Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach. Bustamante S et al. (2019) Nicotinamide adenine dinucleotide (NAD+) is an essential pyridine nucleotide that serves as a key hydride transfer coenzyme for several oxidoreductases. It is also the substrate for intracellular secondary messenger signalling by CD38 glycohydrolases, DNA repair by poly(adenosine diphosphate ribose) polymerase, and epigenetic regulation of gene expression by a class of histone deacetylase enzymes known as sirtuins. The measurement of NAD+ and its related metabolites (hereafter, the NAD+ metabolome) represents an important indicator of cellular function. A study was performed to develop a sensitive, selective, robust, reproducible, and rapid method for the concurrent quantitative determination of intracellular levels of the NAD+ metabolome in glial and oocyte cell extracts using liquid chromatography coupled to mass spectrometry (LC/MS/MS). The metabolites were separated on a versatile amino column using a dual HILIC-RP gradient with heated electrospray (HESI) tandem mass spectrometry detection in mixed polarity multiple reaction monitoring mode. Quantification of 17 metabolites in the NAD+ metabolome in U251 human astroglioma cells could be achieved. Changes in NAD+ metabolism in U251 cell line, and murine oocytes under different culture conditions were also investigated. This method can be used as a sensitive profiling tool, tailoring chromatography for metabolites that express significant pathophysiological changes in several disease conditions and is indispensable for targeted analysis.//////////////////
Follicle stages
Comment
Phenotypes
Mutations
1 mutations
Species: mouse
Mutation name: type: null mutation fertility: fertile Comment: Mice deficient for the ecto-nicotinamide adenine dinucleotide glycohydrolase CD38 exhibit altered humoral immune responses. Cockayne DA et al. (1998) CD38 is a membrane-associated ecto-nicotinamide adenine dinucleotide (NAD+) glycohydrolase that is expressed on multiple hematopoietic cells. The extracellular domain of CD38 can mediate the catalysis of NAD+ to cyclic adenosine diphosphoribose (cADPR), a Ca2+-mobilizing second messenger, adenosine diphosphoribose (ADPR), and nicotinamide. In addition to its enzymatic properties, murine CD38 has been shown to act as a B-cell coreceptor capable of modulating signals through the B-cell antigen receptor. To investigate the in vivo physiological function(s) of this novel class of ectoenzyme we generated mice carrying a null mutation in the CD38 gene. CD38-/- mice showed a complete loss of tissue-associated NAD+ glycohydrolase activity, showing that the classical NAD+ glycohydrolases and CD38 are likely identical. Although murine CD38 is expressed on hematopoietic stem cells as well as on committed progenitors, we show that CD38 is not required for hematopoiesis or lymphopoiesis. However, CD38-/- mice did exhibit marked deficiencies in antibody responses to T-cell-dependent protein antigens and augmented antibody responses to at least one T-cell-independent type 2 polysaccharide antigen. These data suggest that CD38 may play an important role in vivo in regulating humoral immune responses.//////////////////