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

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, 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 samemore » 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.« less

Authors:
; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
944326
Report Number(s):
LLNL-JRNL-400434
TRN: US200902%%688
DOE Contract Number:  
W-7405-ENG-48
Resource Type:
Journal Article
Journal Name:
Seperation Science, vol. 31, N/A, August 8, 2008, pp. 00-00
Additional Journal Information:
Journal Volume: 31
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 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

Citation Formats

Sporty, J, Kabir, M M, Turteltaub, K, Ognibene, T, Lin, S, and Bench, G. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae. United States: N. p., 2008. Web.
Sporty, J, Kabir, M M, Turteltaub, K, Ognibene, T, Lin, S, & Bench, G. Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae. United States.
Sporty, J, Kabir, M M, Turteltaub, K, Ognibene, T, Lin, S, and Bench, G. Thu . "Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae". United States. https://www.osti.gov/servlets/purl/944326.
@article{osti_944326,
title = {Single sample extraction and HPLC processing for quantification of NAD and NADH levels in Saccharomyces cerevisiae},
author = {Sporty, J and Kabir, M M and Turteltaub, K and Ognibene, T and Lin, S and Bench, G},
abstractNote = {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.},
doi = {},
journal = {Seperation Science, vol. 31, N/A, August 8, 2008, pp. 00-00},
number = ,
volume = 31,
place = {United States},
year = {2008},
month = {1}
}