NADH – reduced β-nicotinamide adenine dinucleotide, disodium salt, CAS 606-68-8 – is the reducing coenzyme in the largest group of routine clinical chemistry assays and one of the two workhorses of enzymatic reduction in industry. It is also one of the least forgiving materials in the catalogue: it oxidises in air, degrades in light and acid, and loses activity through repeated freeze-thaw. This article covers what it is used for, what the specification should contain, and how to keep it alive long enough to be useful.
1. Identity and what makes it different from NAD
| Property | NADH (606-68-8) | NAD (53-84-9) |
|---|---|---|
| Oxidation state | Reduced – donates electrons | Oxidised – accepts electrons |
| Formula / MW (disodium salt) | C21H27N7Na2O14P2 / 709.4 | C21H27N7O14P2 / 663.4 (free acid) |
| Absorption maximum | 340 nm (oxidised form has no such peak) | 260 nm |
| Role in a coupled assay | Consumed – signal decreases at 340 nm | Generated in situ from the dehydrogenase reaction |
| Storage emphasis | Oxygen, light, moisture and freeze-thaw | Moisture and heat |
The 340 nm absorbance is the reason NADH dominates diagnostics: its molar absorptivity at 340 nm is about 6.22 x 103 M-1cm-1, so a change in absorbance converts directly into a quantity of substrate consumed. Every reagent whose result is expressed as “units per litre” through a dehydrogenase relies on that constant.
2. The main diagnostic uses
| Analyte | Enzyme chain | What is measured |
|---|---|---|
| ALT (alanine aminotransferase) | ALT generates pyruvate; lactate dehydrogenase converts it to lactate while oxidising NADH | Rate of absorbance decrease at 340 nm |
| AST (aspartate aminotransferase) | The same coupling through LDH and NADH | Rate of absorbance decrease at 340 nm |
| LDH (lactate dehydrogenase) | Pyruvate to lactate, NADH oxidised directly | Rate of absorbance decrease at 340 nm |
| Urea / BUN | Glutamate dehydrogenase: alpha-ketoglutarate + ammonium + NADH | Rate of absorbance decrease at 340 nm |
| GLDH (glutamate dehydrogenase) | Direct NADH-linked reaction | Rate of absorbance decrease at 340 nm |
In all of these the NADH is a reagent, consumed stoichiometrically, and its own purity directly biases the result. A lot that is 96% NADH with 4% already oxidised to NAD delivers less reducing capacity than a 99% lot, and the difference appears as a calibration shift rather than as an obvious failure.
3. The main biocatalytic uses
- Ketoreductases (KRED) – asymmetric reduction of ketones to chiral alcohols; the largest industrial use of NADH/NAD+ recycling.
- Alcohol dehydrogenases (ADH) – used in reverse for reduction, with isopropanol as the sacrificial substrate.
- Ene-reductases – activation of C=C bonds in the synthesis of chiral intermediates.
- Amino acid dehydrogenases – reductive amination of keto acids to L-amino acids, usually with ammonium formate or glucose as the regeneration substrate.
In industry NADH is rarely dosed stoichiometrically. It is charged catalytically and regenerated by a second enzyme – glucose with glucose dehydrogenase, formate with formate dehydrogenase, or isopropanol with the same alcohol dehydrogenase driving the reverse reaction. That changes the economics completely: the coenzyme is an inventory item, not a consumable per mole of product, which is why the specification should focus on stability and on how much of the material survives in-process rather than only on the assay figure.
4. Stability: where NADH is usually lost
| Stress | Effect | Control |
|---|---|---|
| Oxygen in solution | Slow oxidation to NAD+; the 340 nm signal decays even without enzyme | Dissolve fresh; keep solutions on ice; consider degassing for long runs |
| Light | Accelerated degradation | Amber glassware, foil-wrapped vessels, dark storage |
| Acid | Hydrolysis of the nicotinamide-ribose bond | Work at pH 7.0-8.0; the disodium salt helps here |
| Freeze-thaw cycles | Moisture migration and slow decomposition | Aliquot on receipt into single-use portions |
| Moisture in the powder | Hygroscopic; caking and hydrolysis | Equilibrate to room temperature before opening; reseal immediately |
| Trace metals | Catalytic oxidation | Use good-quality buffer water and clean glassware |
A practical working rule: weigh and dissolve what the shift will use, keep it cold and dark, and do not hold a prepared solution overnight expecting the same calibration.
5. What the specification should contain
| Item | Why it matters for NADH specifically |
|---|---|
| Assay, with the method named | HPLC and enzymatic assay answer different questions: HPLC measures the molecule, the enzymatic assay measures usable reducing capacity. Ask for both on critical lots. |
| Water content | Hygroscopic; a damp lot delivers less coenzyme per gram weighed. |
| NAD+ content (oxidation product) | Directly reduces the reducing capacity you paid for. Rarely stated unless requested. |
| Residual solvents | Typically ethanol from the isolation step; matters for pharmaceutical customers. |
| Sodium content | Confirms the disodium salt rather than a partly exchanged form. |
| Endotoxin / microbial | Required for cell-based work and for IVD reagents; not stated by default. |
6. Packaging, storage and cold chain
NADH ships as a powder in foil-lined bags or small bottles for reagent use, and in bulk for manufacturing. The recommended storage is sealed at -20 °C or below, protected from light and moisture; shipment at ambient temperature is usually acceptable for a short transit because the material is dry, but if your incoming goods procedure requires a cold chain, say so when requesting the quotation. Each lot arrives with a certificate of analysis and a safety data sheet, and third-party testing can be arranged. Bulk packs should be split into single-use aliquots on receipt – the single most effective step for protecting the assay figure you paid for.
7. Common incoming-goods failures
- A certificate with only a purity figure. Purity by HPLC can be high while oxidised NAD content is also high.
- Free acid supplied against a disodium specification. Different CAS, different dissolution behaviour, different sodium load.
- Long ambient transit in summer. Dry powder tolerates it; a solution would not – know which you ordered.
- Repackaged material with no lot traceability. Insist on the manufacturer’s lot number on the COA.
- No stated storage instruction. If the supplier does not specify -20 °C for long-term storage, ask why.
Related reading
- NAD, NADH, NADP and NADPH: the whole coenzyme family on one page
- NADP salt forms: free acid, monosodium or disodium
- NADH disodium salt (CAS 606-68-8) product page
Products in this family
- NADH disodium salt (CAS 606-68-8) – assay 98% min
- NAD (CAS 53-84-9) – 85% min solution, 98% min and 99% min powders
- NADPH tetrasodium salt (CAS 2646-71-1) – assay 95% min
PolyBlueChem supplies NADH disodium salt (CAS 606-68-8), assay 98.0% min, with per-batch COA and SDS and FOB/CIF delivery. Send your assay requirement, quantity, required documentation and destination, and we will confirm the grade and the shipping arrangement.