ИСТИНА

The blood measurement of cardiac troponin T (cTnT) is one of the most reliable methods of acute myocardial infarction (AMI) diagnosis. cTnT is known to be susceptible to the proteolytic degradation, but there is still no consistent data describing the level and sites of degradation of cTnT molecules that are present in the blood of AMI patients. Meanwhile, degradation might have a significant influence on the precise immunodetection of cTnT. In this study we aimed to border the cTnT fragments present in the blood of AMI patients and to quantify their relative abundance at different time periods after AMI. Serial heparin plasma samples were collected from AMI patients over a period of 3-31 hours following the onset of AMI. cTnT and its fragments were studied by means of Western blotting and sandwich immunofluorescent analysis (IFA) that utilized monoclonal antibodies (mAbs) which were specific to different portions of the cTnT molecule. In the blood of AMI patients we were able to discriminate approximately 23 proteolytic fragments of cTnT with relative molecular masses of 10-30 kDa, along with “full-size” 2-287 amino acid residues (aar) cTnT of ~37 kDa. Three major regions of cTnT degradation were observed: The first one was between aar 68 and 69, the second one – near aar 164 and the third one (which contained several sites of degradation) was between aar 190 and 223. Immunostaining with mAbs that were specific to the N-terminal and central parts of cTnT revealed the formation of fragments with relative molecular masses of 25-29 kDa (the most abundant fragments were ~2-164, ~2-190 aar, ~2-223 aar and 69-287 aar) and of 12-20 kDa (the most abundant fragments were ~69-164, ~69-190 to ~69-223 aar). Immunostaining of cTnT with mAbs that interact with the C-terminal part of cTnT (epitopes lay within 223-288 aar) showed the presence of seven C-terminal fragments of 8-14 kDa that appeared as a result of cleavage at several sites located between aar 190 and 223. Immunostaining of cTnT from serial samples of AMI patients with the mAb 329 that was specific to the central part of the molecule (epitope 119-138 aar) revealed that the ratio of the full-size cTnT (2-287 aar) decreased by more than one half from ~65% at the early time point (5±1.7 hours after AMI, mean±SD) to ~25% at the late time point (29±3.2 hours after AMI). The ratio of the 25-29-kDa fragments remained unchanged and represented ~15% of all detected cTnT. The ratio of the 12-20 kDa central fragments increased from ~10% to ~55% (among those the ratio of the shortest fragment (aar 69-164) increased from ~5% to ~15%). Taken together, the data suggest that the region located approximately between aar 69 and 164 of cTnT is a promising target for the antibodies to be used in cTnT immunoassays.