Nicotinamide Adenine Dinucleotide Redox Research Articles

NMNAT2 supports vesicular glycolysis via NAD homeostasis to fuel fast axonal transport

BackgroundBioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer’s, Parkinson’s, and Huntington’s disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function.MethodsWe generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos.ResultsWe provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide “on-board” ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons.ConclusionNMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.

Tachycardia-induced metabolic rewiring as a driver of contractile dysfunction.

Prolonged tachycardia-a risk factor for cardiovascular morbidity and mortality-can induce cardiomyopathy in the absence of structural disease in the heart. Here, by leveraging human patient data, a canine model of tachycardia and engineered heart tissue generated from human induced pluripotent stem cells, we show that metabolic rewiring during tachycardia drives contractile dysfunction by promoting tissue hypoxia, elevated glucose utilization and the suppression of oxidative phosphorylation. Mechanistically, a metabolic shift towards anaerobic glycolysis disrupts the redox balance of nicotinamide adenine dinucleotide (NAD), resulting in increased global protein acetylation (and in particular the acetylation of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), a molecular signature of heart failure. Restoration of NAD redox by NAD+ supplementation reduced sarcoplasmic/endoplasmic reticulum Ca2+-ATPase acetylation and accelerated the functional recovery of the engineered heart tissue after tachycardia. Understanding how metabolic rewiring drives tachycardia-induced cardiomyopathy opens up opportunities for therapeutic intervention.

An enhanced direct electron transfer-type NAD+/NADH regenerating system using the diaphorase subunit of formate dehydrogenase 1

Nicotinamide adenine dinucleotide redox couple (NAD+/NADH) is a significant coenzyme in the catabolism of organisms. Coupling NAD-dependent enzymatic reaction and NAD+/NADH regenerating systems can be utilized for biomimetics-associated applications, such as the production of bioreactors, biofuel cells, and biosensors. Direct electron transfer (DET)-type bioelectrocatalysis, in which an enzyme directly exchanges electrons with the electrode, can produce an efficient, simple, and biocompatible system owing to a mediator-free configuration. Formate dehydrogenase 1 from Methylorubrum extorquens AM1 (FoDH1) has been reported to have diaphorase and DET-type activity. The β subunit (diaphorase subunit) of FoDH1 is an attractive variant for constructing an efficient regenerating system due to downsizing effects and efficient interfacial electron transfer. In this study, we attempt to construct a homologous expression system to establish a recombinant FoDH1 (rFoDH1) expression system and to obtain the β subunit variant (FoDH1B). Their bioelectrochemical properties and the ET pathways of DET-type reactions have been elucidated. Finally, we demonstrated the coupling of an efficient DET-type NAD+/NADH regenerating system with NAD-dependent enzymes.

The Effect of L-Glutamine on Pain Crisis Reduction in Patients with Sickle Cell Anemia and Sickle β°-Thalassemia

Background: A low level of L-glutamine, a precursor of nicotinamide adenine dinucleotide (NAD) in red blood cells (RBCs), is identified as an underlying mechanism for the potential decrement of the NAD redox and the incidence of pain crisis in sickle cell anemia (SCA). The aim of this study is to assess the impact of oral L-glutamine therapy on pain crisis reduction in patients with SCA and sickle β°-thalassemia.
 Materials and Methods: In this pilot clinical trial, 15 patients with SCA and sickle β°-thalassemia with the mean age of 17.2 ± 6.07 were examined to evaluate the efficacy of L-glutamine therapy in pain crises. All the subjects received oral L-glutamine (0.3 gr /kg of body weight) every day for 8 weeks. With respect to the effects of L-glutamine on pain crisis, the subjects were in contact with the physicians at least once or more per week. Blood samples were taken at different follow-up times to evaluate the laboratory characteristics of the patients.
 Results: Pain crises occurred for 1-3 times (mean: 1.29 ± 1.05) during 12 months before the L-glutamine therapy. With no significant change, the patients had lower numbers of painful crises (mean: 0.80 ± 0.58) at the end of a 4-week period compared to the period before the L-glutamine therapy (P = 0.466). Fewer pain crises with a mean of 0.33 ± 0.51 occurred during 8 weeks of L-glutamine therapy than in the pre-treatment period (P = 0.038).
 Conclusion: The results of the present study showed that oral L-glutamine can mitigate a pain crisis and improve the sickle RBCs and reticulocyte count in SCA. This suggests that L-glutamine therapy can increase the antioxidant potential of sickle RBCs.

