NAD+ vs Glutathione
Research Comparison

Research Overview

NAD vs glutathione compares two redox systems built on different chemistry. NAD+ (nicotinamide adenine dinucleotide) is a pyridine nucleotide coenzyme that carries electrons as the NAD+/NADH pair in oxidoreductase reactions, and NAD+ is also used up as a substrate for ADP-ribosylation reactions and Sir2 family deacetylases, the sirtuins (Pollak et al., 2007). Glutathione is a tripeptide and the most abundant non-protein thiol in mammalian tissues (Lu, 2013); its reduced form, GSH, and its disulfide, GSSG, make up the most abundant redox couple in a cell (Schafer and Buettner, 2001).

Glutathione vs NAD is not a choice between two versions of one molecule. The NAD+/NADH pair carries electrons as a hydride; glutathione gives them up from a cysteine thiol. The systems meet at NADPH, which glutathione reductase uses to turn GSSG back into GSH (Giustarini et al., 2013). Disguised Alpha supplies NAD+ as buffered lyophilized vials (500MG or 1000MG) and a 200MG/ML liquid (20ML), the NAD+ Aerosol Spray (1,000MG per bottle), and L-glutathione as a 1500MG lyophilized powder and a 200MG/ML liquid (20ML), for laboratory research use only.

Structure and Class

NAD+ is a dinucleotide, not a peptide: adenosine and nicotinamide riboside joined by a diphosphate bridge. Product page identity: C21H27N7O14P2, 663.43 g/mol, matching the formula for PubChem CID 5892. In NADH (CID 439153), the nicotinamide ring carries the added hydride at carbon 4. NADP+ (CID 5885) adds a third phosphate on the adenosine ribose, and NAD kinase, which makes it from NAD+, is the sole NADP+ biosynthetic enzyme (Ohashi et al., 2012).

Glutathione is gamma-L-glutamyl-L-cysteinyl-glycine: glutamate joins cysteine through its side-chain carboxyl. Product page identity: C10H17N3O6S, 307.33 g/mol, matching PubChem CID 124886. Oxidation links two GSH molecules by a disulfide into GSSG (C20H32N6O12S2, 612.6 g/mol, CID 65359).

In Escherichia coli glutathione reductase, specificity for NADP(H) is built into the protein: a set of side chains in its NADP+-binding domain confers it, and replacing them left substrate specificity unchanged but gave the enzyme a marked preference for NAD+ (Scrutton et al., 1990). Cells usually keep the NADPH/NADP+ ratio high, as a pool of reducing equivalents against oxidative damage (Pollak et al., 2007). Keep NAD+/NADH and NADP+/NADPH apart in notes and figures.

Side by Side

Research Context: NAD+

In freeze-clamped rat liver, free NAD+/NADH ratios calculated from dehydrogenase substrates were about 725 in the cytoplasm and about 8 in mitochondria of well-fed rats, and free ratios differed greatly from total ones because much more NADH than NAD+ is protein-bound (Williamson et al., 1967). Model: rat liver.

In living cells, Peredox, a GFP-based sensor built on the bacterial NADH-binding protein Rex, reports cytosolic NADH:NAD+ ratios and can be calibrated with exogenous lactate and pyruvate; with it, primary mouse astrocytes and neurons showed different redox states (Hung et al., 2011). Models: cultured and primary cells.

NAD+ also feeds the NADP pool. C5orf33, a human protein of previously unknown function, showed NAD kinase activity and localized to the mitochondria of human HEK293A cells, a likely source of human mitochondrial NADP+ (Ohashi et al., 2012). Models: activity assays and human cell culture.

Research Context: Glutathione

Glutathione is a key determinant of redox signaling and vital in the detoxification of xenobiotics. Its synthesis in the cytosol is tightly regulated: cysteine availability and the activity of glutamate-cysteine ligase, the rate-limiting enzyme, are the key determinants; glutathione synthetase is the second enzyme, and Nrf2, AP-1 and NF-kappaB regulate the genes (Lu, 2013). Model: review.

The GSSG/2GSH half-cell reduction potential, estimated with the Nernst equation, depends on pH and the concentrations of the species, and appears to correlate with cell status: about -240 mV in proliferating, -200 mV in differentiating and -170 mV in apoptotic cells (Schafer and Buettner, 2001). Grx1-roGFP2, human glutaredoxin-1 fused to roGFP2, equilibrates specifically with the glutathione couple, imaged its redox potential in different cellular compartments and detected nanomolar GSSG changes against millimolar GSH (Gutscher et al., 2008). Models: redox theory and live-cell imaging.

