NAD/NADH and NADP/NADPH

Nicotinamide adenine dinucleotide (NAD+) and NAD+ phosphatase (NADP+) are coupled redox cofactors associated with maintaining chemical equilibrium in the cellular environment with active roles in many signal pathways, including cellular metabolism. NAD(P)+ are critical in managing oxidative and reducing stress, and are classified under coenzymes in the oxidoreductase family. NAD+ reduces to NADH through dehydrogenases, and phosphorylates to NADP+ through associated kinases, where each play a role in glycolysis and mitochondrial function. NAD+ is synthesized through various methods in the cytosol, nucleus and mitochondria, though reactions are highly compartmentalized within the microbiome due to limited bioavailability of precursors and partnered enzymes.

There exists recent evidence to support that NAD(P)+ can transport across cell membranes, and many NAD(P)+ dependent enzymes are linked to post-chemical nucleotide modification. Researching and understanding how cells manage to maintain NAD+ and complementary analyte pools may then better shed light on associated illnesses, including metabolic diseases, cancers, and neurological disorders.

Pathways of NAD(P)+

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De novo Synthesis

Tryptophan (Trp) can be converted to NAD+ by the kynurenine pathway (KP) which is activated by one of two dioxygenase molecules, abbreviated TDO or IDO. Hydroxylated kynurenine undergoes three more enzymatic conformations before becoming its final form; first firming an unstable amine (ACMS), then Quinolinic Acid (QA), next nicotinic acid (NA) mononucleotide (NAMN), and finally to NA adenine dinucleotide (NAAD), before becoming NAD+. The final conversion of NAAD to NAD+ is mediated by ATP-dependent NAD+ synthetases (NADSYNs) which are found readily in the liver, kidney, and small intestine.

Assaywise Letters:	Application Notes:
NAD+ Integration in Cellular Pathways	Activation of the Mitochondrial Apoptotic Pathway Produces Reactive Oxygen Species and Oxidative Damage in Hepatocytes That Contribute to Liver Tumorigenesis

The Preiss-Handler and Salvage Pathways

The Priess-Handler pathway is a three step process where NAD+ is produced with the addition of ATP and glutamine as a nitrogen donor, involving NAMN and NAAD as intermediate metabolites. This pathway requires NA as starting material, commonly known as the vitamin niacin, which is the limiting substrate. The salvage pathway creates NAM mononucleotide (NMN) from either Nicotinamide (NAM) or NAM riboside (NR) along with the aid of ATP. NMN adenylyltransferases (NMNATs) then catalyzes the conversion of this intermediate into NAD+, also involved in the De novo synthesis platform, that has three associated isoforms, each with distinct localizations in the cell.

Application Notes:

Photometric Detection of Nicotinamide Adenine Dinucleotides

NADP+ Biosynthesis

NADP+ is synthesized by a phosphate rearrangement from ATP to an adenosine ribose moiety of NAD+ which is initiated by NAD+ kinase (NADK). NADK is extremely specific, and requires the prevalence of bivalent cations like magnesium, calcium and manganese in both pro- and eukaryotes. NADK was recently found to also exist in mitochondrial form (MNADK), and variants of this biomolecule have been associated with hyperlysinemia and diseases involving polyunsaturated fatty acid (PUFA) metabolism. MNADK has been exploited in some research studies, where enzymatic overexpression restored functional ability by failing dienoyl-CoA reductase (DECR, another critical metabolite for cellular homeostasis), suggesting therapeutic potential.

 

NAD(P)+ Regulation

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Certain consuming enzymes, some examples include PARPs, SIRTs, CD38 and SARM1, break down NAD+ and in turn generate NAM and ADPribose (ADPR), each with a particular set of functions. PARPs are associated with DNA and RNA repair and processing, respectively, SIRTs are involved with mitochondrial metabolism, and both impact epigenetic activity and circadian rhythm. SARM1 plays a role in inflammation and cell adhesion, while CD38 mediates Ca signaling.

Assaywise Letters:	Datasets:
A Look at NAD-Dependent SIRT-1	Mono [ADP-ribose] polymerase PARP16 Inhibitors (IC50, Ki)

NAD(P)+ Physiology

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NAD(P)+ pathways are also inter connected in a number of other signaling pathways. The conversion of NAD+ to NADH stimulates glycolysis, fatty acid oxidation and the tricarboxylic acid cycle. NADH altering back to NAD+ affects lactic acid degeneration, PUFA desaturation, and the mitochondrial electron transport chain. NADP+ conversion to NADPH vitalizes the pentose phosphate pathway, which when transformed back into the monovalent cations NADP+ is involved in antioxidant defense, anabolic reactions and NOX signaling.

