Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, Buffalo, NY 14260, USA.
J Pharm Biomed Anal. 2011 Dec 15;56(5):1127-31. doi: 10.1016/j.jpba.2011.08.017. Epub 2011 Aug 12.
The utility of an LC-MS/MS assay for nitrite determination in studying L-arginine (ARG) cellular action was examined in vitro. EA.hy926 human endothelial cells or cellular fractions (membrane and cytosol) were exposed to 0-500 μM of (15)N(4)-ARG for 2 h. (14)N-nitrite and (15)N-nitrite in the cell lysate and in the incubation medium were derivatized with 2,3-diaminonaphthalene (DAN) to form (14)N- and (15)N-naphthotriazole (i.e., (14)N-NAT and (15)N-NAT). Peak responses of (14)N-NAT and (15)N-NAT were analyzed by LC-MS/MS with 1H-naphth[2,3-d]imidazole as an internal standard. The calibration curves of DAN-derivatized (14)N-NAT and (15)N-NAT from (14)N-nitrite and (15)N-nitrite were linear. Intra- and inter-day variability of the quantification was below 14.2% in quality control samples. Following incubation of EA.hy926 cells with (15)N(4)-ARG, saturable increases of (15)N-nitrite accumulation with increasing (15)N(4)-ARG exposure were observed clearly. This increase however could not be detected by the classical fluorescence method, nor were changes in (14)N-nitrite level observed. When cellular fractions were exposed to (15)N(4)-ARG, (15)N-nitrite formation was only observed in the membrane fragments. The sensitive and selective LC-MS/MS method reported here can be applied to quantify accumulated nitrite levels in human endothelial cells. The selectivity of this stable-isotope labeled LC-MS/MS method offers an advantage over other traditional methods for elucidating cellular ARG action when its stable isotope is employed as a substrate.
本研究旨在探讨 LC-MS/MS 法测定亚硝酸盐在研究 L-精氨酸（ARG）细胞作用中的应用。将 EA.hy926 人内皮细胞或细胞成分（膜和胞质）暴露于 0-500μM 的（15）N（4）-ARG 中 2 小时。细胞裂解液和孵育培养基中的（14）N-亚硝酸盐和（15）N-亚硝酸盐用 2,3-二氨基萘（DAN）衍生化形成（14）N-和（15）N-萘三唑（即（14）N-NAT 和（15）N-NAT）。用 1H-萘[2,3-d]咪唑作为内标，通过 LC-MS/MS 分析 DAN 衍生化的（14）N-NAT 和（15）N-NAT 的峰响应。（14）N-亚硝酸盐和（15）N-亚硝酸盐衍生的 DAN 衍生化（14）N-NAT 和（15）N-NAT 的校准曲线呈线性。质量控制样品的日内和日间变异系数低于 14.2%。在孵育 EA.hy926 细胞与（15）N（4）-ARG 后，观察到（15）N-亚硝酸盐的积累与（15）N（4）-ARG 暴露的增加呈饱和性增加。然而，这种增加不能通过经典的荧光法检测到，也不能观察到（14）N-亚硝酸盐水平的变化。当细胞成分暴露于（15）N（4）-ARG 时，仅在膜片段中观察到（15）N-亚硝酸盐的形成。本文报道的灵敏和选择性 LC-MS/MS 方法可用于定量人内皮细胞中积累的亚硝酸盐水平。当使用其稳定同位素作为底物时，该稳定同位素标记的 LC-MS/MS 方法的选择性为阐明细胞 ARG 作用提供了优于其他传统方法的优势。
