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Diagram of the Pentose Phosphate Pathway and NADPH Consumption Scheme represents the metabolic pathways normally operating in human RBCs to maintain the reduced forms of NADP and GSH. Peroxides exposure induces the oxidation of GSH to form GSSG. The pentose phosphate pathway is accelerated to maintain the levels of NADPH and GSH. See Discussion for details.
Source publication
Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the principal source of reducing power in numerous processes of physiological importance. We examined the influence of oxidative stress on the relative amounts of NADPH in human red blood cells (RBCs). To determine the homeostasis of the NADPH existing in the reduced form following oxid...
Contexts in source publication
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... of BCNU Treatment on the NADPH Ratio Fol- lowing Exposure of the RBC Suspension to t-BHP When GR is inhibited or the glucose concentration is low, reduction of GSSG and the regeneration of NADPH via PPP might not occur as shown in Fig. 4. We used BCNU, a specific inhibitor of GR, 21) to inhibit the consumption of NADPH via the re- duction of GR-dependent GSSG. After treatment with 1 mM BCNU and 10 mM glucose, the RBC suspension was exposed to t-BHP and incubated for 1 h in the presence of glucose. Figure 5 shows the observed change in GSH level (Fig. 5A) and NADPH ...
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... and that the ratio did not recover immedi- ately in human RBCs in vitro. We previously demonstrated that GSH and GSSG levels in intact RBCs transiently de- creased and increased upon exposure to t-BHP, respectively but rapidly recovered within 1 h. 36) These observations sug- gest that t-BHP is scavenged via a GPX-catalyzed reaction with GSH (Fig. 4). Additionally, a decrease in GSH levels ...
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... current study, a clear decrease in the NADPH ratio was observed following exposure to t-BHP under low levels of glucose in RBCs, and the recovery of the ratio was signifi- cantly impaired (Fig. 3). These findings indicate that regener- ation of NADPH is dependent upon reactions with G6PDH and 6-phosphogluconate dehydrogenase via PPP, as shown in Fig. 4. However, it is also well known that exposure to t-BHP induces the formation of methemoglobin. 37) Consequently, glucose in t-BHP-exposed RBCs metabolize via glycolysis in order to maintain a balance of NADH, which is a required cofactor in the reduction of methemoglobin by methemoglo- bin reductase. 38) The acceleration of glycolysis ...
Citations
... G6PD deficiency leads to a defect in the PPP in red blood cells [5]. G6PD plays a critical role in RBCs' metabolism because erythrocytes rely solely on PPP to generate sufficient molecules of reduced nicotinamide adenine dinucleotide phosphate (NADPH), the concentration of which in human RBCs has been reported to range from 16 to 44.9 µM [6,7]. For the obtention of the cellular NADPH pool, G6PD catalyzes the oxidation of glucose-6-phosphate (G6P) to glucose-6-phosphogluconate with the concomitant production of one molecule of NADPH, which plays an essential role in redox homeostasis and is used in RBCs as a substrate for two enzymes: glutathione reductase (GR) and thioredoxin reductase (TR). ...
Glucose-6-phosphate dehydrogenase (G6PD) deficiency, affecting an estimated 500 million people worldwide, is a genetic disorder that causes human enzymopathies. Biochemical and genetic studies have identified several variants that produce different ranges of phenotypes; thus, depending on its severity, this enzymopathy is classified from the mildest (Class IV) to the most severe (Class I). Therefore, understanding the correlation between the mutation sites of G6PD and the resulting phenotype greatly enhances the current knowledge of enzymopathies’ phenotypic and genotypic heterogeneity, which will assist both clinical diagnoses and personalized treatments for patients with G6PD deficiency. In this review, we analyzed and compared the structural and functional data from 21 characterized G6PD variants found in the Mexican population that we previously characterized. In order to contribute to the knowledge regarding the function and structure of the variants associated with G6PD deficiency, this review aimed to determine the molecular basis of G6PD and identify how these mutations could impact the structure, stability, and function of the enzyme and its relation with the clinical manifestations of this disease.
... GSH and GSSG conversion is mediated by hydroxy peroxide and GSH reductase [41][42][43]. Pentose phosphate pathway (PPP)derived NADPH supplies electrons to GSSG via GSH reductase [44,45]. ...
