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Deuterium Tracing to Interrogate Compartment-Specific NAD(P)H Metabolism in Cultured Mammalian Cells

  • Esther W. Lim
  • Seth J. Parker
  • Christian M. MetalloEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)

Abstract

Oxidation–reduction (redox) reactions are ubiquitous in biology and typically occur in specific subcellular compartments. In cells, the electron transfer between molecules and organelles is commonly facilitated by pyridine nucleotides such as nicotinamide adenine dinucleotide phosphate (NADPH) and nicotinamide adenine dinucleotide (NADH). While often taken for granted, these metabolic reactions are critically important for maintaining redox homeostasis and biochemical potentials across membranes. While 13C tracing and metabolic flux analysis (MFA) have emerged as powerful tools to study intracellular metabolism, this approach is limited when applied to pathways catalyzed in multiple cellular compartments. To address this issue, we and others have applied 2H (deuterium) tracers to observe transfer of labeled hydride anions, which accompanies electron transfer. Furthermore, we have developed a reporter system for indirectly quantifying NADPH enrichment in specific subcellular compartments. Here, we provide a detailed description of 2H tracing applications and the interrogation of mitochondrial versus cytosolic NAD(P)H metabolism in cultured mammalian cells. Specifically, we describe the generation of reporter cell lines that express epitope-tagged R132H-IDH1 or R172K-IDH2 and produce (d)2-hydroxyglutarate in a doxycycline-dependent manner. These tools and methods allow for quantitation of reducing equivalent turnover rates, the directionality of pathways present in multiple compartments, and the estimation of pathway contributions to NADPH pools.

Key words

Deuterium Redox metabolism Stable isotope tracing NADH NADPH Metabolite extraction Metabolic flux analysis Mammalian cell culture Isotopomer spectral analysis 

Notes

Acknowledgments

We thank members of the Metallo Lab for their feedback and discussion. C.M.M. is funded by NIH (R01 CA188652) and NSF CAREER (#145442) grants. E.W.L. is supported by a training grant from the NIH (T32 EB009380).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Esther W. Lim
    • 1
  • Seth J. Parker
    • 2
  • Christian M. Metallo
    • 1
    • 3
    • 4
    • 5
    Email author
  1. 1.Department of BioengineeringUniversity of California San DiegoLa JollaUSA
  2. 2.Department of Radiation Oncology, Perlmutter Cancer CenterNew York University School of MedicineNew YorkUSA
  3. 3.Moores Cancer CenterUniversity of California San DiegoLa JollaUSA
  4. 4.Diabetes and Endocrinology Research CenterUniversity of California San DiegoLa JollaUSA
  5. 5.Institute of Engineering in MedicineUniversity of California San DiegoLa JollaUSA

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