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The minor fraction of methylglyoxal that is not metabolized in healthy humans reacts with macromolecules to form AGEs. In diabetics, the formation of MG is accelerated; its level may be enhanced multifold. The glyoxalase enzymes responsible for the regular and effective clearance of excess methylglyoxal may become defective in diabetes mellitus leading to its retention in cells and plasma. The methylglyoxal-modified-HSA was prepared, characterised by multiple biophysical techniques and biochemical (s) and its damaging effect was examined on embryonic kidney cell line HEK 293. The UV results showed hyperchromicity in MG-modified-HSA while nitroblue tetrazolium and fluorescence data suggested AGEs formation in comparison to control HSA. Upward shift of negative peaks in CD suggested reduction in α-helicity. Accelerated mobility and diffused broad bands observed in native and SDS polyacrylamide gel, respectively suggest neutralization of some of the positive charges on MG-modified-HSA as well as generation of cross-links. As observed by trypan blue assay, MTT, LDH activity assay, acridine orange, propidium iodide, ethidium bromide, 4',6-diamidino-2-phenylindole (DAPI) staining and ROS measurements, the MG-HSA AGEs caused damage to human embryonic kidney cells. The data suggest that MG-HSA AGEs may trigger powerful inflammatory responses at cellular level which might set the stage for nephrotoxicity in diabetics. |
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