Evaluating Red Blood Cells' Membrane Fluidity in Diabetes: Insights, Mechanisms, and Future Aspects

ABSTRACT

Aims

This review evaluates the mechanisms underlying red blood cell (RBC) membrane fluidity changes in diabetes mellitus (DM) and explores strategies to assess and address these alterations. Emphasis is placed on developing a comprehensive index for membrane fluidity to improve monitoring and management in diabetic patients.

Materials and Methods

We reviewed current literature on RBC membrane fluidity, focussing on lipid composition, glycation, oxidative stress, and lipid transport alterations in diabetic patients. Key methodologies include lipidomics, multi‐scale probe assessment, and machine learning integration for standardized fluidity measurement.

Results

Diabetic RBCs exhibit increased membrane fluidity, primarily due to oxidative stress, increased glycation, and dysregulated lipid composition. These alterations contribute to vascular complications and impair RBC functionality. Assessing membrane composition as a nutritional marker provides insights into the metabolic impacts of glycaemic management.

Conclusions

There is a critical need for a unified and comprehensive membrane fluidity index in DM, which could support personalised interventions through dietary, medicinal, and lifestyle modifications. Future research should prioritise standardising measurement techniques and integrating lipidomic data with machine learning for predictive modelling, aiming to enhance clinical outcomes for diabetic patients.

1. Introduction

The term ‘fluidity’ is broadly used to describe the physical state of biological membranes. This fluidity is influenced by several factors such as membrane structure, curvature, microviscosity, phase, lipid structure, packing, and composition [1]. Artificial membrane bilayers made from a single type of fatty acid can exist in either liquid (fluid) or gel‐like state depending on the temperature. At low temperatures, the fatty acid chains of phospholipids are tightly packed and laterally ordered, forming a gel‐like phase (Lb). When the temperature rises above a certain threshold (melting temperature, Tm), which varies based on the types of fatty acids in the membrane, the membrane transitions to a fluid state (La). In this fluid state, the polar heads of phospholipids occupy a larger area. Lipid mixtures that resemble the outer leaflet of the plasma membrane (PM) exhibit more complex phase behaviour, with multiple phases and a mosaic of gel and fluid domains coexisting within the bilayer [1].

Membrane fluidity of human cells is influenced by several factors, including the lipid composition, temperature, and proteins, along with their modifications due to processes like glycation and oxidation [2]. The fluidity of cell membranes is mainly influenced by the types of fatty acids they contain. Saturated fatty acids, with their straight structures, pack tightly together, reducing membrane fluidity. In contrast, unsaturated fatty acids have conformational irregularities due to double bonds, preventing tight packing and thus increasing fluidity [2]. This balance of fatty acids is crucial for maintaining the flexibility of red blood cells' (RBCs') membranes, which is essential for their ability to deform and pass through narrow capillaries [3]. Temperature also plays a role in modulating membrane fluidity by affecting lipid composition. At low temperatures, cellular membranes increase their unsaturated lipid content to counteract reduced fluidity, while high temperatures can decrease unsaturated lipid content to manage increased fluidity. However, excessive fluidity at elevated temperatures can compromise membrane integrity, potentially causing cell damage [4, 5]. Proteins embedded in the membranes also significantly modulate fluidity. Integral membrane proteins can act as barriers to lipid movement, decreasing fluidity. Conversely, membrane‐bound enzymes can alter lipid composition, impacting fluidity [6]. In RBCs, the interaction between proteins and lipids is particularly important for maintaining membrane stability and flexibility, which are critical for their oxygen‐carrying function and mechanical resilience. Protein modifications, such as glycation and oxidation, further influence membrane fluidity and overall cell function. In literature, it was deeply explored the role of RBC membrane fluidity as a metabolic biomarker of pathological states in diabetes mellitus (DM) [7]. In DM, alterations in lipid composition and increased oxidative stress can significantly affect membrane fluidity, leading to complications such as impaired microcirculation and increased haemolysis [7, 8, 9, 10].

Understanding the factors that affect membrane fluidity in RBCs in patients with DM is crucial for developing therapeutic strategies to mitigate the adverse effects of metabolic diseases. By elucidating the mechanisms behind fluidity alterations, researchers can better target interventions to restore normal membrane function and improve clinical outcomes in patients with DM and other metabolic disorders [11]. This review aims to synthesise current knowledge on the determinants of RBC membrane fluidity in DM, evaluate the implications of altered fluidity on disease pathology, and discuss potential therapeutic strategies to restore normal membrane function. The review also emphasises the need for standardized assessment techniques to develop comprehensive monitoring indices that can guide the effective management of DM‐related complications.

2. Methods

This review was conducted by searching databases, including PubMed, Scopus, ScienceDirect, and Google Scholar using the term ‘RBC membrane fluidity DM’. The search continued until no new significant results emerged, indicating saturation. The inclusion criteria for this review were studies that specifically investigated changes in RBC membrane composition and fluidity in the context of Type 1 DM (T1DM) and Type 2 DM (T2DM), published within the last 44 years (1980–2024), using various methodologies to assess membrane properties. To ensure a comprehensive analysis, both reviews and original research articles were critically reviewed to extract data on the mechanisms underlying changes in membrane fluidity, such as lipid composition, glycation, oxidative stress, fatty acid composition, and inflammatory lipid profiles. The methodologies employed in these studies, including fluorescence spectroscopy, electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), X‐ray diffraction, and lipidomics, were noted to understand the diverse techniques used in this field. The collected data were synthesised to identify trends and insights into the impact of DM on RBC membrane fluidity and to suggest future research directions in this area.

3. Methods for Studying Membrane Fluidity

The methods used to monitor membrane fluidity can be broadly categorised into two groups: those that assess membrane state by probing physical properties such as hydration (e.g., Laurdan and 1,6‐diphenyl‐1,3,5‐hexatriene (DPH)), diffusion (e.g., EPR) or molecular packing (DSC, X‐ray diffraction), and those that determine its molecular composition (e.g., NMR spectroscopy, and lipidomics).

