Assessment of NAD + metabolism in human cell cultures, erythrocytes, cerebrospinal fluid and primate skeletal muscle

Anal Biochem. 2019 May 1;572:1-8. doi: 10.1016/j.ab.2019.02.019. Epub 2019 Feb 27.

Abstract

The reduction-oxidation state of NAD+/NADH is critical for cellular health with NAD+ and its metabolites playing critical roles in aging and pathologies. Given the inherent autooxidation of reduced dinucleotides (i.e. NADH/NADPH), and the well-established differential stability, the accurate measurement of NAD+ and its metabolites is technically challenging. Moreover, sample processing, normalization and measurement strategies can profoundly alter results. Here we developed a rapid and sensitive liquid chromatography mass spectrometry-based method to quantify the NAD+ metabolome with careful consideration of these intrinsic chemical instabilities. Utilizing this method we assess NAD+ metabolite stabilities and determine the presence and concentrations of NAD+ metabolites in clinically relevant human samples including cerebrospinal fluid, erythrocytes, and primate skeletal muscle.

Keywords: LC-MS/MS; Metabolomics; NAD+ metabolome; NAD+/NADH metabolism.

Publication types

  • Research Support, N.I.H., Intramural

MeSH terms

  • Acrylamides / pharmacology
  • Animals
  • Chromatography, High Pressure Liquid
  • Erythrocytes / cytology
  • Erythrocytes / drug effects
  • Erythrocytes / metabolism*
  • HEK293 Cells
  • Humans
  • Metabolome / drug effects
  • Muscle, Skeletal / cytology
  • Muscle, Skeletal / drug effects
  • Muscle, Skeletal / metabolism*
  • NAD / analysis
  • NAD / cerebrospinal fluid
  • NAD / metabolism*
  • Niacinamide / analogs & derivatives
  • Niacinamide / pharmacology
  • Piperidines / pharmacology
  • Primates
  • Tandem Mass Spectrometry*

Substances

  • Acrylamides
  • N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide
  • Piperidines
  • nicotinamide-beta-riboside
  • NAD
  • Niacinamide