Feasibility of Non-invasive Measurement of Tumour NAD(H) by In Vivo Phosphorus-31 Magnetic Resonance Spectroscopy.

Importance of the redox status of nicotinamide adenine dinucleotide (NAD), including its oxidized (NAD+) and reduced (NADH) forms, has been shown in many biological processes. However, NAD(H) redox status assessment is traditionally limited to biochemical assays in vitro or optical redox imaging (ORI) for superficial tissues in vivo and for deep tissues ex vivo. In recent years, phosphorous-31 magnetic resonance spectroscopy (31P-MRS) was utilized to quantify NAD+, NADH, and the redox ratio NAD+/NADH in normal tissues in vivo. The quantification is based on the spectral fitting of the upfield shoulder of the αATP peak that contains signals of NAD+ (a quartet) and NADH (a singlet), assuming pH-independence of peak positions. To evaluate the feasibility of measuring tumour NAD(H) redox status in vivo, we fitted single voxel 31P-MR spectra of subcutaneous mouse xenografts of human breast cancer cell lines acquired on a 9.4-T horizontal bore preclinical MR scanner. We found larger variations in the chemical shift offsets of NAD+ and NADH from αATP in these tumours than the literature values of normal tissues. Furthermore, our 31P-MR spectra of αATP, NAD+, and NADH solution phantoms indicated that the chemical shift of αATP and thus the offsets between NAD(H) and αATP were pH dependent. Therefore, whether tumour pH should be incorporated into the spectral fitting model should be further evaluated. Additionally, spectral resolution and signal-to-noise ratio should be improved by optimising 31P-MRS protocols, increasing data acquisition time, and using a more sensitive coil for signal detection.

Selective mitochondrial antioxidant MitoTEMPO reduces renal dysfunction and systemic inflammation in experimental sepsis in rats

BackgroundExcess mitochondrial reactive oxygen species (mROS) in sepsis is associated with organ failure, in part by generating inflammation through the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome. We determined the impact of a mitochondrial-targeted antioxidant (MitoTEMPO) on mitochondrial dysfunction in renal proximal tubular epithelial cells, peritoneal immune cell function ex vivo, and organ dysfunction in a rat model of sepsis. MethodsThe effects of MitoTEMPO were assessed ex vivo using adenosine triphosphate and lipopolysaccharide-stimulated rat peritoneal immune cells and fresh rat kidney slices exposed to serum from septic rats. We assessed mROS production and phagocytotic capacity (flow cytometry), mitochondrial functionality (multiphoton imaging, respirometry), and NLRP3 inflammasome activation in cell culture. The effect of MitoTEMPO on organ dysfunction was evaluated in a rat model of faecal peritonitis. ResultsMitoTEMPO decreased septic serum-induced mROS (P<0.001) and maintained normal reduced nicotinamide adenine dinucleotide redox state (P=0.02) and mitochondrial membrane potential (P<0.001) in renal proximal tubular epithelial cells ex vivo. In lipopolysaccharide-stimulated peritoneal immune cells, MitoTEMPO abrogated the increase in mROS (P=0.006) and interleukin-1β (IL-1β) (P=0.03) without affecting non-mitochondrial oxygen consumption or the phagocytotic-induced respiratory burst (P>0.05). In vivo, compared with untreated septic animals, MitoTEMPO reduced systemic IL-1β (P=0.01), reduced renal oxidative stress as determined by urine isoprostane levels (P=0.04), and ameliorated renal dysfunction (reduced serum urea (P<0.001) and creatinine (P=0.05). ConclusionsReduction of mROS by a mitochondria-targeted antioxidant reduced IL-1β, and protected mitochondrial, cellular, and organ functionality after septic insults.

