AAT Bioquest


Chemical Structures of NAD+, NADP+, NADH, NADPH
Chemical Structures of NAD+, NADP+, NADH, 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)+

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:

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:

NADP+ Biosynthesis

Chemical structures of oxidized and reduced NADP(H)
Chemical structures of oxidized and reduced NADP(H) as associated with the ReadiUse™ NADP Regenerating Kit.
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.

Table 1. Biochemical assays for measuring ATP activity, formation or depletion.

Ex/Abs (nm)
Em (nm)
Cutoff (nm)¹
Microplate Type
Unit Size
Cat No.
PhosphoWorks™ Fluorimetric ATP Assay Kit540590570Solid black100 tests21620
PhosphoWorks™ Colorimetric ATP Assay Kit570--Clear bottom100 tests21617
PhosphoWorks™ Luminometric ATP Assay Kit *Maximized Luminescence*---Solid white1 plate21610
PhosphoWorks™ Luminometric ATP Assay Kit *Maximized Luminescence*---Solid white10 plate21621
Cell Meter™ Live Cell ATP Assay Kit---Black wall/clear bottom100 Tests23015

NAD(P)+ Regulation

Chemical structure for ADP-ribose-pNP
Chemical structure for ADP-ribose-pNP, a colorimetric substrate for assessing activity of poly(ADP-ribose)polymerase (PARP) enzymes.
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.

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NAD(P)+ Physiology

NAD+ to NADH redox reaction
Illustration of NAD+ to NADH redox reaction. Made in BioRender.

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

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 images of NADH/NADPH in HeLa cells
Fluorescence images of NADH/NADPH in HeLa cells using Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit.

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

Table 2. NAD/NADH & NADP/NADPH Assay Comparison

Product Name
Ex (nm)
Em (nm)
Detection Limit
Dynamic Range
Unit Size
15259Amplite® Fluorimetric Total NADP and NADPH Assay Kit *Red Fluorescence*5405900.01 µM 0-3 µM 400 Tests
15260Amplite® Colorimetric Total NADP and NADPH Assay Kit575 0.1 µM 0-3 µM 400 Tests
15262Amplite® Fluorimetric NADPH Assay Kit *Red Fluorescence*5715851 µM 0-100 µM 400 Tests
15264Amplite® Fluorimetric NADP/NADPH Ratio Assay Kit *Red Fluorescence*5715850.01 µM 0-3 µM 250 Tests
15272Amplite® Colorimetric NADPH Assay Kit460 3 µM 1-200 µM 400 Tests
15274Amplite® Colorimetric NADP/NADPH Ratio Assay Kit460 0.03 µM 0.03-1 µM 250 Tests
15276Amplite® Colorimetric Total NADP and NADPH Assay Kit *Enhanced Sensitivity*460 0.03 µM 0.03-1 µM 400 Tests
15281Amplite® Fluorimetric NADP Assay Kit *Blue Fluorescence*4224660.03 µM 0.03-1 µM 200 Tests
15257Amplite® Fluorimetric Total NAD and NADH Assay Kit *Red Fluorescence*5715850.1 µM 0-3 µM 400 Tests
15258Amplite® Colorimetric Total NAD and NADH Assay Kit575 0.3 µM 0-10 µM 400 Tests
15261Amplite® Fluorimetric NADH Assay Kit *Red Fluorescence*5715851 µM 0-100 µM 400 Tests
15263Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*5715850.1 µM 0-3 µM 250 Tests
15271Amplite® Colorimetric NADH Assay Kit460 3 µM 1-200 µM 400 Tests
15273Amplite® Colorimetric NAD/NADH Ratio Assay Kit460 0.1 µM 0.1-10 µM 250 Tests
15275Amplite® Colorimetric Total NAD and NADH Assay Kit *Enhanced Sensitivity*460 0.1 µM 0.1-10 µM 400 Tests
15280Amplite® Fluorimetric NAD Assay Kit *Blue Fluorescence*4224660.03 µM 0.03-10 µM 200 Tests



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