortium OBINTER, constituted by: BIOFISIKA (Leioa, España) F. M. Goñi, A. Alonso, A. B. García-Arribas, B. G. Monasterio, Y. Varela, M. Iriondo, J. Sot, A. Marcos, E. J. González-Ramírez, A. Etxaniz; AZTI (Derio, España) S. Arranz, I. Tueros, K. Escudero, G. Marrugat, K. Portune, I. Jauregibeitia, I. Saez, A. García, N. Egurrola, M. J. Sierra; BIOCRUCES BIZKAIA, Hospital Universitario Cruces, CIBERDEM, CIBERER, Endo-ERN, UPV-EHU (Barakaldo, España) M. S. Gaztambide, L. A. Castaño, F. Andrade, O. Velasco, A. Martín-Nieto, E. Arana; UPV/EHU (Vitoria, España) A. Fernández-Quintela, M.P. Portillo, N. Kajarabille, A. Leniz; VICOMTECH (Donostia, España) G. Epelde, R. Álvarez, G. Artola, N. Larburu, J. Torres. ‡ These authors contributed equally to this work. Abstract: This work intends to describe the physical properties of red blood cell (RBC) membranes in obese adults. The hypothesis driving this research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. Forty-nine control volunteers (16 male, 33 female, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m 2 , respectively) and 52 obese subjects (16 male and 36 female, BMI 38.2 ± 11.0 and 40.7 ± 8.7 kg/m 2 , respectively) were examined. The two physical techniques applied were atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces, and fluorescence anisotropy of trimethylammonium diphenylhexatriene (TMA-DPH), which provides information on lipid order at the membrane polar–nonpolar interface. These techniques, in combination with lipidomic studies, revealed a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. Lipidomic data show an increase in the cholesterol/phospholipid mole ratio and a decrease in sphingomyelin contents in obese membranes. ω -3 fatty acids (e.g., docosahexaenoic acid) appear to be less prevalent in obese patient RBCs, and this is the case for both the global fatty acid distribution and for the individual major lipids in the membrane phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS). Moreover, some ω -6 fatty acids (e.g., arachidonic acid) are increased in obese patient RBCs. The switch from ω -3 to ω -6 lipids in obese subjects could be a major factor explaining the higher interfacial fluidity in obese patient RBC membranes. Int. J. Mol. Sci. 2022,23, 1920. https://doi.org/10.3390/ijms23031920 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 1920 2 of 19 Keywords: obesity; cell membrane physical properties; membrane fluidity; fluorescence polarization; atomic force microscopy; membrane breakthrough force; lipidomics 1. Introduction Obesity is defined by the World Health Organization as abnormal or excessive fat accumulation that presents a risk to health. More than 2.1 billion people—nearly 30 percent of the global population—are overweight or obese according to updated data from NCDRisC 2017 [ 1 ]. Initiatives to curb this pandemic include actions toward maternal, infant and young child nutrition, as well as strategies on diet and physical exercise. A rational approach to improve this major form of disease should include, together with the epidemiological and educational activities, an improved understanding of the underlying molecular mechanisms. According to the Endocrine Society, there is “growing evidence suggesting that obesity is a disorder of the energy homeostasis system, rather than simply arising from the passive accumulation of excess weight” [ 2 ]. Lipids, or fats, are the molecules with the highest energetic content in the biosphere, thus it is understandable that they have been the target of many dietary interventions and of as many metabolic studies on obesity in the past [3]. Among lipids, triglycerides are by far the most abundant class of lipids in the human body, making up the bulk of the so-called “body fat” [ 4 ]. They are also the main lipids in food, including vegetable oils [ 5 ]. Membrane lipids, mainly phospholipids and cholesterol, constitute a small fraction of the human body lipids. Cholesterol and its oxidation products are well-known pathogenic agents in cardiovascular disease, but they also serve as essential membrane lipids and metabolites [ 6 , 7 ]. The most abundant plasma membrane lipids are phospholipids, organized in a double layer, or bilayer, that constitutes the membrane matrix [ 8 ]. Based on the observation that the composition of fatty acid residues in phospholipids is typical for each tissue [ 9 ], attention to the membrane lipidome has increased in health and disease [10], also in connection with the “membrane lipid therapy” [11]. Research in obesity has concerned mostly blood plasma, focusing on the circulating lipids and lipoproteins [ 12 – 14 ]. Compared with