3.1. State‐Based Techniques

Fluorescence spectroscopy utilises fluorescent probes such as Laurdan [12] and DPH [13]. Laurdan inserts into membrane phospholipids, and its fluorescence intensity and polarization are sensitive to changes in hydration states, allowing it to monitor both liquid‐ordered and liquid‐disordered phases. DPH is a rigid molecule that inserts into the hydrophobic core of the membrane, and its fluorescence anisotropy decreases as the membrane becomes more fluid. These techniques rely on the different hydration states of the fluorescent probe, with greater hydration in the fluid phase, and are commonly used due to their simplicity and cost‐effectiveness.

EPR is another technique used to monitor changes in membrane fluidity [14]. EPR measures the resonance of unpaired electrons in a sample, with spin‐labelled lipids acting as probes. This technique is based on the diffusion of these spin‐labelled lipids within the membrane. As membrane fluidity increases, the mobility of the spin labels also increases, leading to changes in the EPR signal. By analysing the rotational and lateral diffusion of the spin labels, EPR provides information about the dynamic properties of the membrane. Although highly sensitive, this technique requires specialised equipment and expertise.

DSC measures the transition temperature of lipid bilayers [15]. This technique works by measuring the amount of heat required to increase the temperature of a membrane sample, which allows it to detect the phase transition from a more ordered gel phase to a disordered liquid phase. This principle is based on the fact that the transition from gel to liquid corresponds to a significant increase in molecular motion, reflecting a change in membrane fluidity. DSC monitors changes in the physical state of the membrane (gel vs. liquid phase) rather than its molecular composition. Changes in transition temperature directly reflect alterations in membrane fluidity, as a more fluid membrane will undergo this phase transition at a lower temperature.

X‐ray diffraction monitors changes in the packing and order of lipid molecules in a membrane [16]. This technique works by directing X‐rays at the membrane and measuring how they are scattered by the lipid molecules. The resulting diffraction patterns provide information about the arrangement and spacing between the lipid molecules in the membrane. As membrane fluidity increases, the lipid molecules become less tightly packed, and the diffraction pattern changes accordingly. X‐ray diffraction assesses the physical organisation and structural order of the lipids within the membrane rather than their chemical composition. Changes in lipid spacing and packing are directly linked to alterations in membrane fluidity, with more fluid membranes showing greater spacing between lipid molecules.

3.2. Composition Based Techniques

NMR spectroscopy, on the other hand, is employed to determine the molecular composition of the membrane [17]. NMR works by measuring the magnetic properties of atomic nuclei, particularly the way they resonate in a magnetic field. In membrane studies, deuterium‐labelled lipids are often used as probes to observe the orientation, mobility, and interactions of specific lipid molecules within the membrane. By analysing these resonances, NMR can provide detailed information about the types of lipids present, their relative abundance, and how they are arranged within the bilayer. This level of detail makes NMR a powerful tool for studying the molecular architecture of membranes, although its complexity and the need for specialised equipment limit its accessibility.

Lipidomics provides detailed information about the lipid composition of membranes [18, 19]. This technique uses advanced analytical methods, such as mass spectrometry and chromatography, to identify and quantify the diverse lipids present in a membrane. It analyzes the types, structures, and proportions of lipids, including fatty acids, phospholipids, sphingolipids, and cholesterol. By profiling the complete lipidome, lipidomics offers a comprehensive view of the membrane's molecular makeup, revealing how specific lipid species contribute to its structural and functional properties.

The lipid profiles it generates help explain how variations in lipid types—such as changes in cholesterol levels or shifts in the saturation and length of fatty acid chains—indirectly affect membrane fluidity. These compositional insights are crucial for understanding the molecular mechanisms that regulate membrane dynamics and properties.

3.3. Pros and Cons of Monitoring Membrane Fluidity: Comparing State‐Based Techniques With Lipidomics and Other Composition‐Based Methods

Monitoring membrane fluidity with state‐based techniques provides unique and insightful perspectives on the state of cellular membranes, particularly in diseases like DM [20, 21]. However, it has advantages and disadvantages compared with Lipidomics and Other Composition‐Based Methods.

Advantages of state‐based techniques include providing direct functional insight into the physical state and functionality of the cell membrane, which is crucial for understanding how well the cell can perform its functions such as nutrient transport and signal transduction. It also allows real‐time assessment, making it possible to monitor dynamic changes in response to various stimuli or treatments [22, 23, 24].

Despite these advantages, state‐based measurements often lack specificity as they provide an overall picture without identifying specific lipid species or molecular changes responsible for the observed effects, limiting their ability to identify specific lipid alterations or pathways involved in disease processes.

These measurements can be influenced by various factors, including temperature and the presence of proteins, making it challenging to attribute changes to specific pathological conditions. In contrast, composition‐based techniques offer a comprehensive analysis of lipid species and their concentrations, relying on techniques such as NMR spectroscopy and mass spectrometry. Lipidomics provides detailed molecular insights into lipid metabolism and changes associated with diseases [25]. This specificity allows for the identification of biomarkers and potential therapeutic targets. However, lipidomics is more complex, requires advanced equipment and expertise, and is often more expensive and time‐consuming. Moreover, information on the physical state can only be indirectly inferred.

4. Membrane Fluidity Alterations, Diabetes and Complications

The examination of RBC membrane fluidity provides critical insights into the underlying mechanisms of DM [8]. Before delving into the specific mechanisms that can alter membrane fluidity in this disease, it is important to briefly reference some key studies that have contributed to a general understanding of this phenomenon to provide a foundational context for the following analysis on membrane fluidity. We will then explore the potential reasons behind the discrepancies observed in the data reviewed in this study. In paragraph 5, we will focus on the mechanisms through which membrane fluidity can be altered in RBCs in DM patients.

4.1. Key Studies on Membrane Fluidity and Diabetes

Various studies have elucidated the relationship between altered membrane fluidity and the progression of both T1DM and T2DM, highlighting key factors such as lipid composition, glycation, oxidative stress and the lipid transport system (Table 1).