In vivo 31P magnetic resonance spectroscopy study of mouse cerebral NAD content and redox state during neurodevelopment

Nicotinamide adenine dinucleotide (NAD) is an important cofactor of energy-producing pathways. The redox ratio (NAD+/NADH) reflects the cellular oxidoreductive state. Oxidative stress and redox dysregulation have been suggested to contribute to various neurological diseases. The assessment of NAD content has been recently demonstrated in large animals and human brains by 31P magnetic resonance spectroscopy. However, its measurement in small rodents has never been attempted. The purpose of this study was to investigate, in vivo, the NAD content during mouse brain neurodevelopment. 31P-MR-spectra were acquired in the mouse brain at postnatal days P20, P40, P90 and P250 at 14.1 T using a 3D-localization sequence. High spectral quality was achieved at 14.1 T. NAD+ and NADH were quantified with mean Cramér-Rao lower bound of 10% and 14%, respectively. An increase in NAD+/NADH was observed from P20 to P250 due to a decrease in [NADH]. The intracellular pH was significantly reduced with age, while the free [Mg2+] in the brain was significantly increased. This study demonstrates for the first time the feasibility of the measurement of NAD content in vivo in mouse brains during development, which opens the prospect of longitudinally studying energy metabolism and redox dysfunction in mouse models of brain pathology.

Protein cysteine S-nitrosylation provides reducing power by enhancing lactate dehydrogenase activity in Trichomonas vaginalis under iron deficiency

BackgroundIron plays essential roles in the pathogenesis and proliferation of Trichomonas vaginalis, the causative agent of the most prevalent non-viral human sexually transmitted infection. We previously demonstrated that under iron deficiency, the endogenous nitric oxide (NO) is accumulated and capable of regulating the survival of T. vaginalis. Herein, we aim to explore the influence of NO on the activity of the pyruvate-reducing enzyme lactate dehydrogenase in T. vaginalis (TvLDH).MethodsLevels of lactate and pyruvate were detected for determining glycolysis activity in T. vaginalis under iron deficiency. Quantitative PCR was performed to determine the expression of TvLDH. S-nitrosylated (SNO) proteomics was conducted to identify the NO-modified proteins. The activities of glyceraldehyde-3-phosphate dehydrogenase (TvGAPDH) and TvLDH were measured after sodium nitrate treatment. The effects of protein nitrosylation on the production of cellular reducing power were examined by measuring the amount of nicotinamide adenine dinucleotide (NAD) and the ratio of the NAD redox pair (NAD+/NADH).ResultsWe found that although the glycolytic pathway was activated in cells under iron depletion, the level of pyruvate was decreased due to the increased level of TvLDH. By analyzing the SNO proteome of T. vaginalis upon iron deficiency, we found that TvLDH is one of the glycolytic enzymes modified by SNO. The production of pyruvate was significantly reduced after nitrate treatment, indicating that protein nitrosylation accelerated the consumption of pyruvate by increasing TvLDH activity. Nitrate treatment also induced NAD oxidation, suggesting that protein nitrosylation was the key posttranslational modification controlling cellular redox status.ConclusionsWe demonstrated that NO-mediated protein nitrosylation plays pivotal roles in the regulation of glycolysis, pyruvate metabolism, and the activity of TvLDH. The recycling of oxidized NAD catalyzed by TvLDH provided the reducing power that allowed T. vaginalis to adapt to the iron-deficient environment.

Numerical modelling of the effects of cold atmospheric plasma on mitochondrial redox homeostasis and energy metabolism

A biochemical reaction model clarifies for the first time how cold atmospheric plasmas (CAPs) affect mitochondrial redox homeostasis and energy metabolism. Fundamental mitochondrial functions in pyruvic acid oxidation, the tricarboxylic acid (TCA) cycle and oxidative phosphorylation involving the respiratory chain (RC), adenosine triphosphate/adenosine diphosphate (ATP/ADP) synthesis machinery and reactive oxygen species/reactive nitrogen species (ROS/RNS)-mediated mechanisms are numerically simulated. The effects of CAP irradiation are modelled as 1) the influx of hydrogen peroxide (H{}_{2}O{}_{2}) to an ROS regulation system and 2) the change in mitochondrial transmembrane potential induced by RNS on membrane permeability. The CAP-induced stress modifies the dynamics of intramitochondrial H{}_{2}O{}_{2} and superoxide anions, i.e., the rhythm and shape of ROS oscillation are disturbed by H{}_{2}O{}_{2} infusion. Furthermore, CAPs control the ROS oscillatory behaviour, nicotinamide adenine dinucleotide redox state and ATP/ADP conversion through the reaction mixture over the RC, the TCA cycle and ROS regulation system. CAPs even induce a homeostatic or irreversible state transition in cell metabolism. The present computational model demonstrates that CAPs crucially affect essential mitochondrial functions, which in turn affect redox signalling, metabolic cooporation and cell fate decision of survival or death.

