Hemoglobin Structure at Higher Levels of Hemoglobin A1C in Type 2 Diabetes and Associated Complications

Diabetes mellitus, particularly type 2 diabetes, represents a significant global health burden. The disease is characterized by chronic hyperglycemia, which contributes to long-term complications such as cardiovascular diseases, neuropathy, and retinopathy. Central to diabetes management is the monitoring of hemoglobin A1C (HbA1C), a glycated form of hemoglobin that reflects average blood glucose levels over the preceding 2–3 months. While HbA1C is widely used as a diagnostic and prognostic marker, its direct impact on hemoglobin (Hb) structure and subsequent pathophysiological consequences remains understudied. This study employs Fourier transform infrared (FTIR) spectroscopy to investigate structural alterations in hemoglobin at elevated HbA1C levels in type 2 diabetes, providing insights into the molecular mechanisms underlying diabetic complications.

Methodology and Experimental Design

Study Population and Sample Preparation

The study enrolled 53 type 2 diabetic patients from Bahawal Victoria Hospital, Pakistan, categorized into two groups based on HbA1C levels: Group A (6% < HbA1C < 7%; n = 25) and Group B (HbA1C ≥9%; n = 28). A control group (Group N) comprised 20 healthy volunteers from The Islamia University of Bahawalpur. Blood samples were collected between January 2018 and March 2019. Exclusion criteria included chronic infections, cancer, renal disorders, hematological abnormalities, and smoking.

Whole blood samples were centrifuged to isolate erythrocytes, which were washed with 0.9% NaCl and lysed to extract hemoglobin. Purification involved gel filtration chromatography, yielding Hb with >95% purity confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To model hyperglycemia, erythrocytes from healthy donors were incubated with glucose concentrations of 8.5, 15.0, and 30.0 mmol/L for seven days, simulating diabetic conditions.

FTIR Spectroscopy Analysis

FTIR spectroscopy was performed using a Bruker Tensor 27 spectrometer. Hemoglobin samples were mixed with potassium bromide (KBr) at a 1:100 mass ratio and pressed into pellets. Spectra were acquired over 400–4000 cm⁻¹, with 64 scans per sample at 25°C. Background corrections for atmospheric water vapor and CO₂ were applied automatically. Data preprocessing included Savitsky-Golay smoothing and baseline correction. The amide I region (1600–1700 cm⁻¹) was analyzed for secondary structure components (α-helix, β-sheet) using Gaussian curve fitting in PeakFit V4.0 software.

Statistical Analysis

Clinical parameters (body mass index, fasting plasma glucose, HbA1C) and spectral data were compared across groups using ANOVA and rank-sum tests (SPSS v23). Significance was set at P < 0.05.

Key Findings

Structural Changes in Hemoglobin

FTIR spectra revealed distinct differences in hemoglobin secondary structure between diabetic and control groups. In Group N (healthy controls), the amide I band exhibited peaks characteristic of native hemoglobin: α-helix (1654 cm⁻¹), β-sheet (1630 cm⁻¹), and random coils (1645 cm⁻¹). Group A (HbA1C 6–7%) showed minor deviations, with a slight reduction in α-helix content (52% vs. 55% in controls) and a marginal increase in β-sheet structures (28% vs. 25%). These changes were not statistically significant (P > 0.05).

In contrast, Group B (HbA1C ≥9%) demonstrated pronounced structural alterations. The α-helix content dropped to 43%, while β-sheet structures increased to 35% (P < 0.05). Derivative spectra further highlighted these shifts: the negative peak at 1609 cm⁻¹ in controls became positive in diabetic groups, indicating destabilization of hydrogen bonding networks. Peaks at 1670 cm⁻¹ and 1682 cm⁻¹, associated with β-turns and antiparallel β-sheets, intensified in Group B, suggesting progressive protein misfolding.

Erythrocyte Morphology and Functional Implications

Microscopic analysis revealed morphological changes in erythrocytes correlated with HbA1C levels. Control erythrocytes (Group N) displayed typical biconcave morphology, whereas Group A cells showed slight rounding. Group B erythrocytes exhibited marked spherical transformation and reduced deformability, consistent with increased membrane rigidity. These structural changes were mirrored in hyperglycemic models, where erythrocytes incubated with 30 mmol/L glucose lost their flexibility and adopted a circular shape.

Clinical Correlations

HbA1C levels in Group B (11.93 ± 2.10%) were significantly higher than in Group A (6.05 ± 0.66%) and controls (4.69 ± 0.31%). Fasting plasma glucose followed a similar trend: 10.21 ± 1.58 mmol/L (Group B) vs. 7.21 ± 1.34 mmol/L (Group A) and 5.27 ± 0.62 mmol/L (Group N). Body mass index did not differ significantly between diabetic groups, emphasizing glycemic control as the primary variable influencing hemoglobin structure.

Mechanistic Insights and Pathophysiological Relevance

Glycation-Induced Protein Misfolding

The study posits that chronic hyperglycemia promotes non-enzymatic glycation of hemoglobin, leading to the accumulation of advanced glycation end products (AGEs). Glycation alters hemoglobin’s tertiary structure by cross-linking lysine and arginine residues, destabilizing α-helical domains, and promoting β-sheet formation. This structural shift reduces hemoglobin’s solubility and oxygen-binding capacity, impairing erythrocyte function.

Implications for Diabetic Complications

Rigid, spherical erythrocytes in Group B compromise microcirculation, particularly in capillary networks requiring cellular deformability. This hemodynamic impairment exacerbates tissue hypoxia, a key contributor to diabetic retinopathy and nephropathy. Furthermore, glycated hemoglobin may potentiate oxidative stress by generating reactive oxygen species during autoxidation, creating a vicious cycle of cellular damage.

FTIR Spectroscopy as a Diagnostic Tool

The study underscores FTIR spectroscopy’s utility in detecting preclinical structural changes in hemoglobin. Traditional HbA1C assays quantify glycation but provide no information on molecular conformation. FTIR-derived metrics, such as α-helix/β-sheet ratios, could serve as early biomarkers for diabetes progression and therapeutic efficacy.

Conclusion

Elevated HbA1C levels in type 2 diabetes induce significant structural remodeling of hemoglobin, characterized by α-helix reduction and β-sheet proliferation. These changes correlate with erythrocyte dysfunction and microvascular complications, highlighting the importance of stringent glycemic control. FTIR spectroscopy emerges as a powerful tool for elucidating the molecular pathology of diabetes, offering potential applications in personalized medicine and drug development.

doi.org/10.1097/CM9.0000000000000801

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