Measurement of pyridine nucleotides (NMN, NAD+, NADH, NADP, NADPH) in biological samples is vitally important given their pivotal, multifaceted roles in intermediary metabolism. We devised robust sample processing and LC/MS/MS methods to accomplish this challenging task given the inherent instability of these metabolites. Our assay precision and accuracy were enabled by the use of a Precellys Evolution homogenizer and custom-synthesized heavy isotope-labeled internal standards. We used these methods to profile the pyridine nucleotide pool in mouse gastrocnemius.
Pyridine nucleotides in the homogenate supernatants were quantitated using a Dionex 3000 HPLC/ThermoScientific Quantiva triple quadrupole mass spectrometer. The tables below show the comparison between levels of pyridine nucleotides (pmol/mg dry tissue) determined in mouse gastrocnemius homogenized using manual versus Precellys Evolution homogenization.
Table 1. Levels of oxidized pyridine nucleotides (n = 4) obtained from manual and Precellys Evolution homogenization.
Table 2. Levels of reduced pyridine
nucleotides (n = 2) obtained from manual and
Precellys Evolution homogenization.