Files and links (1)
Accepted (AM)Open Access (License Unspecified), Open
Journal article
Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism
Analyst (London), v 142(23), pp 4431-4437
07 Dec 2017
PMID: 29072717
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) are conserved metabolic cofactors that mediate reduction-oxidation (redox) reactions throughout all domains of life. The diversity of synthetic routes and cellular processes involving the transfer of reducing equivalents to and from these cofactors makes the accurate quantitation and metabolic tracing of NAD(H) and NADP(H) of broad interest. However, current analytical techniques typically rely on standard curves that do not incorporate confounding effects of the sample matrix. We utilized the essential requirement of niacin and tryptophan for NAD synthesis in mammalian cells to devise a stable isotope labeling by essential nutrients in cell culture (SILEC) method for efficient labeling of intracellular NAD(H) and NADP(H) pools. Coupling this approach with detection by liquid chromatography-high resolution mass spectrometry (LC-HRMS), we demonstrate the utility of incorporating a [C-13(3) N-15(1)]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [C-13(3) N-15(1)]-nicotinamide and [C-13(11)]-tryptophan, we quantify the relative contribution of salvage and de novo NAD synthesis, respectively, in S. cerevisiae and HepG2 human hepatocellular carcinoma cells under basal conditions. As a further proof-of-principle, we demonstrate an improvement in the linear range for quantification of NAD and apply this method to analysis of NAD(H) in mouse liver. This method demonstrates the generalizability of SILEC, and provides a simple method for generating a library of stable isotope labeled internal standards for quantifying and tracing the metabolism of cellular and tissue NAD(H) and NADP(H).
Metrics
Details
- Title
- Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism
- Creators
- David W. Frederick - University of PennsylvaniaSophie Trefely - Drexel UniversityAlexia Buas - Drexel UniversityJason Goodspeed - Drexel UniversityJay Singh - Drexel UniversityClementina Mesaros - Center for Excellence in Environmental Toxicology, Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania,Philadelphia,USAJoseph A. Baur - University of PennsylvaniaNathaniel W. Snyder - Drexel University
- Publication Details
- Analyst (London), v 142(23), pp 4431-4437
- Publisher
- Royal Soc Chemistry
- Number of pages
- 7
- Grant note
- R01 DK098656 / NIDDK NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) R03 HD092630; R21 HD087866 / NICHD NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) K22 ES026235; P30 ES013508 / NIEHS NIH HHS; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Environmental Health Sciences (NIEHS) R03HD092630 / EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) R01DK098656 / NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) K22ES026235 / NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Environmental Health Sciences (NIEHS)
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute
- Web of Science ID
- WOS:000415866200010
- Scopus ID
- 2-s2.0-85034771848
- Other Identifier
- 991019167855904721
UN Sustainable Development Goals (SDGs)
This publication has contributed to the advancement of the following goals:
InCites Highlights
Data related to this publication, from InCites Benchmarking & Analytics tool:
- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Chemistry, Analytical