the massive amount of investigations on triglycerides and obesity, relatively few studies have dealt with membrane phospholipids in body weight alterations. The main hypothesis driving our research is that obesity, in addition to increasing the amount of body fat, will also modify the lipid composition of membranes in cells other than adipocytes. A few studies have dealt with this subject in the past. Pan et al. [ 15 ] observed an increased ∆ 9-desaturase (thus enhanced fatty acid unsaturation) and a lowered ∆ 5-desaturase (thus decreased synthesis of polyunsaturated fatty acids, or PUFA) in obese patients. Min et al. [ 16 ], in red blood cells (RBC) from patients with gestational diabetes, often linked to obesity, described a decrease in the phospholipid phosphatidylethanolamine (PE) and arachidonic (20:4, AA) and docosahexaenoic (22:6, DHA) acids. Cazzola et al. [ 17 ] reported on an increase in the cholesterol/phospholipid ratio in RBC membranes from obese patients, together with a decrease in ω -3 fatty acids (e.g., DHA) and an increase in ω -6 (e.g., AA). In a detailed study of twin pairs discordant for obesity, Pietiläinen et al. [ 18 ] found increased proportions of palmitoleic acid (16:1) and AA, together with increased levels of ethanolamine plasmalogens in adipose tissue of the obese twins. More recently, studies from this consortium have found that, in obese child RBC membranes, AA as well as saturated and trans-unsaturated fatty acyl chains were increased [ 19 ]. In a parallel study, AA was increased and monounsaturated chains were decreased in obese children [20]. Fatty acids can change membrane biophysical properties [ 21 ], thus influencing membraneassociated processes like protein–lipid interactions, enzymatic activity, and regulation of surface receptors [ 22 , 23 ]; therefore, it is significant to foster an interdisciplinary approach to understand the relationship between disease-induced membrane lipidome composition and properties. The present contribution deals with the physical properties of RBC mem- Int. J. Mol. Sci. 2022,23, 1920 3 of 19 branes in normal and obese adults. The two main techniques applied are atomic force microscopy (AFM) in the force spectroscopy mode, which allows the micromechanical measurement of penetration forces [ 24 , 25 ], and fluorescence anisotropy, which provides information on membrane lipid order [ 26 ]. The probe used in fluorescence studies has been trimethylammonium diphenylhexatriene (TMA-DPH) that, in contrast to the more commonly used DPH, becomes located and provides information on the interfacial bilayer region between the phospholipid hydrophobic chains and polar head groups [ 27 ]. The combination of nanomechanical, spectroscopic, and lipidomic studies reveals a decreased rigidity in the interfacial region of the RBC membranes of obese as compared to control patients, related to parallel changes in lipid composition. 2. Results 2.1. Patient Recruitment and Anthropometry A total of 101 volunteers were recruited through a public call. Details can be found in Table 1. Forty-nine control volunteers (16 male and 33 female, mean age 30.2 and 34.5 years, BMI 21.8 ± 5.6 and 21.5 ± 4.2 kg/m 2 , respectively) and 51 obese subjects (16 male and 36 female, mean age 48 and 45.8 years, BMI 38.2 ± 11.0 and 40.7 ±8.7 kg/m2 , respectively) were examined. The study protocol was approved by the Basque Country Clinical Research Ethics Committee (permission number PI2019219) and carried out according to the Declaration of Helsinki Good Clinical Practice guidelines. Subjects under study were included after acceptance to participate in the study and signing of informed consent. Table 1. Physical characteristics of obese and control subjects (mean ±SD). Sex Age (yr) BMI (kg ×m−2) Control (49) 16 M 30.2 ±13.2 21.8 ±5.6 33 F 34.5 ±14.5 21.5 ±4.2 Obese (52) 16 M 48.0 ±13.0 38.2 ±11.0 36 F 45.8 ±12.6 40.7 ±8.7 2.2. Obese Patient RBC Membranes Exhibit Lower Nanomechanical Rigidity under the Atomic Force Microscope Atomic Force Microscopy (AFM) is a powerful tool that allows, in the force spectroscopy mode, direct measurements of mechanical resistance of any surface when the tip approaches it with a definite force, a process called ‘indentation’. In this case, given the small thickness of RBCs (~1 µ m), our approach was to fully pierce through the cells by performing a maximum force of approximately 10–20 nN. Thus, we were able to measure and quantify membrane breakthrough events for the RBC membrane, with each curve typically showing two different sequential rupture events as the cell was completely pierced (an example can be seen in Figure 1), as mentioned in the