Quantitative analysis of cardiac 3-L-nitrotyrosine during acute allograft rejection in an experimental heart transplantation

Recent studies have shown that nitric oxide interacts with superoxide to form peroxynitrite, a potent oxidant that modifies cellular proteins producing 3-L-nitrotyrosine (N-Tyr). This study was designed to evaluate N-Tyr quantitatively with high-performance liquid chromatography (HPLC) during cardiac allograft rejection. Rat transplanted hearts (allogeneic or syngeneic grafts) were examined with HPLC analysis, immunohistochemistry for N-Tyr, and histological studies on 0, 1, 3, and 7 days after transplantation. No histological rejection was found in syngeneic grafts, or day 0 or 1 allografts. HPLC demonstrated that N-Tyr in allografts increased on day 1 and continued to increase through day 7, while N-Tyr was not detected in any syngeneic grafts. Immunostaining of the allografts did not show N-Tyr on day 1. These results demonstrate that N-Tyr shows a time-dependent accumulation in cardiac allografts during acute rejection. N-Tyr detection using HPLC may be an useful maker for early diagnosis of acute rejection before pathological rejection occurs.