LC/MS Analysis of NAD Biosynthesis Using Stable Isotope Pyridine Precursors

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 μM 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.