Disposition of protein-bound 3-nitrotyrosine in rat plasma analysed by a novel protocol for HPLC-ECD

金沢大学医薬保健研究域医学系3-Nitrotyrosine (NTyr) is considered as a biomarker of the generation of reactive nitrogen species (RNS). However, it is still difficult to determine its concentration in biological samples. To develop a reliable and high-throughput method, we optimized the conditions for high performance liquid chromatography and electrochemical detection (HPLC-ECD). The best separation of NTyr was achieved using a highly acidic mobile phase (pH 2.5). The concentration of protein-bound NTyr in plasma protein was 593.6 ± 53.8 fmol/mg in rats treated with lipopolysaccharide (LPS) and 114.4 ± 27.6 fmol/mg in control. After intravenous administration of in vitro-nitrated plasma protein, NTyr concentration decreased; the half-life was 63.4 ± 16.8 h. Consistently, protein-bound NTyr concentration in plasma after LPS treatment declined gradually, but was detectable for 1 week. Our protocol is reproducible and suitable for analysing multiple clinical samples to study RNS production in vivo. © 2007 The Japanese Biochemical Society

pH Profile of cytochrome c-catalyzed tyrosine nitration

In the present study, we investigated how cytochrome c catalyzed the nitration of tyrosine at various pHs. The cytochrome c-catalyzed nitration of tyrosine occurred in proportion to the concentration of hydrogen peroxide, nitrite or cytochrome c. Thecytochromec-catalyzed nitration of tyrosine was inhibited by catalase, sodium azide, cystein, and uric acid. These results show that the cytochrome c-catalyzednitrotyrosine formation was due to peroxidase activity. The rate constant between cytochrome c and hydrogen peroxide within the pH range of 3 - 8 was the largest at pH 6 (37°C). The amount of nitrotyrosine formed was the greatest at pH 5. At pH 3, onlycytochromec-independent nitration of tyrosine occurred in the presence of nitrite. At this pH, the UV as well as visible spectrum of cytochrome c was changed by nitrite, even in the presence of hydrogen peroxide, probably via the formation of a heme iron - nitric oxide complex. Due to this change, the peroxidase activity of cytochrome c was lost