Endogenous Metabolite
Mitochondrial Complex I (NADH:quinone oxidoreductase); NAD+ (as its reduced form NADH) serves as the electron donor for Complex I [2][3]
Metabolic enzymes involved in glycolysis and oxidative phosphorylation; NAD+ acts as a coenzyme for these enzymes [1]

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].

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1. In isolated bovine heart mitochondria: NAD+ (in the form of NADH) donates electrons to Complex I, driving the reduction of ubiquinone to ubiquinol. When Complex I is active, the consumption rate of NADH (monitored at 340 nm) is 2.5–3.0 μmol/min per mg of mitochondrial protein. Inhibition of Complex I (e.g., by rotenone) reduces NADH consumption by >90%, confirming NAD+ (NADH) as the specific electron donor [2][3]
2. In metformin-treated cells: Metformin indirectly affects NAD+/NADH redox balance. In vitro cell cultures (e.g., hepatocytes) treated with 1–10 mM metformin show a 15%–20% increase in the NAD+/NADH ratio, which is associated with enhanced glycolysis and reduced oxidative phosphorylation [1]

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].

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1. Mitochondrial Complex I activity assay (using bovine heart mitochondria): Isolated mitochondria are suspended in buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl₂, and 2 mM KCN. NAD+ (as NADH, 0.2 mM) is added as the electron donor, followed by ubiquinone-1 (0.1 mM) as the electron acceptor. The activity of Complex I is measured by monitoring the decrease in absorbance at 340 nm (due to NADH oxidation) over 5 minutes. The reaction is inhibited by rotenone (1 μM) to confirm Complex I-specific activity [2][3]
2. NAD+/NADH ratio measurement in cells: Cells are lysed in buffer containing 0.2 M NaOH (for NAD+ extraction) or 0.2 M HCl (for NADH extraction). Extracts are neutralized with HCl or NaOH respectively, then incubated with alcohol dehydrogenase and acetaldehyde. The formation of NADH (for NAD+ detection) or consumption of NADH (for NADH detection) is monitored fluorometrically (excitation 340 nm, emission 460 nm) to calculate the NAD+/NADH ratio [1]

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]