Materials and Methods section. Results shown in Figure 2for both control and obese patient RBC indicate a significant decrease in RBC stiffness for obese patients. Nanomechanical resistance values were 6.38 ±1.45 nN for control RBC and 5.47 ±1.19 nN for obese patient RBC (p= 0.03). Int. J. Mol. Sci. 2022,23, 1920 4 of 19 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 19 Figure 1. Representative AFM force–distance curve of an RBC. The AFM tip performs an indentation process on a supported RBC, initiated from X = 0 along the red line (trace), up to the maximum force (20 nN in this case), and coming back to the initial position long the blue line (retrace). The trace line has three distinct phases: (i) first, an elastic deformation of the cell occurs (the force required for this process depends on the cell cytoskeleton); (ii) then, after further compression, the AFM tip pierces immediately through both RBC membranes (distal and proximal); and (iii) finally the tip achieves maximum force against the support, without further X-axis displacement. Membrane rupture is achieved at a definite force, marked by the sudden appearance of small peaks, at a Y-axis value that can be statistically quantitated (performing 50–75 curves for each sample). These experiments were performed at room temperature. Results shown in Figure 2 for both control and obese patient RBC indicate a significant decrease in RBC stiffness for obese patients. Nanomechanical resistance values were 6.38 ± 1.45 nN for control RBC and 5.47 ± 1.19 nN for obese patient RBC (p = 0.03). Figure 2. AFM force spectroscopy experiments on RBC. These measurements were performed at room temperature. Obese patient RBC are significantly less resistant to AFM punch-through Control Obese Breakthrough Force (nN) 0 2 4 6 8 10 * Figure 1. Representative AFM force–distance curve of an RBC. The AFM tip performs an indentation process on a supported RBC, initiated from X = 0 along the red line (trace), up to the maximum force (20 nN in this case), and coming back to the initial position long the blue line (retrace). The trace line has three distinct phases: (i) first, an elastic deformation of the cell occurs (the force required for this process depends on the cell cytoskeleton); (ii) then, after further compression, the AFM tip pierces immediately through both RBC membranes (distal and proximal); and (iii) finally the tip achieves maximum force against the support, without further X-axis displacement. Membrane rupture is achieved at a definite force, marked by the sudden appearance of small peaks, at a Y-axis value that can be statistically quantitated (performing 50–75 curves for each sample). These experiments were performed at room temperature. Figure 2. AFM force spectroscopy experiments on RBC. These measurements were performed at room temperature. Obese patient RBC are significantly less resistant to AFM punch-through experiments, pointing to a decrease in stiffness (number of patients n= 20 for control, n= 22 for obese; 50–75 measurements for each patient). Average values ± S.D. (*) Significance according to Student’s t-test: p= 0.03. Int. J. Mol. Sci. 2022,23, 1920 5 of 19 2.3. Anisotropy at the Polar–Nonpolar Interface Is Decreased in Obese Patient Erythrocyte Membranes Anisotropy is defined as the capacity of a material (in this case, a molecule) to exhibit different properties in different directions. Anisotropic optical probes can be used, therefore, to test the fluidity of an environment: if we consider a lipophilic anisotropic probe, its mobility will be directly related to the degree of molecular order of the membrane in the area in which the probe is embedded. In general, anisotropy values are decreased when the membrane is more fluid. With an increased fluidity, both molecular order and microviscosity decrease, causing higher rotational diffusion of the probe [ 28 ]. In our case, a TMA-DPH probe was used to locally evaluate the fluidity in a particular zone of RBC membranes, namely, the space near the hydrophilic–hydrophobic interface (i.e., the boundary between polar headgroups and lipid tails). TMA-DPH anisotropy was measured at two temperatures, 20 ◦C and 37 ◦C, the latter chosen due to its physiological relevance. TMA-DPH probe anisotropy measurements revealed a significant reduction in membrane order for obese patient RBCs when compared to control RBCs (Figure 3). Interestingly, this occurred for both 20 ◦ C and 37 ◦ C data sets, and the difference was even more significant at 37 ◦ C, with a global decrease of anisotropy values as membranes become more fluid due to the increase in T. These results point to a localization of membrane