The NADH to NADPH link has been studied in mouse liver mitochondria. Nicotinamide nucleotide transhydrogenase (NNT) reduces NADP+ at the expense of NADH oxidation; mitochondria from C57BL/6J mice with a spontaneous Nnt mutation showed no transhydrogenation between NAD and NADP, spontaneous NADPH oxidation and higher oxidized/reduced glutathione ratios than those of an Nnt wild-type C57BL/6 substrain (Ronchi et al., 2013). Model: mouse liver mitochondria.

Choosing Between Them for a Research Question

Hydride transfer, dehydrogenase kinetics, NAD+/NADH ratios and NAD+-consuming enzymes call for NAD+; thiol-disulfide exchange, GSSG/2GSH potentials and glutathione-dependent detoxification call for glutathione. A question about how one system supplies the other needs NADP+/NADPH in the design and readout, the pair that Escherichia coli glutathione reductase is built to use (Scrutton et al., 1990). In mouse work, record the substrain and its Nnt status; the study's authors caution that the potential drawbacks of using Nnt-mutant mice should not be overlooked (Ronchi et al., 2013).

Measure each pool with methods that keep its forms apart. A validated plate reader method read NAD+, NADP+ and both glutathione forms in HCl extracts of Arabidopsis leaves, and NADH and NADPH in parallel alkaline extracts (Queval and Noctor, 2007). For LC-MS of cultured mammalian cells and mouse tissues, interconversion during extraction was a major barrier to accurate NADH/NAD+ and NADPH/NADP+ ratios; 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid gave the least, followed by immediate neutralization (Lu et al., 2018).

For glutathione in blood and tissues, 5 to 15% of GSH can oxidize during acid deproteinization, markedly overestimating GSSG, unless GSH is first derivatized with N-ethylmaleimide; after excess reagent is back-extracted with dichloromethane, GSSG is read by recycling with glutathione reductase, NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) (Giustarini et al., 2013).

Handling and Storage

The glutathione powder is listed at -20°C as shipped and 2 to 8°C after reconstitution, protected from light, without freeze/thaw cycles. The 200MG/ML glutathione liquid ships in a glass bottle and is listed at 15 to 25°C unopened and 2 to 8°C once opened, protected from light and never frozen; the NAD+ Aerosol Spray lists the same temperatures and is kept upright. For the NAD+ vials and liquid, the product page shows one storage listing for every size; it lists protection from light and 2 to 8°C once the seal is broken.

Prepare GSH solutions fresh and cold, since the thiol oxidizes. Neutralize acid NAD extracts at once to avoid acid-catalyzed degradation (Lu et al., 2018). Aliquot stocks, record the lot number and use the measured content from the COA portal where a certificate reports it, not the label mass.

Questions Researchers Ask

NAD vs glutathione: what is the difference?

NAD+ is a dinucleotide coenzyme that carries electrons as NAD+/NADH and is consumed by ADP-ribosylation reactions and sirtuins (Pollak et al., 2007); glutathione is a cysteine-containing tripeptide whose GSSG/2GSH couple is the most abundant redox couple in a cell (Schafer and Buettner, 2001).

Glutathione vs NAD: which one is a peptide?

Glutathione, a tripeptide made by glutamate-cysteine ligase and glutathione synthetase (Lu, 2013). NAD+ has no amino acids: it is two nucleotides joined through phosphates (PubChem CID 5892).

How are NAD+ and glutathione connected?

Through NADP+ and NADPH. NAD kinase makes NADP+ from NAD+ (Ohashi et al., 2012), glutathione reductase uses NADPH to return GSSG to GSH (Giustarini et al., 2013), and in mitochondria NNT reduces NADP+ at the expense of NADH (Ronchi et al., 2013).

Is NADH the same as NADPH?

No. NADPH (PubChem CID 5884, 745.4 g/mol) carries a third phosphate that NADH (CID 439153, 665.4 g/mol) lacks, and cells usually keep the NADP pool mostly reduced (Pollak et al., 2007). Escherichia coli glutathione reductase is specific for the phosphorylated coenzyme (Scrutton et al., 1990).

How do labs measure NAD+ and glutathione?

With methods that keep oxidized and reduced forms apart: parallel acid and alkaline extracts (Queval and Noctor, 2007), acidic organic extraction for LC-MS (Lu et al., 2018), N-ethylmaleimide for GSH (Giustarini et al., 2013), and Peredox (Hung et al., 2011) or Grx1-roGFP2 (Gutscher et al., 2008) in living cells.