Endogenous Metabolite

NAD+ is a coenzyme made up of pyrophosphate bonds connecting adenosine 5'-phosphate and ribosyl nicotinamide 5'-diphosphate. The oxidized form of NADH is called NAD+ [1]. Widespread throughout nature, NAD+ serves as an electron carrier in numerous enzymatic activities by alternating between oxidation (NAD+) and reduction (Nadide) [2].

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Superoxide generation by complex I is strongly inhibited by NAD+ (Fig. 1A), so the data in Fig. 4 A, B, and D were recorded by using a regenerating system to maintain [NADH] and minimize [NAD+]. Otherwise, NADH is converted to NAD+ at ≈0.2 μM min−1, leading to significant curvature in the assay traces, particularly at low [NADH] (Fig. 4C). For O2•− production KM(app) for NADH is 0.05 μM. Importantly, NAD+ does not exert its inhibitory effect by causing NADH oxidation by complex I to become rate limiting. For example, 30 μM NAD+, which decreases O2•− formation by ≈50%, does not affect the much faster [Fe(CN)6]3−, [Ru(NH3)6]3+, or decylubiquinone reductions. Our observations suggest strongly that a preequilibrium is established between NADH, NAD+, and different states of complex I (oxidized, reduced, nucleotide free, or nucleotide bound), to determine how much of the complex is able to reduce O2 and to thus determine the rate of O2•− formation.
The Molecular Mechanism of Superoxide Formation: Superoxide formation by purified complex I is not affected by decylubiquinone directly or amplified during catalysis (Fig. 1). Therefore, the FMN or one of the FeS clusters is the cofactor that controls O2 reduction, cofactor X. If cofactor X is an FeS cluster, then NAD+ acts, with NADH, to set its equilibrium level of reduction, according to the Nernst equation. If cofactor X is the flavin, then active site occupancy exerts an additional effect on the preequilibrium and is likely to affect the rate of the bimolecular reaction also, as bound nucleotide may preclude or hinder O2 access. Fig. 5A shows the effect of varying the [NADH]/[NAD+] ratio on O2•− production, plotted as a redox titration of cofactor X (Eq. 1) [3].

Oral NAD+ supplements have been utilized to treat energy-draining, unexplained diseases like fibromyalgia and chronic fatigue syndrome, as well as simple weariness [3].

Redox Titrations Using NADH and NAD+. [3]
NADH was repurified in a glovebox (O2 < 2 ppm) by anion exchange chromatography (5-ml HiTrap Q-Sepharose column) to remove contaminating NAD+. After experimentation, the integrity of the NADH stock solution was reevaluated (0.08 ± 0.04% NAD+ formed in 6 h). Typically, redox potentials were set by using 30 μM NADH and a varying amount of NAD+ (Sigma), and the low potential limit was checked by using the NADH regenerating system.

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EPR.[3]
Complex I (10 mg ml−1) was reduced anaerobically by 1 mM purified NADH or by dialysis against purified NADH (≈−0.4 V) or to ≈−0.3 V by using 1 mM NADH and 10 mM NAD+, and frozen immediately. Spectra were recorded on a Bruker EMX X-band spectrometer by using an ER 4119HS high-sensitivity cavity and a ESR900 continuous-flow liquid helium cryostat [3].

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects.[3]

mouse LD50 intraperitoneal 4333 mg/kg Pharmaceutical Chemistry Journal, 20(160), 1986

[1]. Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond), 2012. 122(6): 253-70.

[2]. Energy converting NADH:quinone oxidoreductase (complex I). Annu Rev Biochem, 2006. 75: 69-92.

[3]. The mechanism of superoxide production by NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. Proc Natl Acad Sci U S A, 2006. 103(20): 7607-12.

NAD zwitterion is a NAD. It has a role as a geroprotector. It is functionally related to a deamido-NAD zwitterion. It is a conjugate base of a NAD(+).
A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed)
Nadide has been reported in Homo sapiens with data available.
Nadide is a dinucleotide of adenine and nicotinamide. It has coenzyme activity in redox reactions and also acts as a donor of ADP-ribose moieties.
A coenzyme composed of ribosylnicotinamide 5'-diphosphate coupled to adenosine 5'-phosphate by pyrophosphate linkage. It is found widely in nature and is involved in numerous enzymatic reactions in which it serves as an electron carrier by being alternately oxidized (NAD+) and reduced (NADH). (Dorland, 27th ed)

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Considerable efforts have been made since the 1950s to better understand the cellular and molecular mechanisms of action of metformin, a potent antihyperglycaemic agent now recommended as the first-line oral therapy for T2D (Type 2 diabetes). The main effect of this drug from the biguanide family is to acutely decrease hepatic glucose production, mostly through a mild and transient inhibition of the mitochondrial respiratory chain complex I. In addition, the resulting decrease in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, providing a generally accepted mechanism for the action of metformin on hepatic gluconeogenesis. The demonstration that respiratory chain complex I, but not AMPK, is the primary target of metformin was recently strengthened by showing that the metabolic effect of the drug is preserved in liver-specific AMPK-deficient mice. Beyond its effect on glucose metabolism, metformin has been reported to restore ovarian function in PCOS (polycystic ovary syndrome), reduce fatty liver, and to lower microvascular and macrovascular complications associated with T2D. Its use has also recently been suggested as an adjuvant treatment for cancer or gestational diabetes and for the prevention in pre-diabetic populations. These emerging new therapeutic areas for metformin will be reviewed together with recent findings from pharmacogenetic studies linking genetic variations to drug response, a promising new step towards personalized medicine in the treatment of T2D. [1]