A Novel Photo-Driven Hydrogenation Reaction of an NAD+-Type Complex Toward Artificial Photosynthesis.

The photocatalytic reduction of carbon dioxide (CO2) to value-added chemicals is an attractive strategy to utilize CO2 as a feedstock for storing renewable energy, such as solar energy, in chemical bonds. Inspired by the biological function of the nicotinamide adenine dinucleotide redox couple (NAD+/NADH), we have been developing transition-metal complexes containing NAD+/NADH-functionalized ligands to create electro- and/or photochemically renewable hydride donors for the conversion of CO2 into value-added chemicals. Our previous findings have provided insights for the development of photocatalytic organic hydride reduction reactions for CO2, however, further examples, as well as investigation, of these photo-driven NAD+/NADH-type hydrogenation and organic hydride transfer reactions are required not only to explore the mechanism in detail but also to develop a highly efficient catalyst for artificial photosynthesis. In this paper, we report the synthesis, characterization, and photo-induced NAD+/NADH conversion properties of a new ruthenium(II) complex, [Ru(bpy)2(Me-pn)](PF6)2 (1), which contains a new NAD+-type ligand, Me-pn (2-methyl-6-(pyridin-2-yl)-1,5-naphthyridine). In addition, we have succeeded in the isolation of the corresponding two-electron reduced ruthenium(II) complex containing the NADH-type ligand Me-pnHH (2-methyl-6-(pyridin-2-yl)-1,4-dihydro-1,5-naphthyridine), i.e., [Ru(bpy)2(Me-pnHH)](PF6)2 (1HH), by the photo-induced hydrogenation reaction of 1. Thus, in this study, a new photo-driven NAD+/NADH-type hydrogenation reaction for possible CO2 reduction using the NAD+/NADH redox function has been constructed.

Nicotinamide adenine dinucleotide (NAD) oxidation preserves T cell function under lactic acidosis characteristic of the tumor microenvironment (TME)

Abstract Cancers are adept at employing metabolic events to evade host immunity. The TME is often glucose depleted and enriched with lactic acid, weakening effector T cell (Teff) function and interferon (IFN)-γ production. By contrast, immunosuppressive T-regulatory (Treg) cells are well adapted to low glucose and elevated lactate and enriched in the TME. Overcoming immunosuppressive metabolic conditions in the TME may be an important step towards improving anti-tumor immunotherapy. Here we show that NAD oxidation can protect Teffs from the immunosuppressive effects of lactic acid. We found that Teff proliferation and IFN-γ production can be controlled through NAD redox metabolism and NAD-dependent enzymes, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Metabolomic and bioenergetics studies showed that reducing NAD led to accumulation of pre-GAPDH glycolytic metabolites and impaired glycolytic flux similar to GAPDH inhibition, which was reversed by NAD oxidation. Human and murine Teffs exposed to lactate, oligomycin, rotenone, or antimycin lost GAPDH function despite sufficient glucose, limiting glycolysis and IFN-γ mRNA translation, while the Treg transcription factor Foxp3 was stabilized. Oxidizing NAD (pyruvate, mitochondrial uncoupling, β-lapachone, α-ketobutyrate) rescued Teff glycolysis and restored their function despite ambient lactate, while Foxp3 turnover increased in a poly-(ADP-ribose) polymerase dependent manner, using NAD as substrate. Our results identify a novel therapeutic approach to overcoming metabolic barriers to anti-tumor immunity, and provide new mechanistic insights into the role of NAD redox metabolism in controlling T cell function in the TME.