TABLE 1.

Summary of studies on red blood cells' membrane fluidity in diabetes according to measurement method.

Study (year)	Population (CTRL/DM)	Approach	Method	Main results
State based methods
Birlouez‐Aragon et al. (1990) [26]	21/30	In vitro, incubation with glucose/galactose	DPH polarization assay	Evidence for a relationship between protein glycation and red blood cell membrane fluidity (increase in fluidity)
Watala et al. 1990 [27]	Systematic review	In vivo, cross‐sectional	Several, mainly DPH	Cell membranes of diabetic subjects exhibited variations in membrane fluidity, which can be reduced, normal, or increased. This variability is attributed to differences in measurement methods and number and selection of the populations studied. Membranes of diabetic individuals often had an increased (C/PL) ratio, contributing to alterations in membrane fluidity.
Kamada et al. (1992) [28]	11/15	In vivo, cross‐sectional	DPH polarization assay	Younger RBCs in diabetes mellitus exhibit lowered membrane fluidity. (Decrease in fluidity)
Mazzanti et al. (1992) [29]	23/26	In vivo, cross‐sectional	Laurdan GP microscopy	Diabetes mellitus induces alterations in the RBC membrane possibly affecting the ageing process. (Increase in fluidity)
Maulucci et al. (2017) [20]	8/18	In vivo, cross‐sectional	Laurdan GP microscopy	Phase separation in RBC membranes in type 1 diabetes mellitus correlates with disease progression. (Increase in fluidity)
Cordelli et al. (2018) [9]	27 DM patients	In vivo, cross‐sectional	Laurdan GP microscopy	The decision support system distinguishes DM types based on RBC membrane fluidity. (Increase in fluidity)
Bianchetti et al. (2020) [30]	15/33	In vivo, cross‐sectional	Laurdan GP microscopy	RBC membrane fluidity as a marker of diabetic retinopathy in type 1 diabetes mellitus. (Increase in fluidity)
Bianchetti et al. (2022) [31]	27 DM patients (12 non‐PAD+, 15 in any stage of PAD)	In vivo, cross‐sectional	Laurdan GP microscopy	Spatial reorganization of RBC liquid crystalline domains in type 2 diabetes mellitus with PAD. (Increase in fluidity)
Bianchetti et al. (2023) [11]	234 DM patients with and without previous cardiovascular event	In vivo, longitudinal study	Laurdan GP microscopy	RBC membrane fluidity is associated with residual cardiovascular risk in type 2 diabetes mellitus. (Increase in fluidity)
Bryszewska et al. (1986) [32]	24/20	In vivo, cross‐sectional	Pyrene diffusion rate	Increased microviscosity, elevated C/PL ratio, and higher HbA1C levels in RBC membranes. (Increase in fluidity)
Composition Based Methods
Kostara et al. (2021) [33]	20/20	In vivo, cross‐sectional	NMR‐based lipidomics	Altered RBC membrane lipidome linked to microvascular impairment in type 2 diabetes. (No direct assessment)
Kröger et al. (2015) [34]	362/1378	Longitudinal cohort study	Lipophilic index	Erythrocyte membrane fatty acid fluidity associated with risk of type 2 diabetes. (No direct assessment, indirect assumption of decreased fluidity)

Note: This table summarises key studies investigating RBCs' membrane fluidity in patients with Type 1 and Type 2 diabetes. It includes information on the fluidity changes observed, the type of diabetes, any associated complications, the methods used to estimate fluidity, and changes in lipid composition. The studies are listed in chronological order to provide a clear timeline of the research progress in this field.

Abbreviations: C/PL: Cholesterol/Phospholipid; DPH: 1,6‐diphenyl‐1,3,5‐hexatriene; EPR: Electron paramagnetic resonance; ESR: RBC Sedimentation Rate; GP: Generalised polarization; HbA1C: Haemoglobin A1c; NMR: Nuclear magnetic resonance.

Bryszewska et al. first showed that RBC membranes in T1DM patients exhibit increased microviscosity associated with an elevated cholesterol/phospholipid (C/PL) ratio and increased Haemoglobin A1c (HbA1C) levels by measuring the pyrene diffusion rate (Table 1). According to the authors, this increase in cholesterol and subsequent alteration in lipid composition significantly reduce membrane fluidity [32]. To dissect the differential contributions of glycosylation and altered lipid profiles, Watala et al. explored in vitro the effect of high glucose levels on RBC membrane fluidity through nonenzymatic glycation of membrane proteins. The study demonstrated that even short periods of hyperglycemia could significantly reduce membrane fluidity through increased glycation, highlighting the impact of glycaemic control on membrane properties [27].

Birlouez‐Aragon et al. investigated the relationship between protein glycation and membrane fluidity using a DPH polarization assay (Table 1). Their study showed that incubation of RBC membranes from healthy subjects with glucose or galactose increased both protein glycation and membrane fluidity. However, diabetic subjects' RBC membranes, already exhibiting high glycation levels, showed no further change [26]. This suggests that glycation‐induced alterations in lipid‐protein interactions play a critical role in modifying membrane fluidity, further influenced by lipid composition and oxidative stress. Kamada et al. focused on the impact of RBC age on membrane fluidity in both normal and T1DM subjects (Table 1). Their findings revealed that membrane fluidity decreases with cell age, with diabetic RBCs consistently showing lower fluidity across all age groups compared to normal cells. This reduction in fluidity was associated with increased HbA1C levels and decreased acetylcholinesterase activity, indicating a strong link between glycation and membrane fluidity [28]. Despite this evidence, Mazzanti et al. findings indicated that membrane fluidity and lipid peroxidation increased in T2DM subjects (Table 1). Notably, RBCs from diabetic patients exhibited higher fluidity and lipid peroxidation compared with normal subjects, suggesting that DM accelerates age‐related changes in membrane properties [29]. Muzulu et al. investigated membrane fluidity and calcium pump activity in patients with T2DM. Although membrane fluidity levels were similar to controls, diabetic subjects exhibited significantly reduced basal and calmodulin‐stimulated calcium pump activities [35] (Table 1).

