|Title||Extraction and quantitation of NAD(P)(H).|
|Publication Type||Journal Article|
|Year of Publication||2017|
|Authors||Lu, W, Wang, L, Chen, L, Hui, S, Rabinowitz, JD|
|Journal||Antioxid Redox Signal|
|Date Published||2017 May 12|
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.
|Alternate Journal||Antioxid. Redox Signal.|
|Grant List||DP1 DK113643 / DK / NIDDK NIH HHS / United States |
R50 CA211437 / CA / NCI NIH HHS / United States