Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy

• Innovative approach to study the process of human erythrocytes disintegration was applied.
• Human RBC were studied as consisting of two components: homogeneous interior filled with hemoglobin and closure -the cell membrane.
• 2D Raman correlation method was used to determine the influence of the natural aging process on the human healthy blood cells. The role of the senescent-cell antigen (SCA) was commented.
• 2D correlation EPR spectra of the whole blood show the participation of the metal ion centers in the disintegration process.

The most numerous elements of the blood cells, erythrocytes, consist mainly of two components: homogeneous interior filled with hemoglobin and closure which is the cell membrane. To gain insight into their specific properties we studied the process of disintegration, considering these two constituents, and comparing the natural aging process of human healthy blood cells.MicroRaman spectra of hemoglobin within the single RBC were recorded using 514.5, and 785 nm laser lines. The generalized 2D correlation method was applied to analyze the collected spectra. The time passed from blood donation was regarded as an external perturbation. The time was no more than 40 days according to the current storage limit of blood banks, although, the average RBC life span is 120 days. An analysis of the prominent synchronous and asynchronous cross peaks allow us to get insight into the mechanism of hemoglobin decomposition. Appearing asynchronous cross-peaks point towards globin and heme separation from each other, while synchronous shows already broken globin into individual amino acids.Raman scattering analysis of hemoglobin “wrapping”, i.e. healthy erythrocyte ghosts, allows for the following peculiarity of their behavior. The increasing power of the excitation laser induced alterations in the assemblage of membrane lipids. 2D correlation maps, obtained with increasing laser power recognized as an external perturbation, allows for the consideration of alterations in the erythrocyte membrane structure and composition, which occurs first in the proteins. Cross-peaks were observed indicating an asynchronous correlation between the senescent-cell antigen (SCA) and heme or proteins vibrations.The EPR spectra of the whole blood was analyzed regarding time as an external stimulus. The 2D correlation spectra points towards participation of the selected metal ion centers in the disintegration process.