Eryavuz Onmaz Duygu, Abusoglu Sedat, Yaglioglu Havva, Abusoglu Gulsum, Unlu Ali
Department of Biochemistry, Selcuk University Faculty of Medicine, Konya, Turkey.
Department of Medical Laboratory Techniques, Selcuk University Vocational School of Health, Konya, Turkey.
J Mass Spectrom Adv Clin Lab. 2021 Mar 6;19:34-45. doi: 10.1016/j.jmsacl.2021.02.002. eCollection 2021 Jan.
Nitric oxide (NO) plays an important role in endothelial homeostasis. Asymmetric dimethyl arginine (ADMA), L-N monomethyl arginine (L-NMMA) and symmetric dimethyl arginine (SDMA), which are derivatives of methylarginine, directly or indirectly reduce NO production. Therefore, these metabolites are an important risk factor for various diseases, including cardiovascular diseases. Numerous methods have been developed for the measurement of methylarginine derivatives, but various difficulties have been encountered. This study aimed to develop a reliable, fast and cost-effective method for the analysis and measurement of methylarginine derivatives (ADMA, SDMA, L-NMMA) and related metabolites (arginine, citrulline, homoarginine, ornithine), and to validate this method according to Clinical and Laboratory Standards Institute (CLSI) protocols.
For the analysis of ADMA, SDMA, L-NMMA, arginine, homoarginine, citrulline, ornithine, 200 µl of serum were precipitated with methanol, and subsequently derivatized with a butanol solution containing 5% acetyl chloride. Butyl derivatives were separated using a C18 reverse phase column with a 5 min run time. Detection of analytes was achieved by utilising the specific fragmentation patterns identified through tandem mass spectrometry.
The method was linear for ADMA, SDMA, L-NMMA, ornithine, arginine, homoarginine and citrulline in the ranges of 0.023-6.0, 0.021-5.5, 0.019-5.0, 0.015-250, 0.015-250, 0.019-5 and 0.015-250 µM, respectively. The inter-assay CV% values for all analytes was less than 9.8%.
Data obtained from method validation studies shows that the developed method is highly sensitive, precise and accurate. Short analysis time, cost-effectiveness, and multiplexed analysis of these metabolites, with the same pretreatment steps, are the main advantages of the method.
一氧化氮（NO）在内皮稳态中起重要作用。不对称二甲基精氨酸（ADMA）、L-N-单甲基精氨酸（L-NMMA）和对称二甲基精氨酸（SDMA）作为甲基精氨酸的衍生物，直接或间接减少NO的生成。因此，这些代谢产物是包括心血管疾病在内的各种疾病的重要危险因素。已经开发了许多用于测量甲基精氨酸衍生物的方法，但遇到了各种困难。本研究旨在开发一种可靠、快速且经济高效的方法，用于分析和测量甲基精氨酸衍生物（ADMA、SDMA、L-NMMA）及相关代谢产物（精氨酸、瓜氨酸、高精氨酸、鸟氨酸），并根据临床和实验室标准协会（CLSI）的方案验证该方法。
为了分析ADMA、SDMA、L-NMMA、精氨酸、高精氨酸、瓜氨酸、鸟氨酸，取200μl血清用甲醇沉淀，随后用含5%乙酰氯的丁醇溶液进行衍生化。使用C18反相柱在5分钟的运行时间内分离丁基衍生物。通过利用串联质谱法确定的特定碎片模式实现对分析物的检测。
该方法对ADMA、SDMA、L-NMMA、鸟氨酸、精氨酸、高精氨酸和瓜氨酸的线性范围分别为0.023 - 6.0、0.021 - 5.5、0.019 - 5.0、0.015 - 250、0.015 - 250、0.019 - 5和0.015 - 250μM。所有分析物的批间变异系数（CV%）值均小于9.8%。
方法验证研究获得的数据表明，所开发的方法具有高度的敏感性、精密度和准确性。分析时间短、成本效益高以及对这些代谢产物进行多重分析且预处理步骤相同是该方法的主要优点。
