Abstract / Summary
Introduction: Type 2 diabetes mellitus (T2DM) induces progressive biochemical and morphological alterations in red blood cells (RBCs), driven by hyperglycemia-induced oxidative stress and disrupted lipid–protein homeostasis. These changes compromise membrane integrity and serve as sensitive molecular indicators of disease severity. Yet, a non-invasive strategy for capturing such alterations at the molecular level remains an unresolved challenge. Objectives: This study employs Fourier Transform Infrared Spectroscopy (FT-IR) and Raman Spectroscopy (RS) to uncover sex- and age-specific molecular alterations in RBCs induced by T2DM. By integrating classical and advanced analytical techniques, we identify novel spectrochemical markers of disease progression, offering a non-invasive, label-free framework for staging and monitoring diabetic pathology at the molecular level. Methods: A total of 120 db/db and C57BL/6J mice were tested at 7, 12 and 24 weeks of age. Standard hematological and biochemical analyses were combined with vibrational spectroscopy (FT-IR, RS) and atomic force microscopy (AFM), to assess RBC morphology and molecular composition. Spectral data was analyzed using univariate and multivariate statistics to identify disease-specific spectral markers. Results: Partial Least Squares Discriminant Analysis reliably distinguished diabetic and control RBC profiles, confirming the presence of robust, disease-specific molecular patterns. Integration with hematological and biochemical parameters further validated the diagnostic relevance of this label-free, non-invasive approach. Structural protein analysis revealed a consistent decline in α-helical content and an increase in β-sheet and β-turn structures, reflecting protein misfolding and aggregation, particularly in older diabetic females. Alterations in disulfide bonding, hydrated β-sheets and H-bonded antiparallel β-sheets highlighted oxidative stress-mediated membrane destabilization. Additionally, elevated plasma triglyceride levels and increased saturation of RBC membrane lipids indicate impaired lipid handling. Conclusion: Vibrational spectroscopy enables non-invasive, molecular-level profiling of diabetes-induced RBC alterations, revealing sex- and age-dependent signatures of disease progression. This approach offers a promising platform for precise staging and stratification in metabolic disorders.