In a review by C. Watala explored the structural and dynamic changes in blood cell membranes in individuals with DM. According to the review, the cell membranes of diabetic subjects (both T1DM and T2DM were considered) exhibited variations in membrane fluidity, which can be reduced, normal, or increased. This variability is attributed to differences in measurement methods and the number and selection of the populations studied [36]. However, the main technique used was DPH fluorescence polarization measured in cuvette experiments. The review thus concluded that there was no direct evidence that membrane fluidity is consistently decreased in diabetic subjects. However, the membranes of diabetic individuals often had an increased (C/PL) ratio, contributing to alterations in membrane fluidity. Additionally, there were changes in the composition of phospholipids and levels of polyunsaturated fatty acids (PUFAs), with specific variations depending on the type of cells studied, such as RBCs and platelets [36]. Nonenzymatic glycation was more frequent in membrane proteins of diabetic subjects compared with controls, and this glycation could negatively impact membrane fluidity and protein function [36].

Kröger et al. assessed the lipophilic index, a measure of RBC membrane fatty acid fluidity, in relation to T2DM incidence. They found a positive association between a high lipophilic index, reflecting lower membrane fluidity, and an increased risk of T2DM [34] (Table 1). However, in this case, the fluidity of RBC membranes was assessed indirectly by only considering an index referring to the average saturation degree.

Maulucci et al. studied the modulation of membrane fluidity by fatty acids using Laurdan GP microscopy to measure fluidity. This technique allowed us to assess the extent of fluid domains directly on the membranes [1]. In a subsequent study, the same authors observed phase separation and, overall, an increased fluidity in the plasma membranes of RBCs from Type 1 diabetic patients, correlating these patterns with disease severity and complications [20] (Table 1). Cordelli et al., using the same microscopy‐based method, developed a decision support system (DSS) that utilised dual‐channel analysis of RBC membrane fluidity to distinguish between healthy subjects, T1DM patients, and those with complications. The DSS, leveraging advanced statistical measures, achieved high accuracy, outperforming traditional HbA1C tests [9] (Table 1). Bianchetti et al. studied RBC membrane fluidity as a marker of diabetic retinopathy in T1DM. They found that RBC membranes of diabetic retinopathy patients analysed with Laurdan GP microscopy were more fluid compared to non‐retinopathy patients, again suggesting that membrane fluidity could serve as a complementary index to HbA1C for detecting microvascular complications [30] (Table 1). Kostara et al. investigated the structural disturbances in the membranes of RBCs in patients with T2DM. This study employs an NMR‐based lipidomic approach to analyse the lipid composition of RBC membranes in 20 newly diagnosed T2DM patients compared with 20 healthy controls. The main findings revealed significant alterations in the lipid species of RBC membranes in T2DM patients. Specifically, there is an increase in cholesterol, total sphingolipids, sphingomyelin, and glycolipids. Conversely, there is a decrease in total phospholipids, particularly phosphatidylethanolamine, total ether glycerolipids, and plasmalogen‐phospholipids. These changes resulted in a higher cholesterol‐to‐phospholipid molecular ratio compared to the control group [33] (Table 1). Bianchetti et al. studied the spatial reorganization of liquid crystalline (LC) domains in RBC membranes of T2DM patients with peripheral artery disease (PAD) using Laurdan GP microscopy. They found that RBC membranes in PAD patients were significantly more fluid (lower GP values) compared with non‐PAD patients. These findings suggest that changes in membrane fluidity and LC domain organisation could serve as early markers for PAD in T2DM patients [31] (Table 1). The same authors investigated RBC membrane fluidity as a potential new biomarker for residual cardiovascular risk in T2DM. The researchers assessed the membrane fluidity of RBC in 234 T2DM patients using Laurdan GP microscopy. They found that lower GP values, indicating more fluid membranes, were associated with a higher occurrence of major cardiovascular events. This study suggests that changes in the fatty acid composition of RBC membranes, particularly an increase in pro‐inflammatory omega‐6 fatty acids, contribute to this increased risk. These findings imply that erythrocyte membrane fluidity could serve as a valuable biomarker for cardiovascular risk assessment in T2DM, potentially leading to more personalised treatment strategies [11] (Table 1).

4.2. Analysis of Methodological Approaches to Membrane Fluidity

The discrepancies in the results of the studies reviewed can largely be attributed to the diverse techniques employed to measure membrane fluidity. Techniques such as fluorescence spectroscopy, EPR, and NMR spectroscopy, while each providing valuable insights, often measure different parameters under the broad term ‘fluidity’. Fluorescence spectroscopy typically assesses the mobility of lipid molecules using probes like Laurdan or DPH, whereas EPR focuses on the behaviour of spin‐labelled lipids, and NMR examines the magnetic properties of deuterium‐labelled lipids. Each of these techniques targets different aspects of membrane dynamics. For instance, Laurdan and DPH detect changes in the hydration state or molecular packing of lipids, while NMR and EPR offer insights into molecular‐level interactions and lipid compositions. However, while composition‐based techniques, like lipidomics, do not directly measure membrane fluidity, they provide crucial support in understanding the biochemical changes that influence it, particularly in terms of lipid composition and saturation levels.

The consistency of results depends on the method employed. A notable observation from Table 1 is that the results obtained using DPH fluorescence vary significantly across studies, potentially due to the sensitivity of the probe to larger‐scale phenomena and environmental factors such as temperature or protein presence. In contrast, Laurdan microscopy, which measures membrane fluidity by detecting phase separations at a higher spatial resolution, produces more consistent results across studies. This consistency is likely due to the technique's ability to detect fine‐scale domain separations in the membrane, which are highly sensitive to pathological conditions like diabetes.