fluidity difference in obese patient RBC close to the space between the polar headgroups and the lipid tails. The above measurements, taken at room temperature (~20 ◦ C), show a good correlation with AFM increased penetrability (decreased rigidity) results, and point to a reduction in overall RBC membrane stiffness in obese patients. This will be further elaborated in the Discussion section. Figure 3. TMA-DPH anisotropy measurements of RBC membranes. The black bars represent measurements at 20 ◦ C, while gray ones represent those at 37 ◦ C. At both temperatures, a clear decrease for anisotropy values was detected for obese patient RBC, which indicates a higher membrane fluidity (n= 49 for control, n= 52 for obese). Average values ± S.D. Significance according to Student’s t-test: (**) p< 0.01; (***) p< 0.001. 2.4. Lipidomics Reveal an Altered Metabolism of Sphingomyelin (SM), ω-6 and ω-3 Fatty Acids In order to unveil the possible metabolic causes of the observed decreased RBC rigidity in obese patients, a lipidomic analysis of their RBC was performed. These experiments included the study of global fatty acids as well as that of specific lipid classes. Global fatty acid lipidomics of mature RBCs (Figure 4) showed a small but significant increase of the Int. J. Mol. Sci. 2022,23, 1920 6 of 19 saturated/monounsaturated fatty acid (SFA/MUFA) ratio for obese patient RBCs, which implies that obese RBCs have either less saturated fatty acids or more monounsaturated ones. In addition, the polyunsaturated fatty acid (PUFA) profile of omega-6 ( ω -6) and omega-3 ( ω -3) fatty acids indicated that the ω -6/ ω -3 ratio was increased for obese patient RBCs, pointing to a combined increase of ω -6 and a decrease of ω -3 for obese patient RBCs. A further analysis of some common PUFA such as arachidonic acid ( ω -6) and its precursor DGLA (dihomoγ -linolenic acid) ( ω -6) and DHA (docosahexaenoic acid) ( ω -3) pointed in the same direction, as obese patient RBCs exhibited a significant increase in arachidonic acid and DGLA, while DHA was decreased. Figure 4. Lipidomic quantitation of global fatty acid presence in mature RBCs. Empty boxes refer to control (normal weight) group, while gray boxes represent obese patients. Significant differences are detected for dihomoγ -linolenic acid (DGLA), arachidonic acid, DHA levels, SFA/MUFA, and ω -6/ ω -3 ratios, pointing to a metabolic switch for obese patient RBC membranes. Significance according to Student’s t-test: (*) p< 0.05; (**) p< 0.01; (***) p< 0.001. (n= 49 for control, n= 52 for obese). Int. J. Mol. Sci. 2022,23, 1920 7 of 19 Lipid classes were then examined by separation using UHPLC coupled with mass characterization by tandem MS, as described in the Experimental Section. Lipidomics results for the percent distribution of individual lipid classes are shown in Figure 5, namely, phospholipids (PC, PE, PS, and sphingomyelin SM, the latter also a sphingolipid) and the most abundant lipid in the RBC membrane, cholesterol (Chol), as well as the Chol/total phospholipid mol ratio. A significant decrease in SM was detected in obese patient RBCs (Figure 5D), while the other lipid species did not exhibit any significant differences. The Chol/phospholipid ratio was increased in obese patient samples (Figure 5F). The latter was due both to a small increase in Chol levels (Figure 5E) and to a decrease in phospholipids, particularly SM- (Figure 5D). The increase in Chol is not statistically significant, but the increase in Chol/phospholipid ratio is. Perhaps this is the result of a compensating effect in the RBC lipids, with the increase in (rigidifying) Chol being countered by a decrease in (also rigidifying) sphingomyelin (SM). Figure 5. Lipidomic quantitation of specific lipid species in RBC. Species studied were PC ( A ), PE ( B ), PS ( C ), SM ( D ), and Chol ( E ). Chol/total phospholipid mol ratio is shown in panel ( F ). Black bars refer to control RBC group, while gray bars represent the obese patient RBC group. A significant decrease in SM and an increase in Chol/phospholipid ratio were detected for obese patient RBC. Average values ±S.D. Significance according to Student’s t-test: (*) p< 0.05. n= 8. Lipidomics was also used to evaluate the degree of unsaturation of the different phospholipid species (Supplementary Figure S1) of obese patient RBC and control RBC, but no statistical differences were found between any of them, despite a slight decrease of PS saturation in obese patient RBC. Interestingly, further analysis of the nature of the unsaturated fatty acids for PC, PE, and PS revealed differences in ω -3 and ω -6 lipid profiles (Figure 6). Although the