Hou Yongqing, Jia Sichao, Nawaratna Gayan, Hu Shengdi, Dahanayaka Sudath, Bazer Fuller W, Wu Guoyao
Hubei Key Laboratory of Animal Nutrition and Feed Science, Hubei Collaborative Innovation Center for Animal Nutrition and Feed Safety, Wuhan Polytechnic University, Wuhan, 430023, China.
Amino Acids. 2015 Sep;47(9):2005-14. doi: 10.1007/s00726-015-1962-9. Epub 2015 Mar 28.
L-Homoarginine (hArg) may play a role in regulating the metabolism of its structural homologue L-arginine via multiple pathways (including nitric oxide synthase) in animals. Accurate measurement of hArg is essential for studying its synthesis and utilization by cells and the whole body. Here, we describe a simple, sensitive and automated method for analysis of hArg in biological samples by high-performance liquid chromatography involving precolumn derivatization with o-phthalaldehyde (OPA) and N-acetyl-L-cysteine (NAC) as the thiol. The hArg-OPA-NAC derivative was separated at 25 °C on a reversed-phase C18 material and detected by fluorescence at excitation and emission wavelengths of 340 and 450 nm, respectively. The total running time for one sample (including the time for column regeneration) was 20 min, with the retention time for hArg being 10.03 min. The limit of detection was 188 fmol hArg, which was equivalent to 12 nM hArg in the 160-µl assay mixture. The assay was linear between 1.0 and 80 pmol hArg injected into the HPLC column (equivalent to 0.0625 and 5 µM hArg in the 160-µl assay mixture, respectively). The precision (relative deviation, %) and bias (relative error, %) of the HPLC method were 0.52-1.16 and 0.42-1.12, respectively, for aqueous solutions of hArg and for various biological samples (e.g., plasma, liver, brain and kidney). This is a highly sensitive, accurate, efficient and easily automated method for analysis of hArg in biological samples and provides a useful tool for studying the biochemistry, nutrition, physiology and pharmacology of hArg and arginine in animals and humans.
L-高精氨酸（hArg）可能通过动物体内的多种途径（包括一氧化氮合酶）参与调节其结构类似物L-精氨酸的代谢。准确测定hArg对于研究其在细胞和全身的合成与利用至关重要。在此，我们描述了一种简单、灵敏且自动化的方法，用于通过高效液相色谱分析生物样品中的hArg，该方法采用邻苯二甲醛（OPA）和N-乙酰-L-半胱氨酸（NAC）作为硫醇进行柱前衍生。hArg-OPA-NAC衍生物在25℃下于反相C18材料上分离，并分别在激发波长和发射波长为340和450nm处通过荧光检测。一个样品的总运行时间（包括柱再生时间）为20分钟，hArg的保留时间为10.03分钟。检测限为188 fmol hArg，在160μl测定混合物中相当于12 nM hArg。该测定在注入HPLC柱的1.0至80 pmol hArg之间呈线性（在160μl测定混合物中分别相当于0.0625和5μM hArg）。对于hArg水溶液以及各种生物样品（如血浆、肝脏、大脑和肾脏），HPLC方法的精密度（相对偏差，%）和偏差（相对误差，%）分别为0.52 - 1.16和0.42 - 1.12。这是一种用于分析生物样品中hArg的高灵敏、准确、高效且易于自动化的方法，为研究动物和人类中hArg及精氨酸的生物化学、营养、生理学和药理学提供了有用的工具。