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NADH:quinone oxidoreductase (complex I) pumps protons across the inner membrane of mitochondria or the plasma membrane of many bacteria. Human complex I is involved in numerous pathological conditions and degenerative processes. With 14 central and up to 32 accessory subunits, complex I is among the largest membrane-bound protein assemblies. The peripheral arm of the L-shaped molecule contains flavine mononucleotide and eight or nine iron-sulfur clusters as redox prosthetic groups. Seven of the iron-sulfur clusters form a linear electron transfer chain between flavine and quinone. In most organisms, the seven most hydrophobic subunits forming the core of the membrane arm are encoded by the mitochondrial genome. Most central subunits have evolved from subunits of different hydrogenases and bacterial Na+/H+ antiporters. This evolutionary origin is reflected in three functional modules of complex I. The coupling mechanism of complex I most likely involves semiquinone intermediates that drive proton pumping through redox-linked conformational changes. [2]

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NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a significant contributor to cellular oxidative stress. Here, we describe the kinetic and molecular mechanism of superoxide production by complex I isolated from bovine heart mitochondria and confirm that it produces predominantly superoxide, not hydrogen peroxide. Redox titrations and electron paramagnetic resonance spectroscopy exclude the iron-sulfur clusters and flavin radical as the source of superoxide, and, in the absence of a proton motive force, superoxide formation is not enhanced during turnover. Therefore, superoxide is formed by the transfer of one electron from fully reduced flavin to O2. The resulting flavin radical is unstable, so the remaining electron is probably redistributed to the iron-sulfur centers. The rate of superoxide production is determined by a bimolecular reaction between O2 and reduced flavin in an empty active site. The proportion of the flavin that is thus competent for reaction is set by a preequilibrium, determined by the dissociation constants of NADH and NAD+, and the reduction potentials of the flavin and NAD+. Consequently, the ratio and concentrations of NADH and NAD+ determine the rate of superoxide formation. This result clearly links our mechanism for the isolated enzyme to studies on intact mitochondria, in which superoxide production is enhanced when the NAD+ pool is reduced. Therefore, our mechanism forms a foundation for formulating causative connections between complex I defects and pathological effects. [3]

C21H27N7O14P2

663.43

663.109

C, 38.02; H, 4.10; N, 14.78; O, 33.76; P, 9.34

53-84-9

NAD+-13C5 ammonium;NAD+-d4;NAD+-13C5-1;1859096-06-2;NAD+ lithium;64417-72-7; 53-84-9 (free acid); 20111-18-6 (sodium); 58-68-4 (reduced)

5892

White to off-white solid

140 - 142ºC

Gut microbial/endogenous metabolite

-5.72

7

18

11

44

1120

8

P(=O)(O[H])(OP(=O)([O-])OC([H])([H])[C@]1([H])[C@]([H])([C@]([H])([C@]([H])([N+]2=C([H])C([H])=C([H])C(C(N([H])[H])=O)=C2[H])O1)O[H])O[H])OC([H])([H])[C@]1([H])[C@]([H])([C@]([H])([C@]([H])(N2C([H])=NC3=C(N([H])[H])N=C([H])N=C23)O1)O[H])O[H]

BAWFJGJZGIEFAR-NNYOXOHSSA-N

InChI=1S/C21H27N7O14P2/c22-17-12-19(25-7-24-17)28(8-26-12)21-16(32)14(30)11(41-21)6-39-44(36,37)42-43(34,35)38-5-10-13(29)15(31)20(40-10)27-3-1-2-9(4-27)18(23)33/h1-4,7-8,10-11,13-16,20-21,29-32H,5-6H2,(H5-,22,23,24,25,33,34,35,36,37)/t10-,11-,13-,14-,15-,16-,20-,21-/m1/s1

1-((2R,3R,4S,5R)-5-((((((((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methoxy)(hydroxy)phosphoryl)oxy)oxidophosphoryl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-3-carbamoylpyridin-1-ium

β-DPNNSC-20272; Nadide; NSC 20272; NSC20272; nadide; coenzyme I; 53-84-9; beta-NAD; Codehydrogenase I; diphosphopyridine nucleotide; Codehydrase I; nicotinamide adenine dinucleotide; beta-NAD; beta NAD; Enzopride Nadida; Codehydrase I; nadide; 53-84-9; NAD+; coenzyme I; beta-NAD; Codehydrogenase I; diphosphopyridine nucleotide; beta-nicotinamide adenine dinucleotide;NAD; NAD+; Nadidum; Nicotinamide; adenine dinucleotide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~41.67 mg/mL (~62.81 mM)

Solubility in Formulation 1: 100 mg/mL (150.73 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)