Efficient extraction of pyridine nucleotides from mouse muscle - Bertin Instruments

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Efficient extraction of pyridine nucleotides from mouse muscle

Sources: Sanford-Burnham-Prebys Medical Discovery Institute, Orlando, Florida, USA

Context

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.

Results

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.

Tableau_1

Table 1. Levels of oxidized pyridine nucleotides (n = 4) obtained from manual and Precellys Evolution homogenization.

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Table 2. Levels of reduced pyridine
nucleotides (n = 2) obtained from manual and
Precellys Evolution homogenization.

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