NAD+ is also involved in RNA capping in many species, and aids in DNA ligation and repair through adenylation. Besides simply maintaining redox homeostasis, NAD(P)+ also donate electrons in reactive oxygen species (ROS) generation and play a part in the creation of antioxidant species. Due to this role, NAD(P)+ is associated with maintaining genomic equanimity and gene expression manipulation. It is apparent that NAD(P)+ is networked to many different signal pathways, issues in NAD(P)+ metabolism could lead to a multitude of potential negative ensuing effects associated with immune and inflammatory disease.

 

Measuring NAD(P)+/NAD(P)H

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Biochemical Techniques

High-performance liquid chromatography (HPLC) can be used to detect and quantify metabolites produced from NAD(P)+, and is usually coupled with mass spectrometry (LC/MS). The idea is that NAD(P)+ biosynthesis, which positively supports body function, can be monitored through NAD(P)+ intermediate production, namely NMN and NR. Intermediates are identified through acid extraction and NAD(P)+ concentration can be obtained. Enzymatic cycling assays, capillary electrophoresis, and isotope-labeling techniques have also been used, usually alongside LC/MS, though these processes measure total, cumulative NAD(P)+ and do not distinguish between subcomponent concentrations. Each method can also quantify the reduced coenzyme forms, with reproducible and rapid results through fragmentation.

Fluorescence

Genetically encoded fluorescent sensors are also utilized to detect NAD(P)+/H changes in real-time, and techniques also target specific redox reactions, thiols, and ROS. These techniques can also be applied in a high-throughput setting, using many available equipment types like microplate readers, flow cytometry, fluorescence microscopy and other optical imaging systems. Benefits to said biosensors are acute fluorescence, rapid excitation, and subcellular organelle targeting where cytosolic, compartmentalized and mitochondrial NAD(P)+ pools can be separately quantified. Fluorescent techniques still have a long way to go, and easily distinguishing between NADH and NADPH levels in samples remains an issue. Certain biosensors are also not totally impassive to alterations in pH, so consideration must be taken in this regard as well.

P-Magnetic Resonance Spectroscopy (P-MRS)

P-MRS techniques have been used to measure NAD+/H levels in vivo by employing an ultra-high magnetic field, specifically in areas of the brain. Results can be interpreted through analysis by charts showing chemical shifts between the resonance frequency of the observed proton and hydrogens on tetramethylsilane. Through this method, ATP and NAD+/H pools can be distinguished in a minimally invasive manner to living tissue. Therapeutic advances so far have been limited to the low-resolution quality provided by low magnetic fields needed for clinical applications; however progress continues to be made on this forefront.

Product Ordering Information

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References

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Xiao, Wusheng, et al. “NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.” Antioxidants & Redox Signaling, vol. 28, no. 3, 2018, pp. 251-272., https://doi.org/10.1089/ars.2017.7216.

Xie, Na, et al. “NAD+ Metabolism: Pathophysiologic Mechanisms and Therapeutic Potential.” Signal Transduction and Targeted Therapy, vol. 5, no. 1, 2020, https://doi.org/10.1038/s41392-020-00311-7.

Kelly, Gregory. “How Is NAD+ Made? Preiss-Handler Pathway.” Neurohacker Collective, 30 Aug. 2019, https://neurohacker.com/how-is-nad-made-preiss-handler-pathway.

Yoshino, Jun, and Shin-ichiro Imai. “Accurate Measurement of Nicotinamide Adenine Dinucleotide (NAD+) with High-Performance Liquid Chromatography.” Sirtuins, 26 Feb. 2014, pp. 203-215., https://doi.org/10.1007/978-1-62703-637-5_14.

Zou, Yejun, et al. “Analysis of Redox Landscapes and Dynamics in Living Cells and in Vivo Using Genetically Encoded Fluorescent Sensors.” Nature Protocols, vol. 13, no. 10, 2018, pp. 2362-2386., https://doi.org/10.1038/s41596-018-0042-5.

Lu, Ming, et al. “In vivo31p MRS Assessment of Intracellular NAD Metabolites and NAD+/NADH Redox State in Human Brain at 4 T.” NMR in Biomedicine, vol. 29, no. 7, 2016, pp. 1010-1017., https://doi.org/10.1002/nbm.3559.