Improved Survival in a Long-Term Rat Model of Sepsis Is Associated With Reduced Mitochondrial Calcium Uptake Despite Increased Energetic Demand

To investigate the relationship between prognosis, changes in mitochondrial calcium uptake, and bioenergetic status in the heart during sepsis. In vivo and ex vivo controlled experimental studies. University research laboratory. Male adult Wistar rats. Sepsis was induced by intraperitoneal injection of fecal slurry. Sham-operated animals served as controls. Confocal microscopy was used to study functional and bioenergetic parameters in cardiomyocytes isolated after 24-hour sepsis. Electron microscopy was used to characterize structural changes in mitochondria and sarcoplasmic reticulum. The functional response to dobutamine was assessed in vivo by echocardiography. Peak aortic blood flow velocity measured at 24 hours was a good discriminator for 72-hour survival (area under the receiver operator characteristic, 0.84 ± 0.1; p = 0.03) and was used in ex vivo experiments at 24 hours to identify septic animals with good prognosis. Measurements from animals with good prognostic showed 1) a smaller increase in mitochondrial calcium content and in nicotinamide adenine dinucleotide fluorescence following pacing and 2) increased distance between mitochondria and sarcoplasmic reticulum on electron microscopy, and 3) nicotinamide adenine dinucleotide redox potential and adenosine triphosphate/adenosine diphosphate failed to reach a new steady state following pacing, suggesting impaired matching of energy supply and demand. In vivo, good prognosis animals had a blunted response to dobutamine with respect to stroke volume and kinetic energy. In situations of higher energetic demand decreased mitochondrial calcium uptake may constitute an adaptive cellular response that confers a survival advantage in response to sepsis at a cost of decreased oxidative capacity.

Extraction and Quantitation of Nicotinamide Adenine Dinucleotide Redox Cofactors.

Accurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms. We compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach. We found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards. Extraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.

Phase 3 Study of L-Glutamine Therapy in Sickle Cell Anemia and Sickle β0-Thalassemia Subgroup Analyses Show Consistent Clinical Improvement

