Simultaneous tracing of carbon and nitrogen isotopes in human cells

Mol Biosyst. 2016 May 24;12(6):1929-37. doi: 10.1039/c6mb00009f.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes / metabolism*
  • Cell Line
  • Energy Metabolism
  • Humans
  • Intracellular Space / metabolism
  • Isotope Labeling
  • Mass Spectrometry
  • Metabolic Networks and Pathways
  • Metabolome
  • Metabolomics* / methods
  • Nitrogen Isotopes / metabolism*

Substances

  • Carbon Isotopes
  • Nitrogen Isotopes