Determination of Reduced Nicotinamide Adenine Dinucleotide Phosphate Concentration Using High-Performance Liquid Chromatography with Fluorescence Detection: Ratio of the Reduced Form as a Biomarker of Oxidative Stress

Ogasawara, Yuki; Funakoshi, Masayo; Ishii, Kazuyuki

doi:10.1248/bpb.32.1819

Cited by 29 publications

(27 citation statements)

References 35 publications

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“…This result indicates that it is essential to maintain the level of reductive GSH in RBCs and that the maintenance mechanism orderly acts in intact RBC suspensions in the presence of sufficient glucose. As shown in the results presented here, two independent pre-treatments aimed at disrupting GSH homeostasis 16) caused clear alterations in redox responses of RBC to BHP. GSH disruptive conditions were produced by pre-treatment with an inhibitor of GR or incubation in glucose-free medium.…”

Section: Discussionsupporting

confidence: 64%

“…Therefore, we designed experiments to disrupt GSH homeostasis. 16) We used two independent treatments aimed at decreasing the level of the reduced form of GSH concretely: 1) pre-incubation with a GR inhibitor and 2) a glucose-free medium to examine the influences of preventing GSHdependent antioxidant and reducing activity to reduce glutathionyl proteins in the cells. When GSH levels failed to recover after tert-butyl hydroperoxide (BHP) exposure, we clearly detected the formation of glutathionyl protein in human RBCs after pre-treatment aimed at disrupting GSH homeostasis and subsequent BHP exposure.…”

Section: 2)mentioning

confidence: 99%

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Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Ogasawara

Komiyama

Funakoshi

et al. 2010

Biological & Pharmaceutical Bulletin

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Proteins are susceptible to attack by reactive oxygen species (ROS). Several types of ROS-induced protein damage have been identified, including amino acid modification, cross-linking, and degradation. 1,2) Glutathione (GSH) is the major intracellular non-protein thiol compound and plays a key role in the protection of cells and tissue structures from oxidative injury. GSH can exist in a reduced form (GSH), oxidized form (GSSG), or as proteinbound GSH (GSSP). GSH acts as an intracellular antioxidant that is involved in preventing protein sulfhydryl groups from oxidizing and cross-linking. In addition, the GSH redox system functions indirectly with glutaredoxin (Grx), glucose 6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GR), which maintain GSH in its reduced form. An increase in oxidative stress leads to the oxidation of GSH to GSSG and, under certain conditions, the formation of glutathionyl proteins. Various glutathionyl proteins are involved in numerous physical processes (e.g., growth, differentiation, cell cycle progression, cytoskeletal functions, and metabolism), suggesting that S-glutathionylation is a general mechanism of redox regulation. [3][4][5] Red blood cells (RBCs) have been used as a model system in numerous studies to determine the significance of the GSH-dependent redox system 6,7) because they have an efficient antioxidant defense system that prevents oxidative damage.8-10) Moreover, RBCs have high levels of GSH and Grx, which are thought to utilize the reducing power of GSH to catalyze disulfide reductions in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and GR. 11)Many lines of evidence have emerged to support a fundamental role for the glutathionylation of certain proteins in normal cellular biochemical and physiological processes. It has been reported that some proteins are capable of reacting to form the glutathionyl protein. In particular, it is well known that actin 12) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) 13) are oxidized to form glutathionyl proteins. Additionally, the formation of glutathionyl proteins in human RBCs has been reported in in vitro studies. These studies indicated that hemoglobin, GAPDH and actin, 14) as well as spectrin and membrane protein band 4.2, 15) are glutathionylated in response to diamide or ROS. Alternatively, GSH is likely to contribute to the protection of all thiol residues in proteins against ROS in the cell. However, the physiological susceptibility of proteins to ROS and the direct effects of GSH on oxidative stress in human RBCs remain poorly understood. Moreover, there is little information on the significance of redox regulation that is dependent on the glutathionylation of individual proteins.The objective of this study was to identify physiologically relevant glutathionyl proteins formed in RBCs by ROS exposure. Therefore, we designed experiments to disrupt GSH homeostasis.16) We used two independent treatments aimed at decreasing the level of the reduced form of GSH concretely: 1) pre-...