Background: Sickle cell disease (SCD) is characterized by continuous oxidative stress through various mechanisms contributing to the pathophysiology and clinical course of sickle cell crises (SCC) and organ damage. L-glutamine is a precursor for the synthesis of essential metabolic redox cofactors including Nicotinamide Adenine Dinucleotide (NAD). Early studies have demonstrated that altered erythrocyte NAD redox potential was improved by oral L-glutamine therapy, suggesting that higher L-glutamine utilization in SCD exceeded de novo synthesis and its depletion played a role in oxidative stress.A Phase 2 study of Pharmaceutical Grade L-glutamine (L-glutamine) versus placebo showed promising results on clinical endpoints. In a Phase 3 study with 230 SCD patients, randomized 2:1 to L-glutamine or placebo, the median number of SCCs was 25% lower for L-glutamine than placebo (L-glutamine 3.0 vs. placebo 4.0). An analysis of SCC events showed significant differences between groups (p < 0.01). Categories for pre-specified subgroups for Age, Gender and Hydroxyurea (HU) use were defined in the statistical plan. HU use was also a randomization stratification factor since HU is the only FDA-approved drug for SCD treatment. Examination of the HU subgroup for differences in L-glutamine effect across categories (HU vs. no HU) was appropriate since approximately 66% of patients remained on HU throughout the Phase 3 study. To investigate the subgroup treatment effect size, treatment by subgroup interaction and consistency of the primary endpoint across the categories of the subgroups, an examination was performed following the guidelines for reporting subgroup analysis.Methods: The Negative Binomial Regression (NBR) model was utilized to generate an estimate of treatment effect and treatment by subgroup interactions of the three pre-specified subgroups. SCC events are most accurately described as counts and NBR is specifically intended for modeling count variables, usually for over-dispersed counts as seen in SCD studies. Rates of SCC for each treatment arm in a subgroup (Rate L-glutamine per 48 weeks / Rate placebo per 48 weeks) provide rate ratios, and an estimate of effect size, with 95% confidence intervals. A rate ratio <1.0 favored L-glutamine. Tests for treatment by subgroup interactions were conducted simultaneously. A significant interaction could be seen if the treatment is better than placebo for one category and worse than placebo for the other. Predefined categories for subgroups with corresponding sample sizes were as follows: Age: 5 - 18 years (n = 118) and ≥ 18 years (n = 111); Gender: Females (n = 124) and Males (n = 105). HU use before and during study: Yes (n = 153) and No (n = 76). Data for this Phase 3 subgroup report were obtained from an integrated dataset and the NBR model with treatment, subgroup, region and HU use as main effects and a treatment by subgroup interaction term was run with log (time on study) as an offset.Results:Age Subgroup:Both the 5 - 18 years and the > 18 years groups had a lower rate of SCCs per 48 weeks in the L-glutamine arm relative to placebo. The rate ratio was 0.93 [0.67 - 1.29] for the 5 - 18 years group and 0.64 [0.45 - 0.89] in the > 18 years group. There was no treatment by Age Group interaction (p = 0.118).Gender Subgroup: Both the Female and the Male groups had a lower rate of SCCs per 48 weeks in the L-glutamine treatment arm. The rate ratio was 0.81 [0.59 -1.12] for the Female group and 0.73 [0.51 - 1.05] for the Male group, indicating consistent treatment effects across genders. There was no treatment by Gender interaction (p = 0.683).Hydroxyurea Use Subgroup: Both HU groups had a lower rate of SCCs per 48 weeks in the L-glutamine arm. The treatment effects were consistent across groups, with a rate ratio of 0.78 [0.51 - 1.20] for the group without HU use and 0.77 [0.58 - 1.03] for the group with HU use. There was no treatment by HU use interaction (p = 0.961). See Figure 1 for a graphic example.Conclusion: For the Age and Gender subgroups, both categories in each subgroup benefited from L-glutamine but to varying degrees noted by the difference in rate ratios. For the HU use subgroup, both categories (on HU and not on HU) benefited from L-glutamine similarly. Lack of interactions in all subgroups indicated that regardless of category within a subgroup, treatment effect by Pharmaceutical Grade L-glutamine was beneficial. [Display omitted] DisclosuresNiihara:Emmaus Medical, Inc.: Employment, Equity Ownership. Viswanathan:Novartis: Speakers Bureau. Razon:Emmaus Medical, Inc.: Employment. Tran:Emmaus Medical, Inc.: Employment, Equity Ownership. Stark:Emmaus Medical, Inc.: Employment, Equity Ownership.

Diabetic complications within the context of aging: Nicotinamide adenine dinucleotide redox, insulin C-peptide, sirtuin 1-liver kinase B1-adenosine monophosphate-activated protein kinase positive feedback and forkhead box O3.

Recent research in nutritional control of aging suggests that cytosolic increases in the reduced form of nicotinamide adenine dinucleotide and decreasing nicotinamide adenine dinucleotide metabolism plays a central role in controlling the longevity gene products sirtuin 1 (SIRT1), adenosine monophosphate‐activated protein kinase (AMPK) and forkhead box O3 (FOXO3). High nutrition conditions, such as the diabetic milieu, increase the ratio of reduced to oxidized forms of cytosolic nicotinamide adenine dinucleotide through cascades including the polyol pathway. This redox change is associated with insulin resistance and the development of diabetic complications, and might be counteracted by insulin C‐peptide. My research and others' suggest that the SIRT1–liver kinase B1–AMPK cascade creates positive feedback through nicotinamide adenine dinucleotide synthesis to help cells cope with metabolic stress. SIRT1 and AMPK can upregulate liver kinase B1 and FOXO3, key factors that help residential stem cells cope with oxidative stress. FOXO3 directly changes epigenetics around transcription start sites, maintaining the health of stem cells. ‘Diabetic memory’ is likely a result of epigenetic changes caused by high nutritional conditions, which disturb the quiescent state of residential stem cells and impair tissue repair. This could be prevented by restoring SIRT1–AMPK positive feedback through activating FOXO3.

