Oosterink J Efraim, Buijs Nikki, van Goudoever Johannes B, Schierbeek Henk
Department of Pediatrics, Emma Children's Hospital, Academic Medical Center, Amsterdam, The Netherlands.
Department of Surgery, VU University Medical Center, Amsterdam, The Netherlands; Department of Surgery, Medical Centre Alkmaar, Trial Centre Holland Health, Alkmaar, The Netherlands.
J Chromatogr B Analyt Technol Biomed Life Sci. 2014 May 1;958:10-5. doi: 10.1016/j.jchromb.2014.03.005. Epub 2014 Mar 15.
Stable isotope studies offer the opportunity to study the in-depth metabolic pathway of glutamine, citrulline, and arginine amino acids involved in NO synthesis. The use of multiple stable isotopes can be used to elucidate the exact transformation of glutamine to citrulline and arginine de novo synthesis. This novel method provides a purification step using cation exchange resin in combination with a rapid and easy derivatization procedure for a precise and robust measurement of the concentration and isotopic enrichments of NO synthesis-specific amino acids using a liquid chromatography mass spectrometry (LC/MS) ion trap system with high sensitivity and selectivity. The ethyl chloroformate derivatization procedure is beneficial in terms of robustness, velocity, simplicity, and derivative stability. In addition, the ethyl chloroformate derivatization can be performed at room temperature in an aqueous environment without incubation and the isolation of the derivatives from the reaction mixture also serves as a purification step. The concentration and enrichment of NO synthesis-specific amino acids as well as phenylalanine and tyrosine to determine protein turnover, were measured with good inter-day precision for the concentration (<7.4%) and enrichment (<12.7%) in plasma samples at low and high levels. The low limit of quantification was 0.2μmol/L for most of the amino acids and the purification method showed to have good recoveries between 78% and 98%. No ion-suppression was observed by post-column infusion experiments. In order to develop new nutritional strategies, this novel method can be used to support the elucidation of the effect of administration of specific supplements on the glutamine-citrulline-arginine pathway by using stable isotope studies.
稳定同位素研究为研究参与一氧化氮（NO）合成的谷氨酰胺、瓜氨酸和精氨酸的深入代谢途径提供了机会。使用多种稳定同位素可用于阐明谷氨酰胺向瓜氨酸的精确转化以及精氨酸的从头合成。这种新方法提供了一个纯化步骤，该步骤使用阳离子交换树脂，并结合快速简便的衍生化程序，以使用具有高灵敏度和选择性的液相色谱质谱（LC/MS）离子阱系统精确且可靠地测量NO合成特异性氨基酸的浓度和同位素丰度。氯甲酸乙酯衍生化程序在稳健性、速度、简便性和衍生物稳定性方面具有优势。此外，氯甲酸乙酯衍生化可在室温下的水性环境中进行，无需孵育，并且从反应混合物中分离衍生物也可作为一个纯化步骤。测量了NO合成特异性氨基酸以及用于确定蛋白质周转率的苯丙氨酸和酪氨酸的浓度和丰度，血浆样品中低水平和高水平的浓度（<7.4%）和丰度（<12.7%）具有良好的日间精密度。大多数氨基酸的定量下限为0.2μmol/L，纯化方法的回收率在78%至98%之间，效果良好。柱后注入实验未观察到离子抑制现象。为了开发新的营养策略，这种新方法可用于通过稳定同位素研究支持阐明特定补充剂给药对谷氨酰胺 - 瓜氨酸 - 精氨酸途径的影响。
