Department of Pharmaceutical Sciences, University at Buffalo, The State University of New York, 547 Hochstetter Hall, Buffalo, NY 14260, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Mar 1;879(7-8):467-74. doi: 10.1016/j.jchromb.2011.01.006. Epub 2011 Jan 14.
L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹⁵N₄-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples.
Concentrations of unlabeled ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C₆-ARG, D₄-CIT and D₇-ADMA were used as internal standards for ARG and ¹⁵N₄-ARG, CIT, and dimethylarginines, respectively.
The calibration curves of ARG, ¹⁵N₄-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹⁵N₄-ARG, which allowed the observation of generation of ¹⁵N₃-CIT and ¹⁵N₃-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats.
An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.
精氨酸（ARG）可被内皮型一氧化氮合酶转化为一氧化氮（NO）和瓜氨酸（CIT），而内皮型一氧化氮合酶可被不对称二甲基精氨酸（ADMA）竞争性抑制。我们已经开发了一种液相色谱-质谱联用方法，用于同时测定生物样品中的内源性 ARG、标记 ARG（¹⁵N₄-ARG）、CIT、ADMA 及其无活性的同分异构体对称二甲基精氨酸（SDMA）。
采用亲水相互作用液相色谱-电喷雾串联质谱法测定 EA.hy926 人内皮细胞裂解液、细胞孵育培养基、大鼠血浆或大鼠尿液中未标记 ARG、¹⁵N₄-ARG、CIT、ADMA 和 SDMA 的浓度。¹³C₆-ARG、D₄-CIT 和 D₇-ADMA 分别作为 ARG 和 ¹⁵N₄-ARG、CIT 和二甲基精氨酸的内标。
ARG、¹⁵N₄-ARG、CIT、ADMA 和 SDMA 的校准曲线均呈线性，且不受多种样品基质的影响。在质控样品中，所有化合物定量的日内和日间变异性均低于 15%。通过将人内皮细胞暴露于 ¹⁵N₄-ARG 进行体外细胞研究，应用该方法测定这些化合物的摄取和外排，可观察到细胞裂解液中¹⁵N₃-CIT 和 ¹⁵N₃-ARG 的生成，从而证明了该方法的应用。使用这些同位素可以深入了解内源性和外源性 ARG 的细胞处理。在大鼠口服补充 ARG 后，对大鼠血浆和大鼠尿液测定进行了该方法的应用。
开发了一种同时定量 6 种 ARG 相关化合物的 LC-MS/MS 方法，使用了 3 种不同的内标。当测量同位素标记的 ARG 和 CIT 时，该测定法可同时监测摄取、外排和代谢过程，并可用于测定大鼠血浆和大鼠尿液中的这些化合物。