Additionally, changes in PUFA composition and increases in the (C/PL) ratio are common findings across studies, reinforcing the idea that alterations in lipid composition play a central role in modulating membrane fluidity. These lipidomic changes, while not directly linked to fluidity measurements, provide valuable context for understanding how cellular environments and membrane properties evolve in metabolic conditions such as diabetes (Section 5.1).

5. Mechanisms of Membrane Fluidity Alteration in Diabetes

Membrane fluidity is crucial for maintaining numerous cellular processes such as signal transduction, nutrient transport, and enzyme activity. In both T1DM and T2DM, alterations in membrane fluidity have been consistently observed (Figure 1). These alterations are primarily often linked to changes in lipid composition oxidative stress. Protein glycation and the lipid transport system, all of which contribute to the complex interplay of metabolic disturbances associated with DM [37]. Here, we explore these mechanisms in detail, integrating recent advances in the chemical and biochemical analysis of RBC membranes.

FIGURE 1.

Mechanisms Contributing to Red Blood Cell Membrane Fluidity‐Measured by Laurdan Generalised Polarization. This figure illustrates the various mechanisms influencing red blood cell membrane fluidity in diabetes mellitus. Several factors contribute to increased membrane fluidity: Chronic hyperglycemia leading to protein glycation and advanced glycation end‐products (AGEs) formation, increased reactive oxygen species (ROS) causing lipid peroxidation and membrane lipid damage, and an altered fatty acid composition with higher levels of saturated fatty acids like omega‐6 (ω‐6). These changes promote pro‐inflammatory lipid profiles and elevate levels of cytokines and lipid mediators such as AA. Fluidity Measurement of RBC membrane using Laurdan fluorescence and generalised polarization (GP) indicates membrane fluidity, ranging from −1 (increased fluidity) to + 1 (decreased fluidity). DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; LA: linoelaidic acids.

5.1. Lipid Composition

One of the primary determinants of membrane fluidity is lipid composition, particularly the balance between saturated and unsaturated fatty acids, as well as the C/PL ratio. Bryszewska et al. (1986) and more recently, Kostara et al. (2021), showed that diabetic patients often exhibit an increased C/PL ratio, resulting in reduced membrane fluidity due to the stiffening effects of excess cholesterol [32, 33]. This is because cholesterol, due to its rigid steroid structure, modulates membrane dynamics by interacting with adjacent fatty acid chains, making them less mobile. However, the influence of cholesterol is nuanced. Cholesterol has a “buffering” effect on membrane fluidity: below the membrane's phase transition temperature (Tm), it increases fluidity by preventing the packing of phospholipids, but above Tm, it restricts the movement of fatty acid chains, thus decreasing fluidity [38]. In diabetes, these regulatory effects of cholesterol may be disrupted due to changes in the overall lipid environment, particularly with respect to the balance of saturated and unsaturated fatty acids.

Kröger et al. (2015) demonstrated an increased ratio of saturated to unsaturated fatty acids in the RBC membranes of T2DM patients. Saturated fatty acids are known to decrease membrane fluidity because they pack tightly together, creating a more rigid membrane structure. Conversely, unsaturated fatty acids, PUFAs, create more fluid membranes due to the presence of kinks in their carbon chains [34]. Moreover, the balance between omega‐3 (n‐3) and omega‐6 (n‐6) PUFAs is critical. Bianchetti et al. (2023) showed that a higher proportion of pro‐inflammatory n‐6 PUFAs, such as arachidonic acid (AA), correlated with increased membrane fluidity and cardiovascular risk in T2DM patients. This effect was linked to the steric hindrance caused by n‐6 fatty acids, which disrupt lipid packing, leading to increased fluidity [11].

In contrast, omega‐3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit anti‐inflammatory properties and reduce membrane fluidity. Therefore, the ratio of n‐6 to n‐3 fatty acids has been shown to directly influence membrane properties [11]. The cumulative effect of these modifications has been phenomenologically correlated with many studies [11, 27, 36]. For example, studies using Laurdan GP microscopy have shown an increase in fluidity correlating with greater sensitivity to variations in PUFA composition, while no significant variations were observed in cholesterol or general phospholipid content, nor in the C/PL ratio [11]. In other studies, performed with DPH fluorescence polarization, there is instead an increase with the C/PL ratio, suggesting a different sensitivity of the probe used to the membrane state [32].

5.2. Oxidative Stress and Lipid Peroxidation

Oxidative stress, driven by chronic hyperglycemia, plays a significant role in the pathogenesis of DM, contributing to various complications. Hyperglycemia enhances the production of reactive oxygen species (ROS), which exceed the body's antioxidant defences, leading to oxidative stress. RBCs are particularly vulnerable due to their constant exposure to oxygen and glucose in the bloodstream. This triggers lipid peroxidation, leading to the production of harmful by‐products such as malondialdehyde (MDA) and hydroxynonenal (HNE). These aldehydes further propagate oxidative damage, altering membrane properties and reducing the deformability of RBCs [39]. To date, only Mazzanti et al. (1992) have reported increased lipid peroxidation in RBCs from T2DM patients. Using DPH fluorescence polarization, the study observed heightened fluidity due to the breakdown of PUFAs, which disrupted the membrane's structural integrity [29]. Although this increase in fluidity may seem paradoxical, it results from the loss of membrane lipids rather than their reorganization. ROS‐induced peroxidation also modifies membrane proteins through carbonylation and cross‐linking, further impairing membrane functionality [39].

Interestingly, the extent of lipid peroxidation appears to be influenced by the oxidation state of haemoglobin. Studies have shown that oxidised haemoglobin, particularly hemichrome, catalyses lipid oxidation, exacerbating membrane damage. In this context, the RBC membrane becomes a site for Fenton‐type reactions, where free iron, reduced by the Steap3 protein, reacts with hydrogen peroxide to generate hydroxyl radicals (HO•), potent mediators of lipid peroxidation. Bianchetti et al. (2023) demonstrated that elevated Steap3 levels in diabetic patients contributed to increased lipid oxidation and haemolysis, further implicating oxidative stress in the pathogenesis of RBC membrane damage in diabetes [11].