L-Glutamine Therapy Reduces Hospitalization for Sickle Cell Anemia and Sickle β°-Thalassemia Patients at Six Months – A Phase II Randomized Trial

Background: Increased oxidant stress plays an important role in the pathophysiology of sickle cell disease. Nicotinamide Adenine Dinucleotide (NAD) is an important anti-oxidant that protects hemoglobin as demonstrated in diseases such as methemoglobinemia. Early in-vitro studies have shown that L-glutamine, a precursor for NAD, reduced oxidant stress via improvement of NAD redox status in red blood cells.Oral administration of L-glutamine in early clinical studies supported in-vitro findings of improving NAD redox potential, therefore, a larger proof of concept clinical trial was designed and conducted. Methods: A Phase II randomized, double-blind, placebo-controlled, parallel-group, multicenter study was conducted to evaluate the safety and efficacy of L-glutamine therapy for patients 5 years or older diagnosed with sickle cell anemia or sickle β°-thalassemia. Eighty one patients were randomized (1:1 ratio) to oral L-glutamine at 0.3 g/kg or placebo twice daily for 48 weeks. The primary endpoint was the frequency of painful crises. Secondary endpoints included the frequency of hospitalization. Results: At Week 24 (6 months), the mean number of painful crises was 2.5 and 5.5 for L-glutamine and placebo groups respectively (p = 0.060). The mean number of hospitalizations was 0.8 and 1.3 for L-glutamine and placebo groups respectively (p = 0.036). Conclusion: At 6 months of therapy, L-glutamine treatment was efficacious in reducing the frequency of hospitalization (nearly 40% reduction) and there was a major trend for the decrease in frequency of painful crises (over 50% reduction) favoring the L-glutamine treatment arm. There was no difference in safety between groups. Based on these findings, a Phase III trial was conducted and results are now available.

A Regulatory Role of NAD Redox Status on Flavin Cofactor Homeostasis inS. cerevisiaeMitochondria

Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) are two redox cofactors of pivotal importance for mitochondrial functionality and cellular redox balance. Despite their relevance, the mechanism by which intramitochondrial NAD(H) and FAD levels are maintained remains quite unclear in Saccharomyces cerevisiae. We investigated here the ability of isolated mitochondria to degrade externally added FAD and NAD (in both its reduced and oxidized forms). A set of kinetic experiments demonstrated that mitochondrial FAD and NAD(H) destroying enzymes are different from each other and from the already characterized NUDIX hydrolases. We studied here, in some detail, FAD pyrophosphatase (EC 3.6.1.18), which is inhibited by NAD+ and NADH according to a noncompetitive inhibition, with Ki values that differ from each other by an order of magnitude. These findings, together with the ability of mitochondrial FAD pyrophosphatase to metabolize endogenous FAD, presumably deriving from mitochondrial holoflavoproteins destined to degradation, allow for proposing a novel possible role of mitochondrial NAD redox status in regulating FAD homeostasis and/or flavoprotein degradation in S. cerevisiae.

Metabolic Fate of Oral Glutamine Supplementation within Plasma and Erythrocytes of Patients with Sickle Cell Disease: Preliminary Pharmacokinetics Results