Glutathione (GSH) is necessary for maintaining physiological antioxidant function, which is responsible for maintaining free radicals derived from reactive oxygen species at low levels and is associated with improved cognitive performance after brain injury. GSH is produced by the linkage of tripeptides that consist of glutamic acid, cysteine, and glycine. The adequate supplementation of GSH has neuroprotective effects in several brain injuries such as cerebral ischemia, hypoglycemia, and traumatic brain injury. Brain injuries produce an excess of reactive oxygen species through complex biochemical cascades, which exacerbates primary neuronal damage. GSH concentrations are known to be closely correlated with the activities of certain genes such as excitatory amino acid carrier 1 (EAAC1), glutamate transporter-associated protein 3–18 (Gtrap3-18), and zinc transporter 3 (ZnT3). Following brain-injury-induced oxidative stress, EAAC1 function is negatively impacted, which then reduces cysteine absorption and impairs neuronal GSH synthesis. In these circumstances, vesicular zinc is also released into the synaptic cleft and then translocated into postsynaptic neurons. The excessive influx of zinc inhibits glutathione reductase, which inhibits GSH’s antioxidant functions in neurons, resulting in neuronal damage and ultimately in the impairment of cognitive function. Therefore, in this review, we explore the overall relationship between zinc and GSH in terms of oxidative stress and neuronal cell death. Furthermore, we seek to understand how the modulation of zinc can rescue brain-insult-induced neuronal death after ischemia, hypoglycemia, and traumatic brain injury.
... The corresponding NAD(P)H concentration was calculated at the basis of a NADPH standard curve obtained from solutions of different concentrations (Saliola et al., 2012). NAD(P) can be converted into NAD(P)H by alcohol dehydrogenase (NAD) or by glucose phosphate dehydrogenase (NADP) [107], thus total and individual amount of NAD(P)H and NAD(P) can be calculated respectively (Wise and Shear 2006;Ogasawara et al., 2009). The spectroscopy based method is fast and easy. ...
... HPLC for NAD(P)H detection has promised an at least five times more sensitive result than enzymatic reaction (Ogasawara et al., 2009). Peaks of NADH and NADPH can be easily seperated by HPLC. ...
... Peaks of NADH and NADPH can be easily seperated by HPLC. Ion-pairing reverse phase HPLC with fluorescent detection (Ogasawara et al., 2009) or UV detection (Yoshino and Imai 2013) are usually applied. Ogasawara et al. detected NADPH and total NADPH (NADP + NADPH) in human red blood cells with HPLC equipped with an fluorescent detector (Ogasawara et al., 2009), the mobile phase consists of 5% methanol and 95% 0.1 M phosphate buffer, a reverse phase RP-C-18 (4.0 mm × 250 mm, 5 μm) column was applied for compounds separation. ...
Food is essential for human survival. Nowadays, traditional agriculture faces challenges in balancing the need of sustainable environmental development and the rising food demand caused by an increasing population. In addition, in the emerging of consumers’ awareness of health related issues bring a growing trend towards novel nature-based food additives. Synthetic biology, using engineered microbial cell factories for production of various molecules, shows great advantages for generating food alternatives and additives, which not only relieve the pressure laid on tradition agriculture, but also create a new stage in healthy and sustainable food supplement. The biosynthesis of food components (protein, fats, carbohydrates or vitamins) in engineered microbial cells often involves cellular central metabolic pathways, where common precursors are processed into different proteins and products. Quantitation of the precursors provides information of the metabolic flux and intracellular metabolic state, giving guidance for precise pathway engineering. In this review, we summarized the quantitation methods for most cellular biosynthetic precursors, including energy molecules and co-factors involved in redox-reactions. It will also be useful for studies worked on pathway engineering of other microbial-derived metabolites. Finally, advantages and limitations of each method are discussed.
... 22 AzA also elevated the levels of 6-phosphogluconate, a key metabolite in the pentose phosphate pathway, which supplies NADPH toanabolism. 23 Succinate is known to be secreted by B. acidofaciens. Trehalose is a stressresistance compound that is synthesized by microorganisms in a stressed environment, such as under oxidative stress and heat shock. ...
