Reactive oxygen species damaged human serum albumin in patients with type 1 diabetes mellitus: biochemical and immunological studies.
The role of hydroxyl radical (.OH) damaged human serum albumin (HSA) in type 1 diabetes has been investigated in the present study. Hydroxyl radical induced modification on HSA has been studied by UV absorption spectroscopy, ANS fluorescence and carbonyl estimation. Hydroxyl radical modified HSA was found to be highly immunogenic in rabbits as compared to native HSA. The binding characteristics of circulating autoantibodies in type 1 diabetes patients against native and modified HSA were assessed. Diabetes patients (n=31) were examined by direct binding ELISA and the results were compared with healthy age-matched controls (n=22). High degree of specific binding by 54.8% of patients sera towards .OH modified HSA, in comparison to its native analogue (p<0.05) was observed. Sera from those type 1 diabetes patients having smoking history, high aging with high degree of disease showed substantially stronger binding to .OH modified HSA over native HSA in particular. Normal human sera showed negligible binding with either antigen. Competitive inhibition ELISA reiterates the direct binding results. Gel retardation assay further substantiated the enhanced recognition of modified HSA by circulating autoantibodies in diabetes patients. The increase in total serum protein carbonyl levels in the diabetes patients was largely due to an increase in oxidized albumin. HSA of diabetes mellitus patients (DM-HSA) and normal subjects (normal-HSA) were purified on a Sephacryl S-200 HR column. Spectroscopic analysis confirmed that the DM-HSA samples contained higher levels of carbonyls than normal-HSA (p<0.001). DM-HSA was conformationally altered, with more exposure of its hydrophobic regions. Collectively, the oxidation of plasma proteins, especially HSA, might enhance oxidative stress in type 1 diabetes mellitus patients.
Rasheed
Zafar
Z
Department of Biochemistry, Faculty of Medicine, J.N. Medical College, A.M.U. Aligarh 202002, India. zrasheed@rediffmail.com
Ali
Rashid
R
D000328:Adult; D000818:Animals; D001323:Autoantibodies / Q000097:blood; D003922:Diabetes Mellitus, Type 1 / Q000097:blood* / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D017665:Hydroxyl Radical / Q000097:blood / Q000276:immunology; D008297:Male; D008875:Middle Aged; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology*; D050050:Protein Carbonylation; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000097:blood* / Q000276:immunology; D012709:Serum Albumin / Q000276:immunology / Q000378:metabolism*
D001323:Autoantibodies; D017382:Reactive Oxygen Species; D012709:Serum Albumin; D017665:Hydroxyl Radical