Sex- and Age-Dependent Red Blood Cell Remodeling in Type 2 Diabetes Revealed by FT-IR and Raman Spectroscopy: Part III

Using Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy, a research team identified sex- and age-specific molecular alterations in red blood cells from diabetic mice. Spectral analysis revealed protein misfolding, oxidative stress–related membrane destabilization, and lipid remodeling, establishing a non-invasive, label-free platform for molecular staging and monitoring of type 2 diabetes progression.

In the last of a three-part series, Spectroscopy spoke to authors of the resulting paper¹ about how FT-IR spectroscopy and Raman spectroscopy can track diabetes progression through spectral markers of protein aggregation and membrane rigidity, account for age- and sex-dependent variations in the db/db mouse model and address translational challenges in adapting murine spectrochemical signatures to human type 2 diabetes diagnostics.

What spectral changes would you expect to observe in FT-IR or Raman spectra as diabetes progresses from early to advanced stages, particularly in relation to protein aggregation and membrane rigidity?

As type 2 diabetes mellitus progresses from early to advanced stages, FT-IR and Raman spectra of RBCs exhibit systematic and coordinated changes reflecting cumulative alterations in protein conformation, lipid remodeling and membrane organization. These spectral modifications arise from chronic hyperglycaemia, oxidative stress and progressive disruption of lipid–protein homeostasis. Importantly, in our study, spectroscopic analyses were systematically correlated with hematological and biochemical measurements obtained from the same animals. This integrated experimental design demonstrates that spectroscopic markers directly reflect systemic metabolic dysregulation, thereby validating their biological relevance.

With respect to protein structure, early stages of diabetes are characterized by relatively preserved α-helical conformations, as reflected by dominant Amide I and Amide II bands associated with native folding states. Nevertheless, even at early time points, moderate increases in β-sheet and β-turn contributions may be detected, indicating the onset of structural remodeling and early misfolding processes. These initial changes suggest that protein destabilization begins before extensive aggregation becomes apparent.

As the disease advances, particularly in older diabetic animals, progressive broadening and shifting of the Amide I band are observed, reflecting increasing conformational heterogeneity and protein aggregation. A pronounced decline in α-helical content accompanied by significant enrichment in β-sheet, hydrated β-sheet, and H-bonded antiparallel β-sheet structures becomes evident. These spectral features are characteristic of aggregation-prone conformations and misfolded protein assemblies.

The accumulation of these aggregative secondary structures leads to redistribution and increased intensity within the 1600–1700 cm⁻¹ region, reflecting the presence of misfolded and aggregated protein populations. These changes are most pronounced in advanced disease stages and are particularly evident in older diabetic females, consistent with sex-dependent susceptibility to protein aggregation.

Alterations in membrane rigidity are primarily reflected in lipid-associated spectral markers. In FT-IR spectra, progressive reductions in lipid unsaturation, as indicated by decreased 3012/2925 ratios, reveal a shift toward more saturated membrane lipids. Concurrent changes in CH₂ and CH₃ stretching bands indicate tighter lipid packing and increased acyl chain ordering. These spectral features reflect reduced membrane fluidity and increased mechanical stiffness, and impaired cellular flexibility.

Raman spectroscopy provides complementary information, particularly through changes in the C–H stretching region and aromatic residue–associated bands, which reflect modifications in lipid saturation, protein packing and local molecular environments. In parallel, enhanced disulfide bond–related signals further indicate oxidative stress–driven modifications and compensatory stabilization mechanisms that accompany late-stage disease progression. Although Raman spectroscopy detects these trends, FT-IR spectroscopy, especially within the amide and lipid regions, offers superior statistical robustness and diagnostic sensitivity for staging disease progression.

Taken together, the spectroscopic transition from early to advanced diabetes is characterized by a gradual shift from predominantly α-helix–rich, moderately fluid membranes toward β-rich aggregated protein conformations and highly saturated, rigid lipid bilayers. These coordinated molecular changes reflect the cumulative effects of chronic metabolic stress, oxidative damage and disrupted lipid–protein interactions on RBC structure and function, enabling vibrational spectroscopy to serve as a powerful tool for molecular staging of diabetes progression.

How might age- and sex-dependent differences in RBC membrane composition influence vibrational spectroscopic signatures, and why is this stratification important in the db/db mouse model?

