Measured Spectrum Of Hemoglobin In Diabetic Microvascular Complications And Analyzed Impact Factor

Hemoglobin?Hb? is an important functional protein in the red blood cells of the circulatory system.Slight changes in the surrounding environment will affect its molecular structure and function.Patients with diabetic microvascular complications have a long-term disturbance of the blood microenvironment,and Hb may be affected.To understanding the changes of hemoglobin comformation is conducive to explored the correlation between Hb and the occurrence and development of diabetic microvascular complications,and also can provide an experimental basis for exploring the pathogenesis of diabetic microvascular complications.A case-control study was used to analyze the structural changes of Hb and its influencing factors in patients with diabetic microvascular complications.Healthy and sex-matched healthy people and diabetic patients were selected as controls;patients with diabetic nephropathy?DN?,diabetic retinopathy?DR?,and diabetic peripheral neuropathy?DPN?as subjects.Collected general demographic indicators,blood lipid and blood glucose levels and measured plasma and red blood cell GSH,SOD,and MDA levels to study the influencing factors of Hb spectra.G-75 sephadex was used to purify Hb,and then the spectroscopy of Hb was detected by ultraviolet-visible absorption spectrum?UV-vis?and Fourier transform infrared spectroscopy?FTIR?.The change of Hb spectrum was analyzed by comparing the absorption intensity,band frequency shift,and characteristic band with control group to infer the conformational change of Hb.Last but not least,correlation analysis was used to study the correlation between glycemic control,blood lipid levels,oxidative stress and Hb spectral absorption intensity and conformational changes.Stratified by HbA1c levels<8%to explore the effect of oxidative stress on Hb spectra in DN,DR,DPN.This study mainly includes two major parts:1.Measured UV-vis spectrum and FTIR spectrum of Hb in H,T2DM,DN,DR and DPN.The differences of Hb UV-vis spectrum and FTIR spectroscopy between each group were compared and analyzed to analyze the changes of corresponding bands.?1?There were 5 peaks in Hb UV-Vis spectra.Compared with H and T2DM,the absorption intensity in patients with DN,DR,and DPN were significantly decreased.The soret peak were blue-shifted that the maximum frequency shift was 6 nm in DN,DR,DPN group.DN group had the lowest absorption intensity of the Hb UV spectrum,while the soret peak had the highest blue shift.These changes indicated that Hb structure were changed in patients with diabetic nephropathy,diabetic retinopathy and diabetic peripheral neuropathy,leading to hydrophobic state changed,heme exposed in solution and hemoglobin disturbed,which resulted Hb oxygen binding function decline.?2?Hb FTIR spectra has 13 absorption peaks.Compared with healthy controls and T2DM,region in 3067cm^-11 which represented N-H group were red-shift to 3062cm^-11 in patients with DN,DR and DPN,while I₃₃₀₄/I₃₀₆₇067 were significantly decreased,indicating the relative change of N-H content and the degree of hydrogen bonding of N-H groups were increased.Both-CH2 symmetrical stretching and CH3symmetrical stretching were red-shifted and I₂₉₆₃/I₂₈₇₆876 significantly reduced,indicating-CH2 relative content increased and-CH3 content relatively decreased,which represented Hb conformation changed.In Amide bands,both 1541cm-1 and 1312cm-1 have a maximum redshift of7cm^-1,and 1655cm^-11 has a slight blue shift of 3cm^-11 as well as I₁₆₅₅/I₁₅₄₁reduced which indicated that the hydrogen bonds of the secondary structure are destroyed and formed which resulted Hb conformation to become Loose and disordered.In the 1165cm-1 band,blue-shifted was observed and I₁₁₆₅/I₁₁₁₁111 shown significantly increased that the hydrogen bonds between amino acid residues and other functional groups were destroyed,leading to the group binding force decline and the structure of amino acid residues transformation and the instability of Hb structure.In Amide I band,the?-helix which represented the ordered structure in the secondary structure was significantly reduced,but the?-sheet,?-turns and random coils were increased which resulted an increase structural instability and aggregation.2.To study the impact factors of Hb spectrum variation,some statistical analysis was used to the effects of blood glucose,blood lipid levels and oxidative stress on the absorption intensity and secondary structure content of Hb spectra.Afterwards,sub-group analysis was used to investigate the effects of glycemic control and oxidative stress on Hb spectrum in DN,DR,DPN patients.?1?Through statistical analysis,FPG,HbA1c,TG,plasma and red blood cell GSH,SOD levels are significantly increased,and plasma and red blood cell MDA levels are significantly decreased compared to group H and T2DM.The absorption intensity of try/tyr,?subunit,and?subunit peaks in Hb UV-vis spectrum are related to the level of Hb A1c?|r|>0.7,P<0.05?,and red blood cell GSH,SOD,MDA?|r|>0.5,P<0.05?.The decreased of?-helix content are associated with the levels of HbA1c,plasma and red blood cell GSH,SOD,MDA?|r|>0.5,P<0.05?which indicated that HbA1c,GSH,SOD and MDA levels were impact factors in Hb spectrum changes in this study.?2?In sub-group analysis,we reported that the level of oxidative stress in patients with diabetic microvascular complications with HbA1c<8%was significantly lower.Compared with those who glycemic control inadequate,adequate glycemic control in patients with DR and DPN had higher intensity of Hb UV-vis spectrum,and also the FTIR peak shift was smaller,and the?-helix content was higher which indicate that the Hb structure associated with HbA1c and oxidative stress.However,blue-shifted were observed in adequate glycemic control patients with DN on soret bands of the Hb UV-vis spectra.But the FTIR bands frequency shift is similarly,and the?-helix content did not observe significant difference,indicating Hb structure is easily affected even though adequate glycemic control,and there may be other factors that cause Hb conformational changes in DN patients.