Thermal and morphological properties of human erythrocytes from patients afflicted with type 1 diabetes mellitus

Abstract

Red blood cells (RBC), are the most unique and abundant cell types. The diameter of RBCs is 7-8 μm. They have an essential role in transporting circulatory oxygen. The RBCs travel through varioussized capillaries throughout the entire body, some significantly smaller than the RBCs due to their incredible deformability. The RBC membrane allows the cell to resist stress as it squeezes the cytoplasm. Permanent stress or altered blood plasma conditions can result in decreased membrane deformability. Type 1 diabetes mellitus (T1DM) is one of the most common chronic diseases. In diabetes, the primary influence is increased glucose levels in the blood plasma that can result in the oxidation of lipids and proteins and the glycation of proteins. The damage changes the conformation and organization of various lipids and proteins, which can result in the loss of function and decreased deformability. Hemoglobin A1c (HbA1c) or glycohemoglobin is a form of hemoglobin found in RBCs. Glucose and fructose can bind to hemoglobin by non-enzymes, and different glycated forms of hemoglobin can be formed. The ratio of glucose-bound (glycated) hemoglobin to total hemoglobin (expressed as a percentage) is a critical laboratory parameter in managing diabetes. It can be used to determine the average blood glucose level of the patient over the past 60-120 days. Here, we investigate the effect of diabetes on RBCs' shape and membrane stability due to microscopy and DSC (Differential Scanning Calorimetry) methods. The comparison of the RBCs from diabetic and non-diabetic patients was classified by the HbA1c, showing that the conditions in diabetes caused atypical cell morphology and then, in a casedependent manner, increased or decreased the thermal stability of cytoplasm or the cell membrane, respectively. It shows the importance of DSC application in routine quality screening of diabetic erythrocytes and that it can be a crucial parameter of T1DM.

Keywords: DSC; Morphology; RBC; Thermal stability; diabetes.

Graphical abstract

Fig. 1

Microscopic images of blood smears. (A) The erythrocytes from patients without diabetes (HbA1c below 5.7 % or 39 mmol/mol). (B) Erythrocytes of patients with mild diabetes (HbA1c 6–8% or 42–64 mmol/mol). (C) Altered morphological presentation of stomatocytes and discocytes from the sample of patients with severe diabetes (HbA1c above 10 % or 86 mmol/mol). Size bars 14 μm. (D) Box-diagram analysis of cell and central concavity diameters based on images from patients without diabetes, patients with mild and severe diabetes cases. Box normal diagrams represent data with ±SD (error bars), average value (points), and median values (lines). Asterisks mean statistically significant differences between groups. Significance was defined as p values < 0.05. (E) The gender of the patients was not considered a factor in the statistical analysis of the data. A high number of plasma-bridges and cell-cell interactions were observable in the case of patients with severe diabetes. Size bar 14 μm.

Fig. 2

Hemoglobin, membrane, and cytoplasmic components. DSC scans of RBCs from patients without diabetes (HbA1c below 5.7 % or 39 mmol/mol) (blue dotted line), patients with mild diabetes (HbA1c 6–8% or 42–64 mmol/mol) (red line) and patients with severe diabetes (HbA1c>10 % or 86 mmol/mol) (green line). The curves are the average of five independent samplend normalized on total sample mass. The patients without diabetes are the controls.

Fig. 3

The modular structure of hemoglobin. (A) The structure of non-glycated hemoglobin (PDB ID: 1XXT) is the composition of four domains α1,2 and β1,2, with the O₂ binding hem complexes localized in all of them. (B) Structural differences between the non-glycated (PDB ID: 1XXT) and glycated (PDB ID: 5HY8) hemoglobin are remarkable. All the valine and lysin residues (magenta) can be target points of glycation. One possible glucose-binding cleft was identified around the residue of Thr38 in β2 chains (PDB ID: 3B75). (lower panel) One structurally sensitive fructose binding cleft was found at Lys138 and neighboring chains in α1, non-glycated (gray), and glycated (pink).