Across all our studies, including investigations on human erythrocytes, we have consistently demonstrated that sex-specific analyses are essential for the correct interpretation of biochemical and spectroscopic data. Our previous work has shown that susceptibility to disease- and stress-related molecular alterations differs markedly between males and females. For example, studies on stored human RBC concentrates² and on atherosclerosis-related pathology³ revealed faster ageing and more pronounced membrane deterioration in male-derived cells. Human male donor RBCs exhibited faster membrane lipid leakage, greater hemolysis, increased swelling and lower deformability throughout storage, whereas female donor cells maintained higher deformability, slower membrane lipid loss and greater resistance to storage-induced membrane deterioration. These pronounced differences indicate that male and female RBCs differ in their responses to biochemical and mechanical stress. Such sex-specific membrane properties have important implications for designing experiments and developing diagnostic spectroscopic biomarkers, as tests and models that do not account for sex may overlook critical differences relevant to personalized diagnosis and treatment in both men and women.

In the present study on type 2 diabetes, we observed a distinct pattern in which diabetes-induced molecular remodeling was more pronounced in RBCs and membranes derived from female db/db mice. Female animals exhibited stronger alterations in protein aggregation (including increased β-sheet content, hydrated β-sheets, and hydrogen-bonded antiparallel β-structures), lipid remodeling and disulfide bonding, particularly at advanced disease stages. This finding highlights that sex-dependent vulnerability is disease-specific and cannot be generalized across pathological conditions.

At the molecular level, age- and sex-dependent differences in RBC membrane composition influence vibrational spectroscopic signatures through variations in their biochemistry including visible by vibrational spectroscopy, protein folding, lipid saturation, lipid and protein content and lipid–protein interactions. Differences in hormonal regulation, oxidative stress responses, and lipid metabolism contribute to distinct trajectories of membrane remodeling in males and females. Age further amplifies these effects through cumulative oxidative damage, glycation, and lipid peroxidation, leading to progressive strengthening of sex-specific spectral patterns. Consequently, vibrational spectra represent integrated readouts of both biological aging and sex-dependent metabolic regulation rather than purely disease-related changes.

Stratification by age and sex is therefore essential in the db/db mouse model. It prevents masking of subgroup-specific effects, improves biomarker sensitivity, enables identification of distinct molecular pathways underlying disease progression, and enhances translational relevance. Our cumulative findings demonstrate that comprehensive age- and sex-specific analysis is indispensable for reliable spectroscopic staging and biomarker development in metabolic disease research.

However, it is worth mentioning that in one of our earlier studies, atherosclerosis-related alterations were investigated exclusively in male animals, based on prior reports suggesting minimal sex-related differences. In retrospect, we consider this approach a limitation. Since then, whenever funding has permitted, we have systematically included both sexes and increased experimental group sizes accordingly.

What are the main limitations of translating spectrochemical findings from the db/db mouse RBCs to human T2DM diagnostics, and how could future spectroscopic studies address these gaps?

The present study focuses exclusively on the db/db mouse model of type 2 diabetes mellitus, which was deliberately selected to enable deep and internally consistent phenotyping across sex and age within a well-controlled cohort. Because our experimental design compares untreated db/db mice with age-matched controls, the reported spectrochemical differences should be interpreted as robust associations between the diabetic metabolic milieu-including poor glycemic control, dyslipidemia, and oxidative stress-and RBC remodeling, rather than as effects attributable solely to hyperglycaemia. By integrating hematological, biochemical, AFM, FT-IR and Raman datasets, this model provides a robust platform for identifying spectrochemical signatures in db/db mice. Processes in db/db mice are broadly relevant to many forms of T2DM, and the core vibrational markers identified here are therefore expected to extend beyond this specific model. This multidimensional dataset therefore provides a solid foundation for subsequent validation studies in human diabetic populations and for assessing broader translational applicability.

Nevertheless, several important limitations constrain direct translation to human diagnostics. The db/db phenotype is driven by a leptin receptor mutation, resulting in a metabolic profile that captures only part of the heterogeneity observed in human T2DM. Consequently, diabetes-induced molecular alterations in mouse RBC membranes-including lipid remodeling, protein aggregation, and oxidative modifications-may not fully reflect those occurring in human erythrocytes. Moreover, fundamental species-specific differences in RBC metabolic activity and lipid handling influence vibrational signatures and complicate direct extrapolation.

Future studies can address these limitations by validating the identified spectrochemical markers in well-characterized human cohorts stratified by age, sex, disease stage and treatment status. Integration of vibrational spectroscopy with complementary approaches, such as proteomics and lipidomics, could additionally elucidate the molecular origins of key spectral features. In parallel, cross-validation in biologically distinct animal models, including ob/ob mice, may strengthen mechanistic understanding and improve robustness.