5.3. Non‐Enzymatic Glycation

Another mechanism‐altering membrane fluidity in diabetes is the non‐enzymatic glycation of membrane proteins. Chronic hyperglycemia leads to the nonenzymatic binding of glucose molecules to amino groups on proteins, initiating a cascade of chemical reactions. Initially, reversible Schiff bases are formed, which then rearrange into stable Amadori products and eventually into advanced glycation end products (AGEs). AGEs irreversibly modify amino groups on proteins, altering their structure and function. In RBCs, critical membrane proteins like spectrin, ankyrin, and band 3 are susceptible to glycation. These proteins are essential for maintaining the cytoskeletal structure and the mechanical stability of the membrane [40].

Watala et al. explored the effects of glycation on membrane fluidity using in vitro models to demonstrate that glycation reduces membrane fluidity by disrupting protein‐lipid interactions. The glycation of band 3 results in its aggregation, which interferes with ion transport functions and contributes to the stiffening of the membrane [27]. Spectrin glycation weakens the cytoskeletal network, leading to a loss of RBC deformability. These findings were corroborated by Kamada et al., who showed that glycation primarily decreased membrane fluidity in diabetic RBCs through the modification of cytoskeletal and membrane‐bound proteins [28].

Moreover, AGEs make membrane proteins more vulnerable to oxidative damage. The combination of glycation and oxidative stress creates a feed‐forward loop where AGEs enhance the production of ROS, which further damages the membrane and exacerbates diabetic complications such as nephropathy, retinopathy, and neuropathy [40].

In summary, a proper evaluation of membrane fluidity requires a multi‐scale and multi‐probe assessment or at least a standardisation of the procedure to better compare how this parameter is modified. Although the parameter is not specific, it integrates membrane composition, which is a nutritional marker, as well as the metabolic effects related to glycaemic management, thus allowing the creation of a more general index that can provide a comprehensive understanding of membrane fluidity changes. Effective management of DM should therefore include strategies to normalise fluidity values through diet, medication, and lifestyle changes, aiming to restore healthy fatty acid composition.

5.4. Alterations in the Lipid Transport System

Membrane fluidity is largely determined by the lipid composition of the membrane, which includes phospholipids, cholesterol, and proteins, and can influence the ability of cells to carry out metabolic processes and respond to hormonal signals such as insulin. Lipoproteins, particularly high‐density lipoprotein (HDL) [41], play a crucial role in modulating membrane fluidity by facilitating the transport and distribution of dietary and endogenously synthesised lipids to cells [42]. HDL is responsible for the transport of cholesterol and other lipids, contributing to the maintenance of membrane lipid balance [43]. This process is essential for the modulation of membrane fluidity, as the lipid composition of cellular membranes is influenced by the availability and incorporation of transported lipids [44].

HDL plays a crucial role in regulating RBC membrane fluidity by facilitating cholesterol efflux, which impacts the lipid composition and mechanical properties of the RBC membrane. Multiple studies have demonstrated how HDL‐mediated cholesterol removal is essential for maintaining RBC flexibility and function, particularly in the microcirculation [45].

For instance, a study by Holm et al. (2002) showed that HDL facilitates cholesterol efflux from RBC membranes via interactions with transporters like ABCA1 and scavenger receptor class B type I (SR‐BI), reducing membrane cholesterol content and increasing fluidity [46]. This process is particularly critical under physiological conditions where RBCs must navigate narrow capillaries.

Further supporting this, Muller et al. (1990) explored the relationship between lipid fluidity and RBC functionality in hyperlipoproteinemia patients. They found that disruptions in lipid transport mechanisms, particularly with abnormal lipoprotein profiles such as hyperlipoproteinemia, resulted in altered RBC membrane fluidity. The research demonstrated that HDL's role in cholesterol transport is essential for maintaining optimal RBC membrane fluidity and preventing rigidity, which can impair RBC deformability [47].

Moreover, a study by Barenholz et al. (1981) on the interaction of cholesterol and phospholipids in the RBC membrane further emphasized the importance of cholesterol‐phospholipid balance, controlled partly by HDL, in determining membrane dynamics. Their work on patients with abetalipoproteinemia, a condition marked by abnormal HDL levels, showed that increased cholesterol in the RBC membrane led to decreased membrane fluidity and DPH fluorescence polarization, contributing to impaired RBC function. The researchers highlighted the critical interplay between HDL, cholesterol, and membrane phospholipids in regulating RBC membrane fluidity [48].

Maulucci et al. explored altered RBC membrane fluidity in genetic mouse models with lipoprotein deficiencies, linking lipidome changes to broader metabolic disorders. Analysis of membrane fluidity of RBC isolated from APOA1 or LCAT deficient mice by Laurdan two‐photon microscopy shows a drastically altered RBC membrane fluidity at baseline than wild type mice. Overall, this research underscores the importance of the membrane lipid transport system in maintaining cellular function and its potential impact on disease development [1]. Understanding the role of HDL in regulating membrane fluidity provides insights into the pathogenesis of insulin resistance, T2DM, and vascular complications associated with impaired RBC function. Therapeutic strategies aimed at improving HDL function or mimicking its effects on membrane fluidity may enhance insulin secretion, insulin sensitivity, and RBC deformability, offering novel avenues for managing T2DM and its complications.

6. Nutritional and Therapeutic Approaches to Restore the Normal RBC Membrane Fluidity

Alterations in RBC membrane fluidity are associated with complications in metabolic disorders such as diabetes. Consequently, restoring membrane fluidity may serve as a therapeutic target to enhance cellular function and mitigate these complications [49]. This restoration necessitates reestablishing the membrane's normal composition with precise ratios of its primary components, including proteins, cholesterol, carbohydrates, and phospholipids (Figure 1).

6.1. Nutritional Interventions to Restore Optimal Lipid Composition

Although there are currently no studies directly monitoring changes in RBC membrane fluidity across different treatment approaches (nutritional and therapeutic), several studies have investigated changes in the composition of RBCs in response to these treatments (Table 2).

