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PubMed1989

Effects of dietary pyrazinamide, tryptophan, or nicotinic acid and gamma-ray irradiation on levels of NAD and NADP in various organs of mice.

The effects of large amounts of tryptophan, pyrazinamide, or nicotinic acid in diets on the contents of total NAD (NAD + NADH) and NADP (NADP + NADPH) of various organs were investigated in mice with or without gamma-irradiation. Female C3H/HeN mice were fed one of the following 4 kinds of experimental diets for one week: 1) control diet (20% casein diet containing 3 mg niacin per 100g diet); 2) diet supplemented by 0.5% L-tryptophan (T-diet); 3) diet supplemented by 0.5% L-tryptophan (T-diet); 4) diet supplemented by 0.1% nicotinic acid (NA-diet). Half of the mice in each group were subsequently irradiated with 8 Gy of gamma-ray (60 Co) after 4 h of fasting. Then, the contents of total NAD and NADP in thymus, spleen, kidney, liver, and blood were determined in all animals. The results indicated that NAD content of spleen was higher in PT-group (21.5%) and NA-group (23.2%) than in that of control group. In thymus, however, NAD content of only the PT-group was significantly greater (13.1%) than control. NAD level of kidney was also significantly higher (32.6%) in PT-group. By gamma-irradiation, NAD contents of thymus and spleen of all groups tended to be decreased, but those of kidney and liver were not always reduced. In the latter two organs, significant NAD reduction was shown only in kidney of PT-group and in liver of PT- and NA-groups. Even after irradiation, NAD levels of spleen and thymus in PT- and NA-groups tended to be kept higher than those in irradiated control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Mapping the role of NAD metabolism in prevention and treatment of carcinogenesis.

Studies presented here show that cellular NAD, which we hypothesize to be the relevant biomarker of niacin status, is significantly lower in humans than in the commonly studied animal models of carcinogenesis. We show that nicotinamide and the resulting cellular NAD concentration modulate expression of the tumor suppressor protein, p53, in human breast, skin, and lung cells. Studies to determine the optimal NAD concentrations for responding to DNA damage in breast epithelial cells reveal that DNA damage appears to stimulate NAD biosynthesis and that recovery from DNA damage occurs several hours earlier in the presence of higher NAD or in cells undergoing active NAD biosynthesis. Finally, analyses of normal human skin tissue from individuals diagnosed with actinic keratoses or squamous cell carcinomas show that NAD content of the skin is inversely correlated with the malignant phenotype. Since NAD is important in modulating ADP-ribose polymer metabolism, cyclic ADP-ribose synthesis, and stress response proteins, such as p53, following DNA damage, understanding how NAD metabolism is regulated in the human has important implications in developing both prevention and treatment strategies in carcinogenesis.

Animals↗
PubMed2000

Human deoxyhypusine synthase: interrelationship between binding of NAD and substrates.

Deoxyhypusine synthase catalyses the NAD-dependent transfer of the butylamine moiety from the polyamine, spermidine, to a specific lysine residue of a single cellular protein, eukaryotic translation-initiation factor 5A (eIF5A) precursor. The native enzyme exists as a tetramer of four identical subunits and contains four binding sites for NAD. The binding of spermidine and NAD was studied by a filtration assay. [(3)H]Spermidine binding to the enzyme was not detectable alone or in the presence of the eIF5A precursor, but was detected only in the presence of NAD or NADH, suggesting that a NAD/NADH-induced conformational change is required for the binding of spermidine. A strong NAD-dependent binding was also observed with a spermidine analogue, N(1)-guanyl-1, 7-diamino[(3)H]heptane (GC(7)), but not with [(14)C]putrescine or [(14)C]spermine. Although [(3)H]NAD binding to the enzyme occurred in the absence of spermidine, its affinity for the enzyme was markedly enhanced by spermidine, GC(7) and also by the eIF5A precursor. The maximum binding for NAD and spermidine was estimated to be approximately 4 molecules each/enzyme tetramer. The dependence of spermidine binding on NAD and the modulation of binding of NAD by spermidine and the eIF5A precursor suggest intricate relationships between the binding of cofactor and the substrates, and provide new insights into the reaction mechanism.

Binding Sites↗
PubMed1985

Increase in anti-carboxylesterase action of organophosphorothioates by nicotinamide adenine dinucleotide (NAD) in vitro.