While the implementation of vibrational spectroscopy in routine clinical practice will require further advances in standardization, instrumentation and data analytics, ongoing developments-including portable platforms and artificial intelligence–enabled analytical pipelines-are rapidly increasing its feasibility. The translational potential of this approach is further supported by our previous studies demonstrating the successful application in different diagnostic areas. This includes the use of conventional Raman spectroscopy and spatially offset Raman spectroscopy (SORS) for non-invasive quality assessment of red blood cell concentrates using spectroscopic markers.^4-9 Furthermore, Raman spectroscopy of RBCs has shown strong potential in the point of care diagnosis and research of protozoan diseases, such as malaria and babesiosis.^10,11

Acknowledgments

This research was partially funded by the Polish National Science Centre, No. UMO-2020/38/E/ST4/00197. Part of the work was carried out at the Laboratory for Biomedical Spectroscopic Applications (LBSA), Faculty of Pharmacy, Jagiellonian University Medical College. The laboratory infrastructure was co-financed by the European Regional Development Fund under the European Funds for Malopolska 2021–2027 programme (project No.FEMP.01.04-IZ.00-0269/24). The methodologies applied in this study are based on spectroscopic approaches developed by the authors and protected by intellectual property rights, including international and national patent applications: PCT/PL2020/050093, P.436054 and P.43217, as well as a registered trademark (No. Z.493251).

Read the first part of the interview here.
Read the second part of the interview here.

References:

1. Wilk, A.; Wilkosz, N.; Rugiel, M. et al. Next-Generation Diabetes Diagnostics: Spectrochemical Staging of Red Blood Cells Using Vibrational Spectroscopy. J. Adv. Res. 2026, S2090-1232 (26), 00077-9. DOI: 10.1016/j.jare.2026.01.052
2. Szczesny-Malysiak, E.; Mohaissen, T.; Bulat, K. et al. Sex-Dependent Membranopathy in Stored Human Red Blood Cells. Haematologica 2021, 106, 2779–2782. DOI: 10.3324/haematol.2021.278895
3. Alcicek, F. C.; Mohaissen, T.; Bulat, K. et al. Sex-Specific Differences of Adenosine Triphosphate Levels in Red Blood Cells Isolated From ApoE/LDLR Double-Deficient Mice. Front Physiol 2022, 13, 839323. DOI: 10.3389/fphys.2022.839323
4. Wajda, A.; Dybas, J.; Bulat, K. et al. Method for Determining the Biochemical State of the Red Blood Concentrate. Red Blood Cells Spectroscopy Project Team. Jagiellonian Centre for Experimental Therapeutics. P.43217, 2019.
5. Blat, A.; Wajda, A.; Szczesny-Malysiak, E. et al. Method for Determining the Biochemical Status of Red Blood Cell Concentrates with the Use of Infrared Absorption Spectroscopy. Red Blood Cells Spectroscopy Project Team. Jagiellonian Centre for Experimental Therapeutics. P.436054, 2020.
6. Dybas, J.; Wajda, A.; Bułat, K. et al. Non–Invasive and Label–Free Method for Evaluation of Biochemical State of Packed Red Blood Cells. Red Blood Cells Spectroscopy Project Team. Jagiellonian Centre for Experimental Therapeutics. PCT/PL2020/050093, 2020.
7. Wajda, A.; Dybas, J.; Bulat, K. et al. Reliability of Tube-Based Quality Assessment of Packed Red Blood Cells: Insights from FTIR and Raman Spectroscopic Analyses. Spectrochim Acta A Mol Biomol Spectrosc 2026, 352, 127564. DOI: 10.1016/j.saa.2026.127564
8. Wajda, A.; Dybas, J.; Kachamakova-Trojanowska, N. et al. Raman Imaging Unveils Heme Uptake in Endothelial Cells. Sci. Rep. 2024, 14 (1), 20684. DOI: 10.1038/s41598-024-71600-2
9. Dybas, J.; Wajda, A.; Alcicek, F. C. et al. Label-Free Testing Strategy to Evaluate Packed Red Blood Cell Quality Before Transfusion to Leukemia Patients. Sci. Rep. 2022, 12, 21849. DOI: 10.1038/s41598-022-26309-5
10. Perez-Guaita, D.; Marzec, K. M.; Hudson, A. et al. Parasites Under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research. Chem. Rev. 2018, 118, 5330–5358. DOI: 10.1021/acs.chemrev.7b00661
11. Ruther, A.; Perez-Guaita, D.; Poole, W. A. et al. Vibrational Spectroscopic Based Approach for Diagnosing Babesia Bovis Infection. Anal. Chem. 2020, 92, 8784–8792. DOI: 10.1021/acs.chemrev.7b00661