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Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae

Journal Article · · Seperation Science, vol. 31, N/A, August 8, 2008, pp. 00-00
OSTI ID:944326
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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, approximately 10{sup 8} yeast cells were harvested by centrifugation and then lysed under non-oxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50-mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH{sub 3}CN + 50-mM ammonium acetate (3:1; v:v) was added to the cell lysates. After sample centrifugation to pellet precipitated proteins, organic solvent removal was performed on supernatants by chloroform extraction. The remaining aqueous phase was dried and resuspended in 50-mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-VIS absorbance detection. Applicability of this procedure for quantifying NAD and NADH levels was evaluated by culturing yeast under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. NAD and NADH contents are similar to previously reported levels in yeast obtained using enzymatic assays performed separately on acid (for NAD) and alkali (for NADH) extracts. 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 (1) applicable to quantification of these metabolites in mammalian and bacterial cell cultures; and (2) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.
Research Organization:
Lawrence Livermore National Laboratory (LLNL), Livermore, CA
Sponsoring Organization:
USDOE
DOE Contract Number:
W-7405-ENG-48
OSTI ID:
944326
Report Number(s):
LLNL-JRNL-400434
Journal Information:
Seperation Science, vol. 31, N/A, August 8, 2008, pp. 00-00, Journal Name: Seperation Science, vol. 31, N/A, August 8, 2008, pp. 00-00 Vol. 31
Country of Publication:
United States
Language:
English

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Related Subjects

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
ACETATES
BIOLOGICAL PATHWAYS
CELL CULTURES
CENTRIFUGATION
CHLOROFORM
DETECTION
EXPLOSIVE FRACTURING
GLUCOSE
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
METABOLITES
NAD
ORGANIC SOLVENTS
PELLETS
PROCESSING
PROTEIN DENATURATION
PROTEINS
REMOVAL
SACCHAROMYCES CEREVISIAE
YEASTS