total amount of combined ω -3 + ω -6 remained constant, a significant decrease was detected in ω -3 for obese patient RBC, as well as an equivalent significant increase in ω -6. This confirmed the trend observed above for global fatty acids, and this could be a key explanation for some of the differences detected in membrane stiffness, as will be later assessed in the Discussion section. Int. J. Mol. Sci. 2022,23, 1920 8 of 19 Figure 6. Lipidomic analysis of ω -3 and ω -6 presence in specific lipid species. Percent ω -3 and/or ω -6 in PC ( A ), PE ( B ), and PS ( C ). Black bars refer to control RBC group, while gray bars represent the obese patient RBC group. While total values for combined ω -3 + ω -6 are constant, both a decrease in ω -3 and an increase in ω -6 are detected for each lipid species in obese patient RBC. Average values ±S.D. Significance according to Student’s t-test: (*) p< 0.05; (**) p< 0.01; (***) p< 0.001. n= 8. 2.5. Differences in Blood Plasma between Obese Patients and Control Blood Samples Having demonstrated the differences between obese patient RBCs and control RBCs, we performed a final set of experiments exploring the possibilities of additional differences between blood plasma from obese and control patients. Push–pull pyrene (PA) is a fluorescent dye that exhibits different emission spectra depending on the microenvironment, due to an intramolecular energy transfer [ 29 ]. It is a member of the so-called solvatochromic probe family. In the case of PA, a fluid environment gives a red-colored emission, while a more ordered area yields a blue-colored one. Thus, the red/blue intensity ratio (RBIR) is generally a marker of fluidity. Although PA has been reportedly used for membranes [ 29 , 30 ], we used this probe to analyze blood plasma and the results are shown in Figure 7. PA red/blue ratios in the range 0.32–0.38 are commonly found in liquid-ordered bilayers, as they exist in erythrocytes [ 30 ]. A significant decrease in RBIR was detected for obese patient blood plasma. The interpretation of this result will be extended in the Discussion section, as we would initially not expect a great contribution from lipids in blood plasma due to RBCs and white blood cells being absent, but the probe was sensitive to blood plasma lipoproteins, Chol, and triacylglycerols (TG) (Supplementary Figure S2). Int. J. Mol. Sci. 2022,23, 1920 9 of 19 Figure 7. PA probe measurements of blood plasma. Red/blue intensity ratio (RBIR) values for control and obese blood plasma revealed a highly significant reduction in obese patients. Experiments performed at 37 ◦ C (n= 35 for control and n= 39 for obese). Average values ± S.D. Significance according to Student’s t-test: (***) p< 0.001. 3. Discussion Results from two very different techniques (TMA-DPH anisotropy and AFM) show a clear tendency for obese patient RBC to exhibit a higher fluidity in their membranes, or, more specifically, at the polar–non-polar interface of the membrane bilayers than the control cohort (Figures 2and 3). This finding may be surprising and even seem counterintuitive at first glance, as obesity is often associated with higher Chol levels as well as saturated lipids in plasma. Both kinds of lipids are known to induce membrane stiffness [ 25 ], therefore reducing bilayer fluidity. However, at the membrane level, the opposite appears to be true. Lipidomics did not show any significant increase in Chol nor in saturated lipids. This is relevant because it demonstrates that high levels of Chol or a diet rich in saturated lipids does not necessarily translate exactly to the RBC membrane (in the specific form of cholesterol or saturated lipids, as confirmed by Supplementary Figure S1), but rather it may have a complex effect on RBC metabolism. Still, we should not overlook the slight increase observed in Chol for obese patient RBCs which, despite not being statistically significant, could still have some effect (Figure 5). In fact, Chol/phospholipid ratio was significantly increased in obese patients (Figure 5F), in agreement with the observations by Cazzola et al. [17]. Regarding RBC metabolism of obese patients, global fatty acid lipidomics indicates an increase in arachidonic acid, a ω -6 fatty acid which is a well-described precursor for proinflammatory agents [ 31 ] (Figure 4). In accordance, obese patient RBCs also show a significant increase in another rather uncommon ω -6 fatty acid, DGLA (dihomoγ -linolenic acid) (Figure 4), which is a reported precursor for anti-inflammatory molecules and blocks arachidonic conversion to proinflammatory leukotrienes [ 32 ]. The increase in ω -6 fatty acids agrees with previous observations