Olfactory receptors are ectopically expressed in extra-nasal tissues. The gut is constantly exposed to high levels of odorants where ectopic olfactory receptors may play critical roles. Activation of ectopic olfactory receptor 544 (Olfr544) by azelaic acid (AzA), an Olfr544 ligand, reduces adiposity in mice fed a high-fat diet (HFD) by regulating fuel preference to fats. Herein, we investigated the novel function of Olfr544 in the gut. In GLUTag cells, AzA induces the cAMP-PKA-CREB signaling axis and increases the secretion of GLP-1, an enteroendocrine hormone with anti-obesity effects. In wild-type (WT) mice injected AzA, GLP-1 plasma levels were elevated. The induction of GLP-1 secretion was negated in cells with Olfr544 gene knockdown and in Olfr544-deficient mice. Gut microbiome analysis revealed that AzA increased the levels of Bacteroides acidifaciens and microbiota associated with antioxidant pathways. In fecal metabolomics analysis, the levels of succinate and trehalose, metabolites correlated with a lean phenotype, were elevated by AzA. The function of Olfr544 in gut inflammation, a key feature in obesity, was further investigated. In RNA sequencing analysis, AzA suppressed LPS-induced activation of inflammatory pathways and reduced TNF-α and IL-6 expression, thereby improving intestinal permeability. The effects of AzA on the gut metabolome, microbiome, and colon inflammation were abrogated in Olfr544-KO mice. These results collectively demonstrated that activation of Olfr544 by AzA in the gut exerts multiple effects by regulating GLP-1 secretion, gut microbiome and metabolites, and colonic inflammation in anti-obesogenic phenotypes and, thus, may be applied for obesity therapeutics.
... 69 It would not be surprising to think that the nondetection of Gor could be lethal due to restraining the NADPH recycling for the pentose phosphate pathway, thereby causing problems in energy metabolism. 70 However, it was shown that E. coli with gor mutations could maintain the healthy growth and had enough reduced glutathione. 71 Therefore, glutathione reduction could be performed by another Gor-independent pathway, providing NADPH for proper energy metabolism. ...
Pseudomonas aeruginosa, a widely distributed opportunistic pathogen, is an important threat to human health for causing serious infections worldwide. Due to its antibiotic resistance and virulence factors, it is so difficult to combat this bacterium; thus, new antimicrobial agents are in search. 3-Hydroxyphenylacetic acid (3-HPAA), which is a phenolic acid mostly found in olive oil wastewater, can be a promising candidate with its dose-dependent antimicrobial properties. Elucidating the molecular mechanism of action is crucial for future examinations and the presentation of 3-HPAA as a new agent. In this study, the antimicrobial activity of 3-HPAA on P. aeruginosa and its action mechanism was investigated via shot-gun proteomics. The data, which are available via ProteomeXchange with identifier PXD016243, were examined by STRING analysis to determine the interaction networks of proteins. KEGG Pathway enrichment analysis via the DAVID bioinformatics tool was also performed to investigate the metabolic pathways that undetected and newly detected groups of the proteins. The results displayed remarkable changes after 3-HPAA exposure in the protein profile of P. aeruginosa related to DNA replication and repair, RNA modifications, ribosomes and proteins, cell envelope, oxidative stress, as well as nutrient availability. 3-HPAA showed its antimicrobial action on P. aeruginosa by affecting multiple bacterial processes; hence, it could be categorized as a multitarget antimicrobial agent.
... On the one hand, it serves as hydrogen and electron donors in reductive biosynthesis of amino acids, lipids, and nucleotides. On the other hand, NADPH provides redox power to protect cells from oxidative damage (Ogasawara et al., 2009;Tan et al., 2009;Ying, 2007). Previously, our studies demonstrated that supplement of exogenous NADPH had a therapeutic effect in rodent and primate models of ischemic stroke (Li et al., 2016). ...
Purpose:
To investigate the effects and mechanisms of NADPH on Kainic acid (KA)-induced excitotoxicity.
Methods:
KA, a non-N-methyl-D-aspartate glutamate receptor agonist, was exposed to adult SD rats via intrastriatal injection and rat primary cortical neurons to establish excitotoxic models in vivo and in vitro, respectively. To determine the effects of NADPH on KA-induced excitotoxicity, neuronal survival, neurologically behavioral score and oxidative stress were evaluated. To explore the mechanisms of neuroprotective effects of NADPH, the autophagy-lysosome pathway related proteins were detected.
