Yanase Sumino, Yasuda Kayo, Ishii Naoki
Department of Health Science, Daito Bunka University School of Sports and Health Science, Higashi-matsuyama, Saitama, Japan.
Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan.
Methods Mol Biol. 2019;1916:123-132. doi: 10.1007/978-1-4939-8994-2_12.
In the nematode Caenorhabditis elegans (C. elegans), monitoring the lactate/pyruvate ratio in cells helps to detect imbalances in age-related energy metabolism. Here, we describe a modified small-scale extraction in C. elegans and measurement of lactate and pyruvate concentrations using colorimetric assay kits. During sample extraction, protein precipitation is the most critical step for precise determination of intracellular metabolites in C. elegans. Moreover, improved sensitivity and accuracy of colorimetric assay kits contributed to measurements of metabolites in samples derived from small-scale extraction. Using these protocols, we recently detected a metabolic alteration that occurs during aging by the monitoring the lactate/pyruvate ratio in a long-lived mutant of the mammalian tumor suppressor p53 ortholog CEP-1 in C. elegans.
在线虫秀丽隐杆线虫中,监测细胞内的乳酸/丙酮酸比值有助于检测与衰老相关的能量代谢失衡。在此,我们描述了一种改良的秀丽隐杆线虫小规模提取物,以及使用比色测定试剂盒测量乳酸和丙酮酸浓度的方法。在样品提取过程中,蛋白质沉淀是精确测定秀丽隐杆线虫细胞内代谢物的最关键步骤。此外,比色测定试剂盒灵敏度和准确性的提高有助于测量小规模提取物样品中的代谢物。使用这些方法,我们最近通过监测秀丽隐杆线虫中哺乳动物肿瘤抑制因子p53直系同源物CEP-1的长寿突变体中的乳酸/丙酮酸比值,检测到了衰老过程中发生的代谢变化。