The pyridine dinucleotide, NAD+, is a substrate of sirtuins and coenzyme for hydride transfer enzymes and other NAD+-dependent ADP ribose transfer enzymes. It is a unique cellular molecule produced from the pyridine mononucleotides nicotinic acid mononucleotide (NaMN) or nicotinamide mononucleotide (NMN). It is exist in either the form NAD+ or NADH. When being cleaved, NAD+ and NADH can convert to nicotinamide (Nam) and acetylated ADP ribose. The acetylated ADP ribose in turn can be deacetylated by sirtuins.
In both prokaryotic and eukaryotic systems, NAD is synthesized through two pathways: de novo pathway and salvage pathway. In the de novo pathway, NAD is generated from tryptophan through quinolinic acid (QA) and nicotinic acid (NA). In salvage pathway, NAD is synthesized by recycling degraded NAD products such as Nam and nicotinamide. Both pathways play essential roles in cell growth. Under normal physiological condition, the salvage pathway plays a more important role than de novo pathway in NAD synthesis. In yeast, the de novo pathway is making of six enzymatic steps and one non-enzymatic reaction. In the last enzymatic reaction, quinolinate is converted to nicotinic acid mononucleotide by a quinolinate phosphoribosyl transferase encoded by the BNA6/ QPT1 gene. This last enzymatic reaction is the converge point of the de novo pathway and the salvage pathway.
NAD takes part in many physiological processes, such as energy metabolism regulation, DNA repair and transcription. Besides acting as a coenzyme, NAD also serves as a substrate such as substrate for NAD-dependent DNA ligases, NAD-dependent oxidoreductases and NAD-dependent deacetylases. At the same time, the reduced form of NAD, NADH, serves as a substrate for the NADH dehydrogenase in the mitochondrial respiratory chain to transfers electrons to coenzyme Q and generate NAD. Calorie restriction accomplished by glucose limitation in wild-type Saccharomyces cerevisiae can extend replicative lifespan of cells and this process relies on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Together with glutathione, the derivative of NAD, the NADPH coenzyme, maintains the intracellular redox state and is also involved in many assimilatory pathways. To maintain the proper redox state, NADH needs to be constantly re-oxidised. Mostly, NAD is converted to NADH mostly in catabolic reactions such as glycolysis and TCA cycle. However, both cytosolic and mitochondrial NADH are re-oxidised mainly by the respiratory chain. Since the inner mitochondrial membrane is impermeable for NAD and NADH, there are several shuttle systems to transport permeable redox equivalents across this barrier. Ethanol-acetaldehyde shuttle is one example.
Though the changes of NAD+ to nicotinamide ratio and the NAD+ to NADH ratio can be anticipated through models to related the effects of calorie restriction. However, the putative alterations of NAD+ metabolism require a sensitive and reliable qualitative and quantitative analysis of NAD+ metabolites.
- Identification and quantification of NAD Metabolites by HPLC-MS/MS
- Normal Volume: 200ul plasma, 200 mg tissue, 2e7 cells
- Minimal Volume: 50ul plasma, 50ul, 50 mg tissue, 5e6 cells
- A detailed technical report will be provided at the end of the whole project, including the experiment procedure, MS/MS instrument parameters
- Analytes are reported as uM or ug/mg (tissue), and CV's are generally<10%
- The name of the analytes, abbreviation, formula, molecular weight and CAS# would also be included in the report.
|NAD Metabolites Quantified in This Service|
|Acetyl-CoA (aCoA)||Adenosine monophosphate (AMP)||Adenosine diphosphate (ADP)|
|Adenosine triphosphate (ATP)||Citrate/Isocitrate combined (Cit/i-Cit)||Erythrose 4-phosphate (E4P)|
|Flavin adenine dinucleotide (FAD)||Fructose-6-phosphate + glucose-6-phosphate (F6P/G6P)||Fructose-bisphosphate (FBP)|
|Glyceraldehyde-3-phosphate (G3P)||Succinate (Suc)||Malate (Mal)|
|nicotinamide mononucleotide (NMN)||nicotinamide (NAM)||Nicotinic acid (NA)|
|Nicotinic acid mononucleotide (NaMN)||Nicotinamide adenine dinucleotide (NAD)||Nicotinamide adenine dinucleotide, reduced (NADH)|
|Nicotinamide adenine dinucleotide phosphate (NADP)||Nicotinamide adenine dinucleotide phosphate, reduced (NADPH)||Phosphoenolpyruvate (PEP)|
|Quinolinic acid (QA)||Ribulose 5-phosphate/Xylulose 5-phosphate/ribose-5-phosphate combined (R5P/X5P/Ru5P)||Sedoheptulose 7-phosphate (S7P)|
|2-Phosphoglycerate/3-Phosphoglycerate combined (2PG/3PG)||6-phosphogluconate (6PG)|
With integrated set of separation, characterization, identification and quantification systems featured with excellent robustness & reproducibility, high and ultra-sensitivity, Creative Proteomics provides reliable, rapid and cost-effective NAD metabolites targeted metabolomics services.