- high-resolution terminology - matching measurements at high-resolution
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, NAD+ and NADH (pyridine nucleotide coenzymes, NAD and NADP), is an oxidation-reduction coenzyme (redox cofactor; compare FADH2). In the NADH electron transfer-pathway state fuelled by type N-substrates, mt-matrix dehydrogenases generate NADH, the substrate of Complex I (CI). The reduced N-substrate RH2 is oxidized and NAD+ is reduced to NADH,
- RH2 + NAD+ → NADH + H+ + R
The mt-NADH pool integrates the activity of the TCA cycle and various matrix dehydrogenases upstream of CI, and thus forms a junction or funnel of electron transfer to CI, the N-junction (compare F-junction, Q-junction). NAD+ and NADH are not permeable through the mt-inner membrane, mtIM. Therefore, an increase of mitochondrial respiration after the addition of NADH may indicate an alteration of the mtIM integrity. Cytosolic NADH is effectively made available for mitochondrial respiration through the malate-aspartate shuttle or glycerophosphate dehydrogenase Complex.
Reference: Gnaiger 2020 BEC MitoPathways
Communicated by Gnaiger Erich 2016-02-12; updated by Doerrier Carolina 2021-01-25.
Application in HRR
- Nicotinadeninedinucleotide, NADH (β-Nicotinamide adenine dinucleotide, reduced potassium salt, C21H27N7O14P2K2); Sigma A 4505, 100 mg, store at -20 °C (old Sigma recommendation: 4-8 °C); M = 742.61 g·mol-1, for important considerations concerning storage of NADH (powder and solutions) and preparation of solutions see the product information from Sigma: . The same precautions are recommended by Sigma for the sodium salt.
- Preparation of NADH to test the integrity of mtIM:
- 2.8 mM is the concentration of NADH in the respirometric chamber described by Hoppel Charles L to assess the integrity of the mtIM (Puchowicz 2004 Mitochondrion; Lai 2018 Acta Physiol (Oxf)).
- For the measurement of NADH autofluorescence: Nicotinadeninedinucleotide, NADH (β-Dihydronicotinamide adenine dinucleotide disodium salt, C21H27N7Na2O14P2); VWR/424237L, 500 mg, store at -20 °C; M = 709.41 g·mol-1.
- Caution: Susceptible to oxidation and light sensitive.
- Preparation of 0.22 mM stock solution for measurement of NADH autofluorescence (dissolved in 10 mM NaOH):
- Prepare fresh every day.
- Weigh 3.90 mg of NADH and add it to 25 mL 10 mM NaOH.
- Protect from light.
- Store cold (on ice and/or at 4 °C) during the experimental day.
- »O2k manual titrations
- Titration volume (2-mL O2k-chamber): 2 µL using a 10 µL Hamilton syringe.
- Final concentration: 0.22 µM.
- DPN+ = NAD+
- DPNH, reduced diphosphopyridine nucleotide = NADH
- TPN+ = NADP+
- TPNH, reduced triphosphopyridine nucleotide = NADPH
MitoPedia topics: Substrate and metabolite