Kuang Liqing, Jing Zhiqiang, Wang Jing, Ma Liyuan, Liu Xiaoqiang, Yang Jin
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China.
Department of Clinical Pharmacy, China Pharmaceutical University, Nanjing 210009, China.
J Pharm Biomed Anal. 2014 Mar;90:1-6. doi: 10.1016/j.jpba.2013.11.003. Epub 2013 Nov 21.
ɛ-N-carboxymethyl-L-lysine (CML) is a stable chemical modification of protein lysine residues resulting from glycation and oxidation reactions and a potential biomarker of oxidative stress caused by sugar and lipid oxidation. In this study, a rapid, simple and sensitive method based on liquid chromatography-tandem spectrometry (LC-MS/MS) for the determination of CML in human plasma has been developed and validated. Sample preparation involved protein precipitation using trichloroacetic acid after addition of deuterated CML as internal standard. Chromatography was performed on an amino column by gradient-elution with a mobile phase containing acetonitrile:ultrapure water (80:20, v/v). CML and CML-d2 were detected by multiple reaction monitoring mode with ion pairs 205.0/130.1 and 207.2/84.1 respectively. The assay was linear in the range 10-1000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL and recovery >90%. Assay validation showed that inter- and intra-day precision and accuracy were satisfactory. The method was applied to compare plasma CML levels in healthy Chinese subjects and patients with diabetes and uremia. In healthy subjects CML concentration (mean±SD) was 16.6±7.8 ng/mL. CML level in diabetic patients was not significantly different from healthy subjects whereas the level in patients with uremia was significantly higher than both healthy subjects and diabetic patients (P<0.001). The method will be useful to assess the value of CML as a biomarker of diabetic vascular complications resulting from elevated oxidative stress.
ε-N-羧甲基-L-赖氨酸（CML）是蛋白质赖氨酸残基经糖基化和氧化反应产生的一种稳定化学修饰，是糖和脂质氧化引起的氧化应激的潜在生物标志物。在本研究中，已开发并验证了一种基于液相色谱-串联质谱（LC-MS/MS）的快速、简单且灵敏的方法，用于测定人血浆中的CML。样品制备包括在加入氘代CML作为内标后，用三氯乙酸进行蛋白质沉淀。在氨基柱上进行色谱分离，采用含乙腈：超纯水（80:20，v/v）的流动相进行梯度洗脱。通过多反应监测模式分别以离子对205.0/130.1和207.2/84.1检测CML和CML-d2。该测定法在10 - 1000 ng/mL范围内呈线性，定量下限（LLOQ）为10 ng/mL，回收率>90%。测定法验证表明，日内和日间精密度及准确度均令人满意。该方法用于比较健康中国受试者与糖尿病和尿毒症患者的血浆CML水平。在健康受试者中，CML浓度（均值±标准差）为16.6±7.8 ng/mL。糖尿病患者的CML水平与健康受试者无显著差异，而尿毒症患者的CML水平显著高于健康受试者和糖尿病患者（P<0.001）。该方法将有助于评估CML作为氧化应激升高导致的糖尿病血管并发症生物标志物的价值。
