Nonenzymatic glycosylation of human serum albumin and its effect on antibodies profile in patients with diabetes mellitusReport as inadecuate




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Background

Albumin glycation and subsequent formation of advanced glycation end products AGEs correlate with diabetes and associated complications.

Methods

Human Serum Albumin HSA was modified with D-glucose for a 40 day period under sterile conditions at 37°C. Modified samples along with native HSA unmodified were analyzed for structural modifications by UV and fluorescence, FTIR, Liquid chromatography mass spectrometry LCMS and X–ray crystallography. New-Zealand white female rabbits immunized with AGEs, represent auto-antibodies formation as assessed by competitive and direct binding enzyme-linked immunosorbent assay ELISA. Neo-epitopesagainst In-vitro formed AGEs were characterized in patients with diabetes mellitus type 2 n = 50, type 1 n = 50, gestational diabetes n = 50 and type 2 with chronic kidney disease CKD with eGFR level 60–89 mL-min n = 50 from serum direct binding ELISA.

Results

Glycated-HSA showed amarked increase in hyperchromicity of 65.82%,71.98%, 73.62% and 76.63% at λ280 nm along with anincreasein fluorescence intensity of 65.82%, 71.98%, 73.62% and 76.63% in glycated-HSA compared to native. FTIR results showed theshifting of Amide I peak from 1656 cm 1 to 1659 cm 1 and Amide II peak from 1554 cm 1 to 1564 cm 1 in glycated-HSA, with anew peak appearance of carbonyl group at 1737 cm-1. LCMS chromatogram of glycated-HSA showed thepresence of carboxymethyl lysine CML at 279.1 m-z. Immunological analysis showed high antibody titre>1:12,800 in theserum of rabbits immunized with glycated-HSA modified with 400 mg-dL glucose and inhibition of 84.65% at anantigen concentration of 20μg-mL. Maximum serum auto-antibody titre was found in T2DM 0.517±0.086, T1DM 0.108±0.092, GDM 0.611±0.041 and T2DM+CKD 0.096±0.25 patients immunized with glycated-HSA modified with 400 mg-dL glucose.

Conclusions

Non-enzymatic glycosylation of HSA manifests immunological complications in diabetes mellitus due to change in its structure that enhances neo-epitopes generation.



Author: Alok Raghav, Jamal Ahmad , Khursheed Alam

Source: http://plos.srce.hr/



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