Chao Mu-Rong, Shih Ying-Ming, Hsu Yu-Wen, Liu Hung-Hsin, Chang Yuan-Jhe, Lin Bo-Huei, Hu Chiung-Wen
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung 402, Taiwan.
Division of Chest Medicine, Department of Internal Medicine, Changhua Christian Hospital, Changhua 500, Taiwan; Department of Public Health, Chung Shan Medical University, Taichung 402, Taiwan.
Free Radic Biol Med. 2016 Apr;93:77-83. doi: 10.1016/j.freeradbiomed.2016.01.025. Epub 2016 Jan 29.
Urinary tract infections (UTIs) are the most common type of nosocomial infection. Traditionally, the presence of white blood cells and microorganisms in the urine provides objective evidence for UTI diagnosis. Here, we describe the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) to measure the nitrite and nitrate levels in urine and investigate the potential of this method for UTI diagnosis. LC-MS/MS analysis was performed in positive electrospray ionization mode. After adding (15)N-labeled internal standards and derivatizing with 2,3-diaminonaphthalene (DAN), the urinary nitrite content was directly analyzed by LC-MS/MS, whereas the urinary nitrate was first reduced to nitrite before derivatization and LC-MS/MS analysis. The derivatization of nitrite and enzymatic reduction of nitrate were optimized. This method was then applied to 241 healthy subjects and 73 UTI patients. Optimization tests revealed that 1 mL of crude urine required at least 6.25 μmol of DAN to completely derivatize nitrite and 2.5 U of nitrate reductase to completely reduce nitrate to nitrite. Urinary analysis showed that the urinary concentration of nitrite and the nitrite/nitrate ratio were higher in UTI patients than in healthy subjects. Compared with the dipstick-based urinary nitrite test and using LC-MS/MS to determine the nitrite concentration (sensitivity: 23-25%), the nitrite/nitrate ratio was significantly more sensitive (95%) and exhibited a satisfactory specificity (91%) in the screening of UTIs. Taken together, the nitrite/nitrate ratio, which reflects the reducing ability of pathogenic bacteria, could be a better method for the diagnosis of UTIs that is not subject to variations in urine specimen quality.
尿路感染（UTIs）是最常见的医院感染类型。传统上，尿液中白细胞和微生物的存在为UTI诊断提供了客观证据。在此，我们描述了使用液相色谱 - 串联质谱法（LC-MS/MS）测量尿液中的亚硝酸盐和硝酸盐水平，并研究该方法用于UTI诊断的潜力。LC-MS/MS分析在正电喷雾电离模式下进行。加入（15）N标记的内标并用2,3 - 二氨基萘（DAN）衍生化后，尿液中亚硝酸盐含量直接通过LC-MS/MS分析，而尿液中的硝酸盐在衍生化和LC-MS/MS分析前先还原为亚硝酸盐。优化了亚硝酸盐的衍生化和硝酸盐的酶促还原。然后将该方法应用于241名健康受试者和73名UTI患者。优化测试表明，1 mL粗尿液至少需要6.25 μmol的DAN才能完全衍生化亚硝酸盐，需要2.5 U的硝酸还原酶才能将硝酸盐完全还原为亚硝酸盐。尿液分析表明，UTI患者尿液中亚硝酸盐浓度和亚硝酸盐/硝酸盐比值高于健康受试者。与基于试纸的尿液亚硝酸盐检测相比，使用LC-MS/MS测定亚硝酸盐浓度（灵敏度：23 - 25%），亚硝酸盐/硝酸盐比值在UTI筛查中显著更敏感（95%），并表现出令人满意的特异性（91%）。综上所述，反映病原菌还原能力的亚硝酸盐/硝酸盐比值可能是一种更好的UTI诊断方法，不受尿液标本质量变化影响。