AbstractAbstract 1636 Introduction:The erythrocyte redox environment may contribute to increased hemolysis and decreased nitric oxide (NO) bioavailability in pulmonary hypertension (PH) of sickle cell disease (SCD). Glutathione (GSH) is the principal thiol redox buffer in erythrocytes and its depletion has been linked to hemolysis. Glutamine plays an additional anti-oxidant role through preservation of the intracellular nicotinamide adenine dinucleotide (NAD) levels, required for reducing GSSG back to GSH. Altered GSH and glutamine metabolism will promote hemolysis and contribute to altered redox homeostasis. Glutamine depletion and low levels of the obligate NO substrate L-arginine are associated with pulmonary hypertension (PH) in SCD. Low arginine bioavailability is also associated with increased mortality risk. Pilot data of L-glutamine therapy has been associated with improved vasculopathy and increased NAD redox potential in SCD. Targeting these deficiencies in SCD has generated interest, however, little information on the pharmacokinetics (pK) is currently available. Methods:We performed pK studies to determine the metabolic fate of glutamine supplementation on plasma and erythrocyte amino acids in patients with SCD. Patients fasting for > 8 hours received 10 grams of L-glutamine powder mixed with Gatorade. Blood was analyzed at baseline, 30, 60, 90, 2hr, 3hr, 4hr and 8hrs after ingesting study drug. A standardized diet was administered to all participants at 3 established time-points (after 2hr, 5hr and 7 hrs). A subset of patients also had pK studies performed without study drug to follow normal diurnal fluctuations in amino acids. Results:We report data on 5 patients with SCD, three of whom performed pK studies both with and without glutamine supplementation. Average age was 50.6 ±5.6 years, 60% were female, 40% SS, 60% SC, and 4 had PH (mean TRV=3.4±0.8m/s, range 3.0–4.6m/s) while one patient had a TRV=2.25 and no history of PH. Diurnal variations in many amino acids were observed. However plasma glutamine levels more than doubled after oral glutamine supplementation, compared to minimal fluctuations with diet. Plasma glutamine concentration peaked within 30 minutes of ingestion (p=0.01) before decreasing to a plateau by 2 hours that remained higher than baseline by 8 hours. Oral glutamine also increased plasma arginine concentration, which peaked by 4 hrs (p=0.03) and remained elevated through 8 hrs (FigB). Global arginine bioavailability (plasma arginine/(ornithine+ citrulline) also increased (0.67± 0.2 to 1.83±0.6, p=0.02). Erythrocyte glutamine levels began to increase by 8 hours, (Fig 1C), while erythrocyte arginine concentration peaked at 4 hours. Anecdotally, the greatest improvement in intracellular arginine bioavailability occurred in our patient with severe PH and a TRV=4.6 (Fig 1D). The erythrocyte glutamine/glutamate ratio peaked at 1–2 hrs, as did the erythrocyte glutathione concentration, although these trends did not reach statistical significance. Conclusions:Oral glutamine supplementation (10 gm) acutely improves glutamine and arginine bioavailability in both plasma and erythrocytes. The clinical implications of these observations remain to be determined, however this represents a promising novel therapy for hemoglobinopathies. Phase II studies of glutamine supplementation targeting PH patients with SCD or thalassemia are ongoing at Children's Hospital & Research Center Oakland. [Display omitted] Disclosures:Off Label Use: L-glutamine - an amino acid that may improve arginine bioavailability. IND held by C. Morris.

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.

Real-Time assessment of organ vitality during the transplantation procedure

Although organ transplantation has become well established as a treatment modality for many patients with organ failure, little attention has been given to the evaluation of organ quality during its various steps. A critical factor in the success of the actual engraftment procedure is the integrity of energy metabolism and oxygen balance (supply/demand) at the microvasculature and intracellular mitochondrial level. The supply of oxygen to the cells is dependent on the saturation of hemoglobin (HbO 2 ), tissue blood flow, and tissue oxygen partial pressure. The mitochondrial reduced nicotinamide adenine dinucleotide redox state represents oxygen balance in the tissue. Although these parameters can be monitored in all tissues of the body, demand for oxygen may be organ-specific. The various steps surrounding transplantation may require different techniques for the evaluation of tissue vitality. Assessment of blood flow or HbO 2 is not possible during preservation of the organ. On the other hand, because extracellular levels of potassium may represent the energy demand processes in many organs, monitoring of extracellular potassium as an indicator of ionic homeostasis may provide important information regarding the quality of the preservation techniques. Although a large number of relevant studies have been performed in small laboratory animals, real-time monitoring in patients needs more practical tools. We present here the principles of multiparametric monitoring by which tissue vitality may be measured in both experimental and clinical situations. Much of the relevant literature on the subject is limited to the monitoring of kidney and liver. There are also some data on the monitoring of skin flaps. We have reviewed the major published reports in which organ and tissue vitality and quality were assessed in real time and will describe tissue and organ oxygen balance, vitality principles, technologic features of the various monitoring techniques, the clinical or experimental tools available and the conceptual and technologic aspects of the multiparametric monitoring concept. We will also discuss both experimental results and preliminary clinical observations by using multiparametric monitoring.