title: The influence of free 3-nitrotyrosine and saliva on the quantitative analysis of protein-bound 3-nitrotyrosine in sputum.
abstract: BACKGROUND
  We have recently developed a new technique for quantitatively measuring protein-bound 3-nitrotyrosine (3-NT), a footprint of nitrosative stress, utilizing high-performance liquid chromatography with an electrochemical detection (HPLC-ECD) system. Using this system, we showed that 3-NT formation was upregulated in the sputum of both COPD and asthmatic patients. However, in order to improve the accuracy of the measurement system, We have to resolve some problems which were the influence of free amino acid form of 3-NT and of salivary contamination.
  
  
  OBJECTIVE
  We initially investigated the amount of the free amino acid form of 3-NT in induced sputum and compared with that of protein-bound 3-NT. Next, we evaluated the concentration of protein-bound 3-NT in saliva and compared with that in induced sputum by means of HPLC-ECD.
  
  
  METHODS
  Five male COPD patients were enrolled. Induced sputum and saliva were obtained from the patients. The free amino acid form of 3-NT in sputum and saliva was measured by HPLC-ECD, and the protein-bound 3-NT and tyrosine in sputum and saliva were enzymatically hydrolyzed by Streptomyces griseus Pronase and measured for the protein hydrolysate by HPLC-ECD.
  
  
  RESULTS
  The mean value of the amount of protein-bound 3-NT was 65.0 fmol (31.2 to 106.4 fmol). On the other hand, the amount of the free amino acid form of 3-NT was under the detection limit (<10 fmol). The levels of both 3-NT (sputum: 0.55 +/- 0.15 pmol/ml, saliva: 0.02 +/- 0.01 pmol/ml, p < 0.01) and tyrosine (sputum: 0.81 +/- 0.43 micromol/ml, saliva: 0.07 +/- 0.04 micromol/ml, p < 0.01) in saliva were significantly lower than in sputum. The percentage of 3-NT in saliva to that in sputum was about 3.1%, and that of tyrosine was about 9.0%.
  
  
  CONCLUSIONS
  The free amino acid form of 3-NT does not affect the measurement of protein-bound 3-NT. Furthermore, the influence of salivary contamination on the measurement of protein-bound 3-NT in induced sputum by means of HPLC-ECD was very small and could be negligible.
authors: Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan.
authors: Kazuhito
authors: K
authors: Ueshima
authors: Yoshiaki
authors: Y
authors: Minakata
authors: Hisatoshi
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authors: Sugiura
authors: Satoru
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authors: Yanagisawa
authors: Tomohiro
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authors: Ichikawa
authors: Keiichirou
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authors: Akamatsu
authors: Tsunahiko
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authors: Hirano
authors: Masanori
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authors: Nakanishi
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authors: Matsunaga
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authors: Ichinose