Endogenous Metabolite

NAD+是由连接腺苷5'-磷酸和核糖基烟酰胺5'-二磷酸的焦磷酸键组成的辅酶。 NADH 的氧化形式称为 NAD+ [1]。 NAD+ 在自然界中广泛存在，通过氧化 (NAD+) 和还原 (Nadide) 之间的交替，在众多酶活性中充当电子载体 [2]。

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复合物I的超氧化物生成受到NAD+的强烈抑制（图1A），因此图4A、B和D中的数据是通过使用再生系统来维持[NADH]并最小化[NAD+]而记录的。否则，NADH在≈0.2μM min−1时转化为NAD+，导致测定痕迹明显弯曲，特别是在低[NADH]时（图4C）。对于O2•−，NADH的KM（app）为0.05μM。重要的是，NAD+不会通过使复合物I的NADH氧化成为限速来发挥其抑制作用。例如，30μM NAD+可减少约50%的O2•−形成，但不会影响更快的[Fe（CN）6]3−、[Ru（NH3）6]3+或癸基泛醌还原。我们的观察强烈表明，NADH、NAD+和复合物I的不同状态（氧化、还原、无核苷酸或核苷酸结合）之间建立了预平衡，以确定复合物中有多少能够减少O2，从而确定O2•−的形成速率。
超氧化物形成的分子机制：纯化络合物I形成的超氧化物不受癸基泛醌的直接影响，也不受催化过程中放大的影响（图1）。因此，FMN或其中一个FeS簇是控制O2还原的辅因子，辅因子X。如果辅因子X是FeS簇，则根据能斯特方程，NAD+与NADH一起作用，以设定其平衡还原水平。如果辅因子X是黄素，那么活性位点的占据会对前平衡产生额外的影响，并可能影响双分子反应的速率，因为结合的核苷酸可能会阻止或阻碍氧气的进入。 图5A显示了改变[NADH]/[NAD+]比对O2•−产生的影响，绘制为辅因子X的氧化还原滴定（方程式1）[3]。

口服 NAD+ 补充剂已被用来治疗能量消耗、不明原因的疾病，如纤维肌痛和慢性疲劳综合症，以及简单的疲倦 [3]。

使用NADH和NAD+的氧化还原滴定。[3]
NADH在手套箱（O2<2ppm）中通过阴离子交换色谱法（5-ml HiTrap Q-Sepharose柱）重新纯化，以去除污染的NAD+。实验后，重新评估NADH储备溶液的完整性（在6小时内形成0.08±0.04%的NAD+）。通常，通过使用30μM NADH和不同量的NAD+（Sigma）来设置氧化还原电位，并通过使用NADH再生系统来检查低电位极限

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EPR。[3]
复合物I（10 mg ml−1）被1 mM纯化的NADH厌氧还原，或通过对纯化的NADH（≈−0.4 V）的透析还原，或使用1 mM NADH和10 mM NAD+还原至≈−0.3 V，并立即冷冻。使用ER 4119HS高灵敏度腔和ESR900连续流液氦低温恒温器[3]，在Bruker EMX X波段光谱仪上记录光谱。

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

340.71

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)

配方 1 中的溶解度: 100 mg/mL (150.73 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。