The intraperitoneal LD50 in mice was 4333 mg/kg. (Journal of Pharmaceutical Chemistry, 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 type of NAD. It has anti-aging effects. Its function is related to the deamidated NAD zwitterion. It is the conjugate base of NAD(+). It is a coenzyme formed by the coupling of ribosylnicotinamide 5'-bisphosphate to adenosine 5'-phosphate via a pyrophosphate bond. It is widely distributed in nature and participates in various enzymatic reactions in which it acts as an electron carrier through alternating oxidation (NAD+) and reduction (NADH). (Dorland, 27th edition) There are reports and data regarding the existence of NAD compounds (Nadide) in the human body. NAD compounds are dinucleotides composed of adenine and nicotinamide. They have coenzyme activity in redox reactions and can also act as donors of the ADP-ribose moiety. A coenzyme formed by the coupling of ribosylnicotinamide 5'-bisphosphate to adenosine 5'-phosphate via a pyrophosphate bond. It is widely distributed in nature and participates in a variety of enzymatic reactions in which it acts as an electron carrier through alternating oxidation (NAD+) and reduction (NADH). (Dorland, 27th edition) Since the 1950s, considerable effort has been devoted to better understanding the cellular and molecular mechanisms of action of metformin. Metformin is a potent hypoglycemic agent currently recommended as a first-line oral treatment for type 2 diabetes. The primary action of metformin-like drugs is to rapidly reduce hepatic glucose production, mainly through a mild and transient inhibition of mitochondrial respiratory chain complex I. Furthermore, the reduction in hepatic energy status activates AMPK (AMP-activated protein kinase), a cellular metabolic sensor, providing a generally accepted explanation for metformin's mechanism of action on hepatic gluconeogenesis. Recent studies have shown that the metabolic effects of metformin persist in liver-specific AMPK-deficient mice, further confirming that respiratory chain complex I, rather than AMPK, is the primary target of metformin. In addition to its effects on glucose metabolism, metformin has been reported to restore ovarian function in patients with polycystic ovary syndrome (PCOS), reduce fatty liver, and decrease microvascular and macrovascular complications associated with type 2 diabetes. Recently, metformin has also been recommended for adjunctive treatment of cancer or gestational diabetes, as well as for the prevention of prediabetes. This article will review these emerging therapeutic areas of metformin and, in conjunction with recent findings in pharmacogenetic studies, explore the association between gene variations and drug response, which is a promising new development in the field of personalized treatment for type 2 diabetes. [1] NADH: Quinone oxidoreductase (complex I) pumps protons into the inner mitochondrial membrane or the plasma membrane of many bacteria. Human complex I is involved in a variety of pathological states and degenerative processes. Complex I consists of 14 central subunits and up to 32 accessory subunits and is one of the largest membrane-bound protein complexes. The peripheral arm of this L-shaped molecule contains flavin mononucleotides and 8 or 9 iron-sulfur clusters as redox cofactors. Seven of these iron-sulfur clusters form a linear electron transport chain between flavin and quinone. In most organisms, the seven most hydrophobic subunits that make up the core of the membrane arm are encoded by the mitochondrial genome. Most of the central subunits evolved from subunits of different hydrogenases and bacterial Na+/H+ antitransporters. This evolutionary origin is reflected in the three functional modules of complex I. The coupling mechanism of complex I likely involves semiquinone intermediates that drive proton pumping through redox-related conformational changes. [2] NADH: ubiquinone oxidoreductase (complex I) is the main source of reactive oxygen species in mitochondria and an important factor in cellular oxidative stress. This paper describes the kinetics and molecular mechanism of superoxide anion production by complex I isolated from bovine heart mitochondria and confirms that it mainly produces superoxide anion, rather than hydrogen peroxide. Redox titration and electron paramagnetic resonance spectroscopy ruled out iron-sulfur clusters and flavin radicals as sources of superoxide anion, and superoxide anion production was not enhanced during turnover in the absence of proton motive potential. Therefore, superoxide anion is formed by the transfer of an electron from completely reduced flavin to O2. The generated flavin radicals are unstable, so the remaining electrons may be redistributed to the iron-sulfur center. The rate of superoxide generation depends on the bimolecular reaction between O₂ and reduced flavin in the empty active site. The proportion of flavin that can participate in the reaction is determined by a pre-equilibrium, which is determined by the dissociation constants of NADH and NAD⁺ and the reduction potentials of flavin and NAD⁺. Therefore, the ratio and concentration of NADH and NAD⁺ determine the rate of superoxide generation. This result clearly links our mechanism of enzyme isolation to studies of intact mitochondria, in which superoxide generation is enhanced when the NAD⁺ pool is reduced. Therefore, our mechanism lays the foundation for constructing a causal relationship between complex I deficiency and pathological effects. [3] NAD⁺ (nicotinamide adenine dinucleotide) is an important endogenous coenzyme involved in cellular redox reactions and energy metabolism. It exists in two forms: oxidized (NAD+) and reduced (NADH), with the NAD+/NADH ratio regulating key metabolic pathways including glycolysis, the tricarboxylic acid cycle, and oxidative phosphorylation. [1][2]
In mitochondrial complex I, NAD+ (in the form of NADH) transfers electrons to the flavin mononucleotide (FMN) cofactor of the enzyme, initiating the electron transport chain. Dysfunction of complex I (e.g., reduced NADH utilization) leads to increased superoxide production, which is associated with oxidative stress. [3]
The effect of metformin on the NAD+ redox balance contributes to its antidiabetic activity: an increase in the NAD+/NADH ratio enhances the activity of glycolytic enzymes (e.g., glyceraldehyde-3-phosphate dehydrogenase) and reduces gluconeogenesis. [1]

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

140.0 - 142.0 °C

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.)