TABLE 2.

Studies investigating various approaches to restore red blood cells' membrane integrity.

Study (year)	Population	Approach & duration	Method	Main results
Cartwright IJ et al. (1985) [50]	Healthy male individuals (n = 5)	Nutritional; effects of a daily dietary supplement of fish oil concentrate (maxEPA) 3 g/day on membrane phospholipids of RBCs.	The phospholipids were analysed by thin layer chromatography	The supplementation of maxEPA significantly altered the fatty acid composition of RBC phospholipids, particularly increasing levels of EPA (20:5w3) and DHA (22:6w3).
		6 weeks.
Kamada T et al. (1986) [51]	Diabetic patients (n = 12) versus age and sex matched healthy individuals (n = 11)	Nutritional; effects of dietary sardine oil 2700 mg/day on RBC membrane fluidity.	Electron spin resonance (ESR) spectroscopy with stearic acid spin labels (SALs)	‐ At baseline: Membrane fluidity and membrane PUFAs were highly correlated and significantly lower in diabetic patients as compared to controls.
		8 weeks.		‐ After dietary consumption:
				A significantly increased membrane fluidity and increased levels of EPA and C22:5 in the phospholipid acyl‐chains of RBCs in both controls and diabetic patients.
Witte TR et al. (2010) [52]	Patients with early‐stage chronic lymphocytic leukaemia (n = 11)	Nutritional; effects of dietary omega‐3 on the fatty acid composition of RBC membrane.	The fatty acid compositions were assessed using gas chromatography.	‐ A linear, dose responsive increase in the fraction of omega‐3 fatty acids in RBC membrane.
		Each patient started with a dose of 3 capsules per day. Dosage was increased to 6 capsules and then to 9 capsules per day at monthly intervals.
McBurney MI et al. (2022) [53]	25,487 healthy adults (≥ 18 years)	Cross‐sectional analysis of blood samples submitted for routine clinical assessment.	RBC structural characteristics (MCV and RDW), Hb, high‐sensitivity CRP, AA, EPA, DHA, and omega‐3 were measured.	A significant inverse association between red blood cell distribution width (RDW) and omega‐3 index.
				Females showed a more consistent decrease in RDW with increasing omega‐3, whereas males exhibited a plateau or modest increase in RDW above 8% omega‐3.
Jain et al. (2000) [54]	Type 2 diabetes patients (n = 29) versus age‐matched nondiabetic siblings (n = 21)	Therapeutic, Vitamin E supplementation (1200 IU/day) to examine its effect on glutathione and lipid peroxidation in RBCs.	Glutathione, malondialdehyde, and vitamin E concentrations in RBC were analysed using high‐performance liquid and a UV/Vis detector.	At baseline: RBCs of diabetic patients had 21% higher (p 0.001) malondialdehyde and 15% lower (p 0.05) glutathione concentrations than healthy subjects.
		4 weeks		After vitamin E supplementation:
				‐ A significant increase in RBCs' vitamin E levels.
				‐ A significant increase in RBCs' glutathione levels.
				‐ A significant decrease in malondialdehyde levels in RBCs.

Note: This table summarises the diverse range of studies and approaches investigated for restoring RBC membrane integrity, including nutritional, lifestyle, supplementation, medication, and therapeutic strategies.

Abbreviations: DHA: docosahexaenoic acid; EPA: eicosapentaenoic acid; PUFAs: polyunsaturated fatty acids; RBC: red blood cells.

It is well established that dietary adjustment to increase the intake of PUFAs, particularly n‐3 fatty acids found in fish oil, flaxseed oil, and walnuts, can enhance cellular function and mitigate complications associated with metabolic disorders such as DM [55]. Improving RBC membrane fluidity may be a critical factor underlying these benefits. Cartwright et al. (1985) found that n‐3 supplementation significantly increased the levels of EPA (20:5w3) and DHA (22:6w3) in RBC phospholipids [50] (Table 2). Kamada et al. (1986) demonstrated that dietary consumption of n‐3 significantly increased membrane fluidity and levels of EPA and C22:5 in the phospholipid acyl‐chains of RBCs in both diabetic patients and healthy individuals [51] (Table 2). Moreover, Witte TR et al. (2010) revealed a linear, dose‐responsive increase in the fraction of n‐3 fatty acids in the RBC membrane upon n‐3 supplementation [52] (Table 2). Recently, McBurney MI et al. (2022) in a huge cross‐sectional study, reported that RBC Distribution Width which is related with membrane fluidity and deformability exhibited a significant inverse association with n‐3 in adjusted models suggesting that higher levels of omega‐3 fatty acids in red blood cell membranes are associated with a more uniform RBC size distribution, and a notable sex differences was observed, particularly in females, suggesting potential implications for establishing a dietary reference intake for EPA + DHA [53] (Table 2).

6.2. Antioxidant Supplementation

Antioxidants have been shown to have a number of beneficial effects, protecting against RBC lipid peroxidation and increasing levels of reduced glutathione (GSH) while reducing levels of ROS [56, 57]. Effects on RBC membrane permeability, however, have been largely unexplored. Antioxidant supplementation with vitamins C and E can reduce oxidative stress and prevent lipid peroxidation, thereby may help in maintaining or restoring membrane fluidity. Jain et al. (2000) demonstrated that vitamin E supplementation significantly increased RBC vitamin E and glutathione levels while reducing malondialdehyde levels in T2DM patients, indicating potential antioxidant benefits in mitigating oxidative stress in RBCs [54] (Table 2). These antioxidants and PUFAs can be consumed through a diet rich in fruits and vegetables or via supplements, highlighting the critical importance of monitoring dietary intake to prevent metabolic disorders such as obesity. Nutrition plays a pivotal role in overall health, influencing conditions from cardiovascular diseases to cancer [58]. Müller et al. first pointed out the effect of treatments on measured membrane fluidity by monitoring changes in the anisotropy of a fluorescent probe, 6‐anthroyloxystearic acid (6‐AS). They found that RBCs' membranes from patients with T2DM treated with metformin were more fluid than those from T2DM patients treated by diet alone or healthy controls [59]. These findings suggest that metformin may enhance membrane fluidity, which could improve the function of membrane‐embedded proteins and receptors, potentially contributing to its antihyperglycemic effects. This research underscores the importance of membrane fluidity in the action of metformin and its potential role in the management of DM.

