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Abstract Background Troponin I (TnI) is a thin filament protein which, together with troponin T (TnT) and troponin C (TnC), forms troponin complex and plays a crucial role in the regulation of muscle contraction. In humans, there are three isoforms of TnI: cardiac TnI (cTnI), which is expressed only in the heart, and two skeletal – fast (fsTnI) and slow (ssTnI) skeletal, which are present in fast-twitch and slow-twitch muscles, respectively. While cTnI is widely used for determination of myocardial damage, skeletal TnIs could be utilized as promising biomarkers of skeletal muscle diseases as in contrast to currently used proteins (creatine kinase and myoglobin) they are strictly specific to skeletal muscles. To develop reliable immunodetection methods, it is important to determine in what forms skeletal TnIs exist in the blood of patients: are they present as free proteins or are complexed with TnC (IC) or with TnC and TnT (ITC). It is also necessary to find out whether skeletal TnIs appear in blood as full-sized proteins or as partially proteolyzed fragments. The aim of this study was to investigate skeletal TnIs present in the blood of patients with skeletal muscle injuries. Methods Sera samples were collected 24 h after hip endoprosthesis surgery and subjected to gel filtration (GF). The fractions obtained were subsequently analyzed with various sandwich fluoroimmunoassays that recognize only fsITC (skTnI14-TnC99A5), only ssITC (TnT111-skTnI38), both free and complexed fsTnI (skTnI89-skTnI6), or free and complexed ssTnI (skTnI58-skTnI27). Skeletal troponins were also extracted from the sera samples using affinity matrix utilizing 24 different mAbs, specific to the various epitopes of fsTnI or ssTnI. Probes were further subjected to western blotting (WB), and troponins were visualized using anti-fsTnI mAbs recognizing central (skTnI89, epitope 86-105) or C-terminal (skTnI11, 142-161) portions of the protein; mAbs that are specific to the central (skTnI77, 44-63) or C-terminal (skTnI38, 170-187) parts of ssTnI; TnT-specific mAb. Results On GF profiles of patients’ sera, major peaks with elution volume of ∼78 ml corresponding to binary fsIC and ssIC were recognized while no fsITC, ssITC or free fsTnI and ssTnI were observed. Both isoforms of skeletal TnI were successfully extracted from patients’ sera and visualized on WB with anti-fsTnI and anti-ssTnI mAbs. Neither fsTnT, nor ssTnT were detected in these probes, confirming the results of GF. FsTnI was presented as a major full-size band and proteolyzed fragments of 12-20 kDa, the latter being detected by mAbs specific both to the central and C-terminal parts of the molecule. SsTnI was also presented by a major full-size band and proteolyzed fragments of 12-20 kDa, but, in contrast to fsTnI, fragments were visualized only by the mAb that was specific to the central part of the molecule. Conclusion 24 h after skeletal muscle injury, both fsTnI and ssTnI are presented in human blood as binary complexes with TnC. Both fsTnI and ssTnI undergo partial proteolysis; while ssTnI is cleaved at the C-terminal part of the molecule, fsTnI is likely to be partially cleaved at its N-terminus.