Quantification of Intracellular Metabolites
Constructs were also harvested to determine potential correlations
between intracellular metabolites and PHD2 activity. Briefly, harvested
constructs were cut from the culture inserts, snap frozen (liquid
N2) and stored at -80°C until analysis. Using a targeted
metabolomics approach, intracellular metabolites were extracted by
KOH/ethanol (0.3 M KOH in 25% ethanol), neutralized with glacial acetic
acid, and then quantified by liquid chromatography tandem quadrupole
mass spectrometry (LC/MS/MS) (Luo et al. , 2007) (Agilent 1100
HPLC/Sciex Q4000 Mass Spectrometer). A total of 14 metabolites (Table
S1), representative of glycolysis, fermentation, TCA, and pentose
phosphate pathways, were quantified with results expressed relative to
DNA content.