The purpose of the present study was to investigate the relationship between chemical structure of organophosphorus insecticides and potentiation of anti-carboxylesterase (CEase) action of these insecticides by NAD in vitro (NAD-effect). Experiments using with three organophosphorothioates having ethoxy group except for diazinon exhibited greater NAD-effect than those having methoxy ones such as methylparathion and fenitrothion. In contrast, none of five organophosphates tested showed NAD-effect. And, 3-acetylpyridine adenine dinucleotide (Ac-py AD) among four derivertives of NAD was also found to have NAD-effect. These results suggested that P = S group in the molecule of organophosphorus insecticides was needed to occur NAD-effect. In addition, the extent of NAD-effect using acetylcholinesterase (AChE) was lesser than that of CEase, therefore, a higher susceptibility of liver microsomal CEase to organophosphorus insecticides may be explained, at least inpart, by NAD-effect.

Animals↗
PubMed1983

[Properties of NAD binding by synaptic membranes of rat brain].

The binding of [14C]NAD to rat brain synaptic membranes is reversible and depends on incubation time, temperature and protein concentration in the reaction mixture. The value of the rate constant for [14C]NAD binding to the synaptic membranes at 24 degrees C (kl) is 1.1 X 10(-6) M-1 S-1, the rate constant for dissociation of the [14C]NAD-receptor complex (k-1) is 3.3 X 10(-3) S-1. The value of the constant for the ligand dissociation from this complex (Kd) is 3.0 nmole. Treatment of the experimental results in the Scatchard plots for the equilibrium binding of [14C]NAD to the synaptic membranes demonstrated that the receptor sites with high and low affinities for the ligand (Kd1 = 3.3 nmol, Kd2 = 14.4 nmole) and with binding capacities of 44 and 77 pmole of [14C]NAD, respectively. It was found that the synaptosomal membrane components which bind the labelled NAD have a protein nature. Data from [14C]NAD and [nicotinamide-3H]NAD binding suggest that brain synaptic membranes bind NAD at the nicotinamide and adenylic moieties.

Animals↗
PubMed1994

Measurement of fluorescence changes of NAD(P)H and of fluorescent flavoproteins in saponin-skinned human skeletal muscle fibers.

Saponin-skinned human muscle fibers from M. vastus lateralis were immobilized in a quartz capillary to detect the fluorescence changes of NAD(P)H and of fluorescent flavoproteins. To get sufficient intense fluorescence signals from a small amount of muscle tissue the NAD(P)H fluorescence was excited by means of an HeCd laser at 325 nm and the flavoprotein fluorescence by an argon-ion laser at 454 nm or by the second wavelength of a HeCd laser at 442 nm. Using this experimental setup the fluorescence spectra of NAD(P)H, of alpha-lipoamide dehydrogenase and of electron-transfer flavoprotein were detected in saponin-skinned human muscle fibers. These fibers behaved identically to isolated mitochondria: (i) The addition of substrates caused an increase in reduction of mitochondrial NAD+, (ii) the addition of ADP caused its reoxidation, and (iii) the addition of respiratory chain inhibitors led to an almost complete reduction of NAD+. It was observed that the redox state of the NAD(P) system and of the alpha-lipoamide dehydrogenase reached after addition of 1 mM ADP correlates with the rate of active state respiration with NAD-dependent substrates. Therefore, this fluorimetric method is suitable to compare the mitochondrial oxidation capacities of NAD-dependent substrates in less then 5 mg wet weight muscle tissue. Moreover, the maximal changes in fluorescence of NAD(P)H and flavoproteins correlate with the amount of mitochondrial marker enzymes per milligram muscle tissue. Using this method a myopathy caused by a diminished content of mitochondria per milligram muscle tissue was observed.

Adenosine Diphosphate↗
PubMed1989

NAD(P)H oxidation elicits anion superoxide formation in radish plasmalemma vesicles.