6.3. Other Treatments

Anti‐glycation agents, such as aminoguanidine, can prevent the formation of AGEs that decrease membrane fluidity, helping maintain membrane integrity and function in diabetic patients [60].

Regular physical activity and lifestyle changes that promote cardiovascular health can positively impact membrane fluidity. Exercise helps reduce oxidative stress and improves lipid profiles, contributing to better membrane properties [61].

Specific drugs designed to modulate membrane fluidity directly can be explored, including new classes of compounds that integrate into lipid bilayers and alter their physical properties beneficially. Lipid modulation therapies that target lipid metabolism and composition, such as enzyme inhibitors or activators influencing the synthesis of membrane lipids, can be used to adjust the fatty acid composition and improve membrane fluidity. Although still largely experimental, gene therapy approaches targeting the regulation of lipid metabolism and oxidative stress at the genetic level could offer long‐term solutions for maintaining healthy membrane fluidity.

Despite the promising potential of these approaches, comprehensive studies specifically targeting the improvement of membrane fluidity through these therapeutic strategies have not yet been published.

7. Future Aspects

The findings of these studies highlight several promising directions for future research on RBCs' membrane fluidity in diabetic patients. There is a pressing need to standardise the measurement techniques for assessing membrane fluidity. This would involve developing universal protocols that harmonise the use of state‐based and composition‐based methods, ensuring consistency and comparability across studies. The integration of advanced technologies such as high‐resolution lipidomics with machine learning models could also help in creating comprehensive indices for membrane fluidity. Standardized procedures will enable the development of a unified biomarker that can be reliably used in clinical practice to monitor and manage complications in diabetic patients, improving diagnostic accuracy and therapeutic interventions [62].

This could involve adjusting dietary recommendations, pharmacological interventions, and lifestyle changes to optimise membrane fluidity and overall cellular function.

Advanced digital medicine, such as the development of Personalised Metabolic Avatars (PMAs) using gated recurrent unit (GRU) neural networks for weight forecasting, showcases the potential of technology in managing and preventing such conditions [63]. Deploying digital twins in clinical practice can aid in providing personalised dietary recommendations and interventions, optimising metabolic health [64]. However, the challenges in maintaining data integrity, model accuracy, and computational efficiency must be addressed. Studies have shown that while GRU and LSTM models provide stable predictive performance and manageable computational times, other models like Transformers, despite their potential, increase computational demands significantly. The consistent performance of PMAs underscores the necessity of leveraging these digital tools to ensure effective dietary monitoring and obesity prevention, emphasising a proactive approach in nutrition management [65].

In the context of future clinical applications, assessing red blood cell membrane fluidity could offer a novel biomarker for predicting the risk of developing diabetes, much like HbA1C is used to reflect past glycaemia over several months. Alterations in membrane fluidity are indicative of underlying metabolic disturbances, such as changes in lipid composition, glycation, and oxidative stress, all of which contribute to the pathophysiology of diabetes.

By integrating membrane fluidity assessments into routine clinical evaluations, it could be possible to identify individuals at higher risk for diabetes before significant metabolic dysfunction occurs, allowing for earlier and more personalised interventions to prevent or delay disease progression.

Further research is needed to explore the therapeutic potential of compounds that can modulate membrane fluidity. Studies on antioxidants like a‐lipoic acid have shown promise, but more comprehensive investigations are required to identify and validate additional agents that can restore or maintain optimal membrane fluidity in diabetic patients.

Additionally, expanding the use of advanced imaging and analytical techniques, such as high‐resolution lipidomics and enhanced spectroscopy methods, can provide deeper insights into the dynamic changes in membrane composition and fluidity [56]. These techniques could uncover new aspects of membrane behaviour under diabetic conditions, offering more detailed targets for intervention.

Collaborative, interdisciplinary research combining biophysics, biochemistry, clinical science, and computational modelling will be essential to advance our understanding of RBCs' membrane fluidity in DM. Integrating data across these fields can lead to a holistic view of the factors influencing membrane dynamics and identify synergistic approaches for therapeutic development.

Finally, longitudinal studies tracking changes in membrane fluidity and lipid profiles over time in diabetic patients can help elucidate the progression of membrane alterations and their relationship with clinical outcomes. Such studies would provide valuable data for refining predictive models and developing targeted treatment strategies to improve patient care.

8. Conclusion

The studies reviewed consistently demonstrate that both T1DM and T2DM often lead to increased membrane fluidity in RBCs. This increase is primarily driven by elevated cholesterol content, protein glycation, oxidative stress and changes in fatty acid composition. These interrelated factors compromise the structural and functional integrity of cell membranes, contributing to the pathophysiology of DM and its complications.

Understanding these mechanisms is crucial for developing therapeutic strategies aimed at restoring normal membrane fluidity and improving cellular function in diabetic patients. Future research should focus on integrating advanced techniques such as lipidomics and machine learning to create comprehensive models that predict changes in membrane fluidity based on lipidomic characteristics. Moreover, addressing the challenges of diverse measurement techniques and focussing on the implications of altered membrane fluidity can enhance our understanding and management of DM‐related complications.

Author Contributions

Conceptualisation, G.M.; methodology, G.M., D.P. and D.H.; writing: original draft preparation, D.P., G.M., and D.H.; review and editing, G.M., D.H., A.R., M.DG., A.R., A.A., C.S., L.T., E.R., L.P., and M.DS. All authors have read and agreed to the published version of the manuscript.

Ethics Statement

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/dmrr.70011.

Data Availability Statement

The data that support the findings of this study are openly available in Pubmed at https://pubmed.ncbi.nlm.nih.gov/.