Ueland Per Magne, Midttun Oivind, Windelberg Amrei, Svardal Asbjørn, Skålevik Rita, Hustad Steinar
LOCUS for Homocysteine and Related Vitamins, University of Bergen, Bergen, Norway.
Clin Chem Lab Med. 2007;45(12):1737-45. doi: 10.1515/CCLM.2007.339.
Derangements of one-carbon metabolism have been related to the development of chronic diseases. Metabolic profiling as part of epidemiological studies in this area should include intermediates involved in the transfer of one-carbon units, cofactors for the relevant enzymes and markers of inflammation, kidney function and smoking.
We established five platforms that measured 6-16 analytes each. Platforms A (gas chromatography-mass spectrometry; GC-MS) and B (gas chromatography-tandem mass spectrometry; GC-MS/MS) involved methylchloroformate derivatization of primary amines, thiols and carboxylic acids. Platform C determined basic compounds by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using an ether-linked phenyl reversed-phase column. Platforms D and E (LC-MS/MS) exploited the efficient ionization and high sensitivity obtained for a wide range of analytes, using a mobile phase containing a high concentration of acetic acid. The chromatographic run times ranged from 3 to 8 min.
The analyte concentrations ranged from 0.2 nmol/L to 400 micromol/L. Platforms A and B both measured methylmalonic acid, total homocysteine and related amino acids. Platform B also included sarcosine, cystathionine, tryptophan and kynurenine. Platform C was optimized for the measurement of choline and betaine, but also included arginine, asymmetric and symmetric dimethylarginine and creatinine. A diversity of low abundance compounds mainly occurring in the nanomolar range were measured on platform D. These were vitamin B(2) and B(6) species, neopterin, cotinine and tryptophan metabolites. Platform E measured folates and folate catabolites.
Approximately 40 analytes related to one-carbon metabolism were determined in less than 1 mL of plasma/serum using five complementary analytical platforms. As a method control, several metabolites were measured on two or more platforms. Logistics and data handling were carried out by specially designed software. This strategy allows profiling of one-carbon metabolism in large-scale epidemiological studies.
一碳代谢紊乱与慢性疾病的发生发展有关。作为该领域流行病学研究一部分的代谢谱分析应包括参与一碳单位转移的中间体、相关酶的辅助因子以及炎症、肾功能和吸烟的标志物。
我们建立了五个平台，每个平台可测量6 - 16种分析物。平台A（气相色谱 - 质谱联用仪；GC - MS）和平台B（气相色谱 - 串联质谱联用仪；GC - MS/MS）涉及对伯胺、硫醇和羧酸进行氯甲酸甲酯衍生化。平台C通过液相色谱 - 串联质谱联用仪（LC - MS/MS），使用醚键连接的苯基反相柱测定碱性化合物。平台D和平台E（LC - MS/MS）利用含有高浓度乙酸的流动相，对多种分析物实现高效电离和高灵敏度检测。色谱运行时间为3至8分钟。
分析物浓度范围为0.2 nmol/L至400 μmol/L。平台A和平台B均测量甲基丙二酸、总同型半胱氨酸及相关氨基酸。平台B还包括肌氨酸、胱硫醚、色氨酸和犬尿氨酸。平台C针对胆碱和甜菜碱的测量进行了优化，但也包括精氨酸、不对称和对称二甲基精氨酸以及肌酐。平台D测量了多种主要存在于纳摩尔范围内的低丰度化合物。这些化合物包括维生素B2和B6种类、新蝶呤、可替宁和色氨酸代谢物。平台E测量叶酸和叶酸分解代谢物。
使用五个互补的分析平台，在不到1 mL的血浆/血清中测定了约40种与一碳代谢相关的分析物。作为方法对照，在两个或更多平台上测量了多种代谢物。物流和数据处理由专门设计的软件进行。该策略可用于大规模流行病学研究中的一碳代谢谱分析。
