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Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

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 [13C315N1]-nicotinamide moiety into a library of NAD-derived metabolites for use as internal standards in matrixed samples. Using a two-label system incorporating [13C315N1]-nicotinamide and [13C11]-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).

SUBMITTER: Frederick DW

PROVIDER: S-EPMC5849469 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

Frederick David W DW Trefely Sophie S Buas Alexia A Goodspeed Jason J Singh Jay J Mesaros Clementina C Baur Joseph A JA Snyder Nathaniel W NW

The Analyst 20171101 23

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 stan ...[more]

PMID: 29072717

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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Dataset Information

Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

Publications

Stable isotope labeling by essential nutrients in cell culture (SILEC) for accurate measurement of nicotinamide adenine dinucleotide metabolism.

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