Radish plasmalemma-enriched fractions show an NAD(P)H-ferricyanide or NAD(P)H-cytochrome c oxidoreductase activity which is not influenced by pH in the 4.5-7.5 range. In addition, at pH 4.5-5.0, NAD(P)H elicits an oxygen consumption (NAD(P)H oxidation) inhibited by catalase or superoxide dismutase (SOD), added either before or after NAD(P)H addition. Ferrous ions stimulate NAD(P)H oxidation, which is again inhibited by SOD and catalase. Hydrogen peroxide does not stimulate NADH oxidation, while it does stimulate Fe2+-induced NADH oxidation. NADH oxidation is unaffected by salicylhydroxamic acid and Mn2+, is stimulated by ferulic acid, and inhibited by KCN, EDTA and ascorbic acid. Moreover, NADH induces the conversion of epinephrine to adrenochrome, indicating that anion superoxide is formed during its oxidation. These results provide evidence that radish plasma membranes contain an NAD(P)H-ferricyanide or cytochrome c oxidoreductase and an NAD(P)H oxidase, active only at pH 4.5-5.0, able to induce the formation of anion superoxide, that is then converted to hydrogen peroxide. Ferrous ions, sparking a Fenton reaction, would stimulate NAD(P)H oxidation.

Cell Membrane↗
PubMed2003

Eukaryotic NAD+ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase.

NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.

Amide Synthases↗
PubMed2005

Characteristics and actions of NAD(P)H oxidase on the sarcoplasmic reticulum of coronary artery smooth muscle.

It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.

Animals↗
PubMed1980

NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.

Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.

Humans↗
PubMed1986

Formation of the N'-methylnicotinamide adenine dinucleotide derivative of NAD in intact rat pituitary tumor GH3 and human promyelocytic leukemia HL-60 cells.

The NAD analog N'-methylnicotinamide adenine dinucleotide (N'AD) is formed in intact human promyelocytic leukemia HL-60 and in rat pituitary tumor GH3 cells during treatment of the cultured cells with the nicotinamide derivative N'-methylnicotinamide (N'CH3NAm). N'AD formation is associated with the induced maturation of HL-60 cells and increased hormone production by GH3 cells during treatment with the nicotinamide derivative. N'AD is detected by HPLC analysis of cytoplasmic extracts as a peak which elutes near NAD. Four facts indicate that this compound is N'AD. First, a compound which elutes with identical time retention is produced by transglycosylation during reaction of NAD with pig brain NAD glycohydrolase in the presence of excess N'CH3NAm. Second, the putative N'AD is degraded by prolonged digestion with the NAD glycohydrolase to ADP-ribose. Third, N'AD formation is prevented by addition of nicotinamide along with N'CH3NAm to compete with binding of N'CH3NAm to the NAD glycohydrolase. Fourth, radioactive precursor labeling demonstrates that it contains adenosine, but it is not labeled by radioactive nicotinamide. The biological relevance of N'AD formation was evaluated. The appearance of N'AD precedes development of HL-60 maturation, and NAD levels increase, not decrease, as observed in other cell types, during treatment with N'CH3NAm. Therefore, we propose that N'AD, not the pyridine base itself, is the active species in inducing maturation. The results provide support of a role for NAD metabolism, probably ADP-ribosylation, in the regulation of HL-60 maturation and in hormone production by pituitary cells.

Adenosine Triphosphate↗
PubMed2000

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart.

The opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD(+) glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca(2+) to isolated rat heart mitochondria resulted in a profound decrease in their NAD(+) content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD(+) depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD(+) available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD(+) content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD(+), which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 microm CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD(+) contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD(+), which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart.

Animals↗
PubMed1982

NAD glycohydrolase activity in the liver of rats fed on excess leucine diet and low or high protein diet.

In rats fed for 2 weeks on a 10% casein diet supplemented with 5% leucine, the hepatic NAD content was lowered when comparing with the control rats pair-fed on the 10% casein diet. The hepatic NAD glycohydrolase [EC 3.2.2.5] activity increased by 25% in the leucine-supplemented diet-fed rats and by 40% in the nicotinic acid-free diet-fed rats. The hepatic NAD glycohydrolase activity in the rats fed on the nonprotein diet freely for 1 week was elevated by 60% compared to the rats fed on the 18% casein diet. The hepatic NAD content in the former animals was significantly lower than that in the latter. The 70% casein diet caused changes in neither hepatic NAD content nor NAD glycohydrolase activity. These results indicated an inverse relationship between hepatic NAD content and NAD glycohydrolase activity.

Animals↗
PubMed1994

Kinetic properties of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase from the host fraction of soybean root nodules.

