Falk Marni J, Rao Meera, Ostrovsky Julian, Daikhin Evgueni, Nissim Ilana, Yudkoff Marc
Department of Pediatrics, The Children's Hospital of Philadelphia, USA.
J Vis Exp. 2011 Feb 27(48):2288. doi: 10.3791/2288.
Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled (13)C-glucose or 1,6-(13)C(2)-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.
长期以来，稳定同位素分析一直允许在细胞和哺乳动物模型中对基因突变和/或药物治疗的代谢后果进行灵敏的研究。在此，我们描述了对线虫秀丽隐杆线虫中间代谢和代谢通量进行稳定同位素分析的详细方法。描述了对暴露于稳定同位素的动物进行全虫游离氨基酸、标记二氧化碳、标记有机酸和标记氨基酸分析的方法，这些动物要么从线虫生长培养基琼脂平板上的早期发育阶段开始暴露，要么从年轻成虫开始，同时在液体培养中接受各种药物治疗。游离氨基酸通过高效液相色谱法（HPLC）在4%高氯酸提取的全虫等分试样中进行定量。普遍标记的（13）C-葡萄糖或1,6-（13）C2-葡萄糖用作稳定同位素前体，其标记的碳通过质谱法在二氧化碳（大气中的和溶解的）以及指示糖酵解、丙酮酸代谢和三羧酸循环通量的代谢物中进行追踪。文中包含代表性结果，以展示同位素暴露时间、各种细菌清除方案以及野生型线虫中替代的虫体破碎方法的影响，以及线粒体复合物III突变体线虫（isp-1（qm150））相对于野生型线虫的同位素掺入相对程度。在活体线虫中应用稳定同位素分析提供了一种新的能力，可在全动物水平研究由个体遗传疾病和/或药物治疗引起的实时代谢改变。