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Section: Discussionsupporting

confidence: 64%

Section: 2)mentioning

confidence: 99%

Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Ogasawara

Komiyama

Funakoshi

et al. 2010

Biological & Pharmaceutical Bulletin

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“…After centrifugation at 14,000g for 5 minutes at 4°C, the acid extracts were adjusted to pH ∼7.4 using 0.2 M tris base, and reduced to NADPH using NADP cycling buffer (0.165 M Tris-HCl (pH 8.0) containing 16.5 mM MgCl 2 , 8.3 mM glucose-6-phosphate, and 8.3 units/ml G6PD (Ogasawara et al, 2009). Then the samples were incubated for 5 minutes at 37°C and heated at 60°C for 30 minutes.…”

Section: Methodsmentioning

confidence: 99%

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy

et al. 2015

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NAD 1 kinase (NADK) is the only known cytosolic enzyme that converts NAD 1 to NADP 1 , which is subsequently reduced to NADPH. The demand for NADPH in cancer cells is elevated as reducing equivalents are required for the high levels of nucleotide, protein, and fatty acid synthesis found in proliferating cells as well as for neutralizing high levels of reactive oxygen species (ROS). We determined whether inhibition of NADK activity is a valid anticancer strategy alone and in combination with chemotherapeutic drugs known to induce ROS. In vitro and in vivo inhibition of NADK with either small-hairpin RNA or thionicotinamide inhibited proliferation. Thionicotinamide enhanced the ROS produced by several chemotherapeutic drugs and produced synergistic cell kill. NADK inhibitors alone or in combination with drugs that increase ROS-mediated stress may represent an efficacious antitumor combination and should be explored further.

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“…Nicotinamide adenine dinucleotide phosphate (NADP) is an important intracellular cofactor. The oxidized form NADP + acts as a precursor for messenger molecules in some organisms , whereas the reduced form NADPH provides reducing power to intracellular antioxidant systems and transfers reduction equivalents from substrates to anabolism . Moreover, there is evidence that NADPH plays a role in processes associated with the metabolic burden that decreases biomass yield and thus the overall product yield in high‐level production of heterologous protein .…”

Section: Introductionmentioning

confidence: 99%

“…Although this approach is used in some protocols , Maharjan and Ferenci found that nucleotides may adsorb to the precipitating salts that are formed during the neutralization step after extraction and would therefore be lost during the subsequent sample preparation steps. Ogasawara et al proposed that a heating step is necessary in an extraction protocol for NADPH, as some of it is protein‐bound and is only released in this step . Gonzalez et al assessed that NADH, which is similarly unstable as NADPH, is stable in boiling ethanol extracts as long as the extraction solvent is buffered to pH 7.5 .…”

Section: Introductionmentioning

confidence: 99%

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†

et al. 2014

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The accurate quantification of the highly unstable intracellular cofactor nicotinamide adenine dinucleotide phosphate in its oxidized and reduced forms demands a thorough evaluation of the analytical workflow and dedicated methods reflecting their solution chemistry as well as the biological importance of their ratio. In this work, we present a workflow for the analysis of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in the yeast Pichia pastoris, including hot aqueous extraction, chromatographic separation in reversed-phase conditions employing a 100% wettable stationary phase, and subsequent tandem mass spectrometric analysis. A thorough evaluation and optimization of the sample preparation procedure resulted in excellent biological repeatabilities (on average <10%, N = 3) without employing an internal standardization approach. As a consequence, the methodology proved to be appropriate for the relative assessment of intracellular levels of oxidized and reduced nicotinamide adenine dinucleotide phosphate in different P. pastoris strains. The ratio of reduced versus oxidized nicotinamide adenine dinucleotide phosphate was significantly higher in an engineered strain overexpressing glucose-6-phosphate dehydrogenase than in the corresponding wildtype strain. Interestingly, a difference was also observed in the nicotinamide adenine dinucleotide phosphate pool size, which was significantly higher in the wildtype than in the modified strain.

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Determination of Reduced Nicotinamide Adenine Dinucleotide Phosphate Concentration Using High-Performance Liquid Chromatography with Fluorescence Detection: Ratio of the Reduced Form as a Biomarker of Oxidative Stress

Cited by 29 publications

References 35 publications

Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†

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Determination of Reduced Nicotinamide Adenine Dinucleotide Phosphate Concentration Using High-Performance Liquid Chromatography with Fluorescence Detection: Ratio of the Reduced Form as a Biomarker of Oxidative Stress

Cited by 29 publications

References 35 publications

Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Disruption of Glutathione Homeostasis Causes Accumulation of <i>S</i>-Glutathionyl Proteins in Response to Exposure to Reactive Oxygen Species in Human Erythrocytes

Suppression of Cytosolic NADPH Pool by Thionicotinamide Increases Oxidative Stress and Synergizes with Chemotherapy

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast†

Contact Info

Product

Resources

About

Sample preparation workflow for the liquid chromatography tandem mass spectrometry based analysis of nicotinamide adenine dinucleotide phosphate cofactors in yeast^†