Nilsson Roland, Jain Mohit
Unit of Computational Medicine, Department of Medicine, Karolinska Institutet, Karolinska University Hospital, SE-17176 Stockholm, Sweden.
Departments of Medicine and Pharmacology, University of California, San Diego, USA.
Mol Biosyst. 2016 May 24;12(6):1929-37. doi: 10.1039/c6mb00009f.
Stable isotope tracing is a powerful method for interrogating metabolic enzyme activities across the metabolic network of living cells. However, most studies of mammalian cells have used (13)C-labeled tracers only and focused on reactions in central carbon metabolism. Cellular metabolism, however, involves other biologically important elements, including nitrogen, hydrogen, oxygen, phosphate and sulfur. Tracing stable isotopes of such elements may help shed light on poorly understood metabolic pathways. Here, we demonstrate the use of high-resolution mass spectrometry to simultaneously trace carbon and nitrogen metabolism in human cells cultured with (13)C- and (15)N-labeled glucose and glutamine. To facilitate interpretation of the complex isotopomer data generated, we extend current methods for metabolic flux analysis to handle multivariate mass isotopomer distributions (MMIDs). We find that observed MMIDs are broadly consistent with known biochemical pathways. Whereas measured (13)C MIDs were informative for central carbon metabolism, (15)N isotopes provided evidence for nitrogen-carrying reactions in amino acid and nucleotide metabolism. This computational and experimental methodology expands the scope of metabolic flux analysis beyond carbon metabolism, and may prove important to understanding metabolic phenotypes in health and disease.
稳定同位素示踪是一种用于探究活细胞代谢网络中代谢酶活性的强大方法。然而，大多数关于哺乳动物细胞的研究仅使用了（13）C标记的示踪剂，并聚焦于中心碳代谢中的反应。然而，细胞代谢涉及其他生物学上重要的元素，包括氮、氢、氧、磷和硫。追踪这些元素的稳定同位素可能有助于阐明人们了解较少的代谢途径。在此，我们展示了使用高分辨率质谱法同时追踪用（13）C和（15）N标记的葡萄糖和谷氨酰胺培养的人类细胞中的碳和氮代谢。为便于解释所产生的复杂同位素异构体数据，我们扩展了当前的代谢通量分析方法以处理多变量质量同位素异构体分布（MMID）。我们发现观察到的MMID与已知生化途径大致一致。虽然测得的（13）C MID对中心碳代谢有参考价值，但（15）N同位素为氨基酸和核苷酸代谢中的含氮反应提供了证据。这种计算和实验方法将代谢通量分析的范围扩展到了碳代谢之外，并可能对理解健康和疾病中的代谢表型具有重要意义。
