RESUMO
Oxidative stress plays an important role in pathogenesis of many diseases. Measurement of 3-nitrotyrosine (NO(2)Tyr), as a potential biomarker for nitric oxide-mediated damage, has recently been the focus of particular attention. We have developed an HPLC method with NBD-F pre-column derivatization followed by C(18) cartridge cleaning. Using this method we achieved limits of detection of 0.5 and 1.1 nm for NO(2)Tyr and tyrosine (Tyr), respectively, close to that achieved by LS-MS/MS. NO(2)Tyr and tyrosine concentrations were linear over the calibration ranges 0.5-100 nm and 1-320 microm, respectively, with correlation coefficients greater than 0.95. To evaluate the utility of this assay in plasma we analysed samples obtained from smokers and non-smoking subjects. Consistent with the presence of elevated oxidative stress, the plasma NO(2)Tyr concentration and NO(2)Tyr:Tyr ratio of smokers were 17.42 +/- 11.6 nm and 0.263 +/- 0.192 nm/microm with 3.8 and 3.9 times higher (both p < 0.05), respectively, than that of non-smoker controls (4.54 +/- 2.75 nm and 0.067 +/- 0.050 nm/microm, respectively). In conclusion, we have developed a novel HPLC assay for NO(2)Tyr without MS detection that is applicable to clinical studies addressing the pathophysiology and importance of oxidative stress.
Assuntos
4-Cloro-7-nitrobenzofurazano/química , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida/classificação , Espectrometria de Fluorescência/métodos , Tirosina/análogos & derivados , Tirosina/sangue , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , Adulto , Humanos , Indicadores e Reagentes , Masculino , Metiltirosinas/sangue , Metiltirosinas/química , Metiltirosinas/normas , Estresse Oxidativo , Plasma/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fumar/sangue , Tirosina/químicaRESUMO
L-3-[123I]iodo-alpha-methyl tyrosine (IMT) is an artificial amino acid suitable for SPECT imaging of various tumours. Manual synthesis of this radiopharmaceutical is reliable, but time-consuming and may require handling of large quantities of radioactivity. We developed an automated IMT synthesis module, which prepares a ready-to-inject product that meets radiopharmaceutical requirements and is identical to the manually synthesised equivalent. Current advantages include decreased operator assistance time and reduced radiation exposure. Application may be extended to other radiopharmaceuticals, including high-dose preparations for therapeutic use.
