Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan.
Department of Occupational Safety and Health, Chung Shan Medical University, Taichung, 402, Taiwan.
Free Radic Biol Med. 2019 Nov 1;143:193-202. doi: 10.1016/j.freeradbiomed.2019.08.005. Epub 2019 Aug 6.
Determination of the modulation of nitrite and nitrate levels in biological samples usually poses a major challenge, owing to their high background concentrations. To effectively investigate the variation of nitrite/nitrate in vivo, in this study, we developed aN-labelled nitrite/nitrate tracer analysis using LC-MS/MS following the derivatization with 2,3-diaminonaphthalene. This method allows for the determination of N-labelled nitrite/nitrate as N-2,3-naphthotriazole (N-NAT) that can efficiently differentiate newly introduced nitrite/nitrate from the background nitrite/nitrate in biological matrices. We also investigated the contribution of background N-NAT isotopomers to N-NAT, which has long been overlooked in the literature. Our results indicated that the contribution of background N-NAT isotopomers to N-NAT is significant. Such contribution is constant (~2.2% under positive ion mode and 1.1% under negative ion mode), and does not depend upon the concentration of N-NAT or the sample matrix measured. An equation has been therefore developed, for the first time, to correct the contribution of background N-NAT isotopomers to N-NAT. With the proposed N-labelled nitrite/nitrate tracer analysis, the amount and percentage distribution of NO and NO, both in urine and feces, after oral administration of N-labelled nitrite/nitrate are clearly demonstrated. The excretions of NO and NO were significantly increased with the increasing dose implying that the dietary nitrite/nitrate intake is an important source in urine/feces. The present method allows for the simple, reliable and accurate quantification of NO and NO, and it should also be useful to trace the biotransformation of nitrite and nitrate in vivo.
测定生物样本中亚硝酸盐和硝酸盐的含量通常是一个主要的挑战，因为它们的背景浓度很高。为了有效地研究体内亚硝酸盐/硝酸盐的变化，本研究采用 2,3-二氨基萘衍生化，结合 LC-MS/MS 开发了一种 N 标记的亚硝酸盐/硝酸盐示踪剂分析方法。该方法可用于测定 N 标记的亚硝酸盐/硝酸盐，即 N-2,3-萘三唑（N-NAT），可有效区分生物基质中引入的新亚硝酸盐/硝酸盐与背景亚硝酸盐/硝酸盐。我们还研究了背景 N-NAT 同量异位素对 N-NAT 的贡献，这在文献中长期被忽视。我们的结果表明，背景 N-NAT 同量异位素对 N-NAT 的贡献是显著的。这种贡献是恒定的（正离子模式下约为 2.2%，负离子模式下约为 1.1%），且不依赖于测定的 N-NAT 浓度或样品基质。因此，首次开发了一种方程来校正背景 N-NAT 同量异位素对 N-NAT 的贡献。通过提出的 N 标记的亚硝酸盐/硝酸盐示踪剂分析，清楚地显示了口服 N 标记的亚硝酸盐/硝酸盐后，尿液和粪便中 NO 和 NO 的量和百分比分布。随着剂量的增加，NO 和 NO 的排泄明显增加，这表明饮食中亚硝酸盐/硝酸盐的摄入是尿液/粪便的一个重要来源。本方法可用于简单、可靠和准确地定量 NO 和 NO，并且也应该有助于追踪体内亚硝酸盐和硝酸盐的生物转化。
