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. 2008 Oct;31(18):3202-11.
doi: 10.1002/jssc.200800238.

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

Jennifer L Sporty  1 , Md Mohiuddin Kabir, Kenneth W Turteltaub, Ted Ognibene, Su-Ju Lin, Graham Bench
Affiliations

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

Jennifer L Sporty et al. J Sep Sci. 2008 Oct.

Abstract

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

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Conflict of interest statement

The authors declared no conflict of interest.

Figures

Figure 1
Figure 1
Diagrammatic representation of the extraction protocol. Cells are pelleted by centrifugation and then lysed by bead beating. Beads are washed and lysates are collected. The sample is then centrifuged and the supernatant is collected. The remaining protein pellet is washed, centrifuged, and the supernatant is collected. The sample undergoes a chloroform extraction and the aqueous phase is removed, lyophilized, and then separated by HPLC.
Figure 2
Figure 2
Typical HPLC chromatograms of 10 μL aliquots of 50 mM ammonium acetate containing (a) 2.50 μg NAD and (b) 2.34 μg NADH (absorbance at 340 nm has been offset vertically by 10 mAu for clarity and an expanded view of the NADH peak is inset). Retention times (tr) of the authentic compounds are: NAD tr = 20.9 min., and NADH tr = 24.4 min.
Figure 3
Figure 3
Standard curves for the chromatographic quantitation of authentic compounds of NAD and NADH. NAD contents range from 0.038 ng to 18.26 μg. NADH contents range from 0.068 ng to 13.87 μg. An expanded view of the standard curves near the origin is inset. Lines represent linear least squares fits to the data. Coefficients of correlation are 0.9975 for NAD and 0.9984 for NADH.
Figure 4
Figure 4
Typical HPLC chromatogram of a 75 μL aliquot of yeast metabolite extracts from approximately 7×107 yeast cells grown in 20 g/L glucose. Retention times (tr) of the examined compounds are: NAD tr = 21.3 min and NADH tr = 24.9 min.
Figure 5
Figure 5
Metabolite concentrations (mM) of yeast grown in synthetic complete media containing 20 g/L glucose (normal) or 5 g/L glucose (CR). Data are presented as mean and SD of seven separate experiments for each culture condition. Student’s t-test assessments were used to determine whether NAD, NADH, and NAD + NADH are similar for the 20 and 5 g/L glucose growth conditions (*p < 0.02, **p < 0.10).
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