The kinetic mechanism of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12] from the host cytosolic fraction of soybean (Glycine max L. Merr. cv. Williams) root nodules has been investigated by steady-state initial velocity studies and inhibition studies with products and product analogs. The results were consistent with the enzyme having a bi-uni-uni-uni ping-pong mechanism in which NAD+ and phosphate interacted sequentially with the enzyme, followed in turn by the release of 1,3-bisphosphoglycerate, the addition of D-glyceraldehyde-3-phosphate, and the release of NADH. At pH 7.2, NAD+ bound to the enzyme in a rapid-equilibrium fashion, whereas at pH 8.8 there was rapid-equilibrium addition of both NAD+ and phosphate to the enzyme. NADH was a strong competitive inhibitor with respect to NAD+ and phosphate. The enzyme was very labile, especially above pH 8, but the rate of loss of activity was considerably reduced in the presence of NAD+ and phosphate. The possible role of these properties in the regulation of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in the host cytosol of soybean nodules is discussed.

Adenosine Monophosphate↗
PubMed1978

NAD in muscle of man at rest and during exercise.

NAD can be used to assess the adequacy of oxygen availability to the respiratory chain. An enzymatic assay was established for NAD in human muscle biopsy samples. It gave reliable, reproducible results. The variation within and between subjects was less than 12%. Muscle NAD and lactate were determined at rest, and after bicycle ergometry work requiring approximately 75 and approximately 100% VO2 max (six subjects, four tests each). A positive (P less than 0.01) linear relationship between resting muscle NAD and percent slow twitch fibers was found, suggesting that fiber types may have different NAD content. Muscle NAD decreased during submaximal and maximal work (P less than 0.05). A large portion (73%) of the NAD reduction could be accounted for by increased muscle water. No relationship could be established between NAD and lactate. The negative linear relationship (P less than 0.01) between the muscle/blood ratio and percent slow twitch fibers is another indication of the fiber having different metabolic responses to the activity.

Adult↗
PubMed1990

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells.

We have determined the ability of UV254nm-irradiated murine lymphoma cells to adapt their NAD+ metabolism to the increased NAD+ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD(+)-utilizing enzyme poly(ADP-ribose) polymerase, NAD+ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV254nm irradiation, a dramatic transient increase of NAD+ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV254nm irradiation, the NAD+ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD+ metabolism to UV254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD+ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.

Adaptation, Physiological↗
PubMed1985

Calmodulin-dependent NAD kinase of human neutrophils.

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.

Adenosine Triphosphate↗
PubMed1987

Changes in pyridine nucleotide levels alter oxygen consumption and extra-mitochondrial phosphates in isolated mitochondria: a 31P-NMR and NAD(P)H fluorescence study.

Isolated rat-liver mitochondria were used to study the relation between mitochondrial NADH levels, oxygen consumption (QO2), and extra-mitochondrial phosphates. Alterations in NADH and QO2 were accomplished by incubating mitochondria with different substrates or varying amounts of exogenous ATPase while monitoring QO2 and NAD(P)H fluorescence. Two sets of conditions were studied: (1) in the presence of excess ADP and inorganic phosphate, an increase in NAD(P)H fluorescence was associated with a linear increase in QO2; (2) when QO2 was driven by the steady-state hydrolysis of ATP by exogenous ATPase, increases in QO2 were associated with proportional decreases in NAD(P)H fluorescence. For all substrates tested this relation was linear; however, the slope was substrate dependent. Different substrates were able to maintain different NAD(P)H levels at the same QO2. To investigate this further, effects of changing substrates at constant QO2 on NAD(P)H and extra-mitochondrial phosphates were determined. Addition of glutamate + malate to mitochondria respiring on citrate caused a 50% increase in NAD(P)H fluorescence, a 41% decrease in ADP, and a 30% decrease in inorganic phosphate. Similar changes for the substrate jump, pyruvate + malate to glutamate + malate were found. Finally, it was determined that a linear relation holds between increases in NAD(P)H fluorescence and increases in QO2 when substrates were varied at constant, physiologic levels of extra-mitochondrial ADP. These results indicate that QO2 depends on NAD(P)H levels as well as on extra-mitochondrial phosphates over a wide range of respiratory rates.

Adenosine Diphosphate↗