A liquid chromatographic-electrospray ionization ion trap mass spectrometry (LC/MS) method has been developed to measure the biosynthetic incorporation of specific precursors into
NAD. The stable
isotope-labeled precursors
tryptophan,
quinolinic acid,
nicotinic acid, and
nicotinamide were added to the media of human liver
tumor cells (SK-HEP) grown in culture. The cells were harvested, the
NAD was extracted, and the ratio of labeled to unlabeled
NAD was measured using the newly developed LC/MS assay. The quantity of
NAD formed from each precursor relative to an internal standard (fully labeled 13C, 15N-labeled
NAD prepared from baker's yeast) was measured. The detection limit (signal-to-noise ratio 5:1) of the LC/MS method was 37 fmol (25 pg) of
NAD and was linear from 20.0 ng to 25 pg. All reported
NAD levels were normalized relative to cellular
protein measurements. At 50 microM precursor concentrations,
nicotinamide was the dominant precursor and
NAD levels in the cell rose well above normal levels. Other precursors were minimally incorporated. The same methods were applied to
NAD biosynthesized by macrophages derived from peripheral blood monocytes. However, the
NAD concentration in macrophages was about 5% of that in SK-HEP cells and the incorporation of stable
isotope-labeled substrates remained below measurable levels.