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.