Extraction and quantitation of NAD(P)(H).

TitleExtraction and quantitation of NAD(P)(H).
Publication TypeJournal Article
Year of Publication2017
AuthorsLu, W, Wang, L, Chen, L, Hui, S, Rabinowitz, JD
JournalAntioxid Redox Signal
Date Published2017 May 12
ISSN1557-7716
Abstract

AIMS: Accurate analysis of dinucleotide redox cofactors NADPH, NADP+, NADH, and NAD+ from biological samples is important to understanding cellular redox homeostasis. Here we aim to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.

RESULTS: We compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues. 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. Innovations: We 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.

CONCLUSIONS: Extraction 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 employed, inclusion of detergents in the aqueous extraction buffer reduces interconversion.

DOI10.1089/ars.2017.7014
Alternate JournalAntioxid. Redox Signal.
PubMed ID28497978
Grant ListDP1 DK113643 / DK / NIDDK NIH HHS / United States
R50 CA211437 / CA / NCI NIH HHS / United States