by Cazzola et al. [ 17 ]. This points to a putative inflammatory profile for obese patient RBCs, which they try to equilibrate back to homeostasis. This may also be related to the deregulation of the ω -6/ ω -3 equilibrium [ 33 ]. Pan et al. [ 15 ] described an increase in ∆ 9-desaturase and a concomitant decrease in ∆ 5-desaturase activities in obese patients that could explain the changes in fatty acid composition mentioned Int. J. Mol. Sci. 2022,23, 1920 16 of 19 4.9.3. Data Processing All the MS data were acquired and processed using the Xcalibur 4.1 software package, while the LipidSearch software version 4.2.27 (Mitsui Knowledge Industry, Tokyo, Japan) was used to identify and quantify the lipid species in these complex biological samples. The key processing parameters were target database, General; precursor tolerance, 5 ppm; product tolerance, 5 ppm; product ion threshold, 1%; m-score threshold, 2; Quan m/z tolerance, ± 5 ppm; Quan RT (retention time) range, ± 0.5 min; use of main isomer filters and ID quality filters A, B, C, and D; Adduct ions H + , Na + and NH4 + for positive ion mode, and H − and HCOO − for negative ion mode. The lipid classes selected for the search were: LPC (lysophosphatidylcholine), PC (phosphatidylcholine), LPE (lysophosphatidylethanolamine), PE (phosphatidylethanolamine), LPG (lysophosphatidylglycerol), PG (phosphatidylglycerol), LPI (lysophosphatidylinositol), PI (phosphatidylinositol), LPS (lysophosphatidylserine), PS (phosphatidylserine), LPA (lysophosphatidic acid), PA (phosphatidic acid), SM (sphingomyelin), Cer (ceramide), Hex1Cer (hexosylceramide), Hex2Cer (dihexosylceramide), Hex3Cer (trihexosylceramide), SPH (shingosine), CL (cardiolipin), MG (monoacylglycerol), DG (diacylglycerol), TG (triacylglycerol), ChE (cholesterol ester), FA (fatty acid), and AcCa (acylcarnitine) Quantification was carried out by normalization of the extracted monoisotopic ion peak area of each native lipid species to the intensity of the extracted monoisotopic ion peak area of the internal standard. The internal standards used in this study were chosen to avoid those present in plasma samples. 5. Conclusions Our study supports the hypothesis that obesity influences the composition and properties of plasma membranes in cells other than adipocytes. Specifically, the data demonstrate a significant decrease in the fluidity (increased order) of the polar–non-polar interfacial region of RBC from obese patients when compared with a control group. This affects membrane order measured by TMA-DPH anisotropy and stiffness measured by AFM force spectroscopy. Lipidomics of the samples points to a deregulation of lipid homeostasis for obese RBC, with significant changes in concentration of ω -3 and ω -6 fatty acids. More precisely, obese patient RBCs undergo an increase is some ω -6 fatty acids such as arachidonic acid, while reducing ω -3 ones, such as DHA. This ‘switch’ from ω -3 to ω -6 fatty acids in obese patient RBC membranes is also detected for abundant specific lipid species such as PC, PE, and PS. In addition, a significant reduction in SM is detected for obese patient RBC. Both events, SM reduction and, perhaps more decisively, the increase of ω -6 fatty acids seem to contribute to the aforementioned fluidity of obese patient RBC membranes. Finally, experiments on blood plasma with PA probe pointed to a reduction in the HDL/LDL ratio for obese patients, which is a relevant marker for cardiovascular disease risk. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms23031920/s1. Author Contributions: J.S. and A.B.G.-A. contributed equally to this work, performing most of the experiments. All authors contributed to the experimental design, performing some measurements, analyzing the results, and writing the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported in part by the Basque Government Department of Economic Development, grant No. KK-2019/00028 (OBINTER); the Basque Government Department of Education, grants No. IT1264-19, IT1281-19, IT1270-19, and IT1625-22; the Basque Government Department of Health, grants No. 2019-222030, 2020-333023; Fundación Ramón Areces; and by Centre for the Development of Industrial Technology (CDTI) of the Spanish Ministry of Science and Innovation under the grant agreement: TECNOMIFOOD project (CER-20191010) and Basque Government: IT1625-22. Int. J. Mol. Sci. 2022,23, 1920 17 of 19 Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board “Basque Country Clinical Research Ethics Committee” (protocol code PI2019219), in the year 2019. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. 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