Results:
In vivo, NADPH (1 mg/kg or 2 mg/kg) diminished KA (2.5 nmol)-induced enlargement of lesion size in striatum, improved KA-induced dyskinesia and reversed KA-induced activation of glial cells. Nevertheless, the neuroprotective effect of NADPH was not significant under the condition of autophagy activation. NADPH (2 mg/kg) inhibited KA (2.5 nmol)-induced down-regulation of TP-53 induced glycolysis and apoptosis regulator (TIGAR) and p62, and up-regulation of the protein levels of LC3-II/LC3-I, Beclin-1 and Atg5. In vitro, the excitotoxic neuronal injury was induced after KA (50 μM, 100 μM or 200 μM) treatment as demonstrated by decreased cell viability. Moreover, KA (100 μM) increased the intracellular levels of calcium and reactive oxygen species (ROS) and declined the levels of the reduced form of glutathione (GSH). Pretreatment of NADPH (10 μM) effectively reversed these changes. Meanwhile NADPH (10 μM) inhibited KA (100 μM)-induced down-regulation of TIGAR and p62, and up-regulation of the ratio of LC3-II/LC3-I, Beclin-1, Atg5, active-cathepsin B and active-cathepsin D.
Conclusions:
Our data provide a possible mechanism that NADPH ameliorates KA-induced excitotoxicity by blocking the autophagy-lysosome pathway and up-regulating TIGAR along with its antioxidant properties.
... Analysis of NADP(H) content by HPLC-FL-UV. NADPH and NADP + were detected as previously described (53,54) with slight modifications. Briefly, hemoglobin and other proteins with MW greater than 30 kDa were removed from RBC lysates using centrifugal filters with a 30-kDa threshold (Merck Amicon Ultra-4), and filtrates were frozen and stored on dry ice. ...
The red blood cell (RBC) storage lesion is a multi-parametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of nicotinamide adenine dinucleotide phosphate [NADP(H)] were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBCs oxidative status after restoration of body temperature provides a sensitive tool to detect storage-related alterations, and has the potential to improve the quality of stored RBCs for transfusion.
... Measurement of NAD(P)H and NAD(P) + has been achieved by autofluorescence [107], genetically encoded fluorescent sensors [108,109], nuclear magnetic resonance spectroscopy [110,111], HPLC with UV or fluorescence detection [112][113][114][115][116], and enzymatic cycling assays with colorimetric, fluorescent, or luminescent detection [117-120] offered by many commercially available kits. Since LC-MS has become the leading analytical technology in untargeted metabolomics and targeted analysis, this technology has been increasingly applied for quantitation of NAD(P)H and NAD(P) + . ...
Redox (portmanteau of reduction-oxidation) reactions involve the transfer of electrons between chemical species in biological processes fundamental to life. It is of outmost importance that cells maintain a healthy redox state by balancing the action of oxidants and antioxidants; failure to do so leads to a multitude of diseases including cancer, diabetes, fibrosis, autoimmune diseases, and cardiovascular and neurodegenerative diseases. From the perspective of precision medicine, it is therefore beneficial to interrogate the redox phenotype of the individual—similar to the use of genomic sequencing—in order to design tailored strategies for disease prevention and treatment. This chapter provides an overview of redox metabolism and focuses on how mass spectrometry (MS) can be applied to advance our knowledge in redox biology and precision medicine.
... Since, under these conditions, NADP + was much more abundant than the newly formed NADPH, the increase in fluorescence due to formation of the mBFP-NADPH complex might be lower than expected. However, it is known that in most cells, under physiological conditions, NADPH is at least ten times more abundant than NADP + [20,21], which minimizes the competition effect. We employed this assay to compare G6PDH activity in different cell lysates, obtained by either chemical (Fig 3C) or physical ( Fig 3D) disruption of the cell wall and membrane. ...
The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) functions as a reducing agent involved in many biosynthetic and antioxidant reactions in cells. Therefore, a lots of detection or assaying method of this cofactor are developed and used broadly in various research and application fields. These detection or assay tools, however, have often some problems, such as the low sensitivity, susceptibility to environmental interference and time-consuming pretreatment steps, remaining hurdle to successful quantification of NADPH or its derivatives accurately and immediately. Herein, we present a rapid (assay time < 30 s) and sensitive (detection limit < 2 pmol) detection method of NADPH using metagenome-derived blue fluorescent protein (mBFP), a protein capable of significantly enhancing NADPH fluorescence upon binding to this cofactor. Our method takes advantage of the high specificity of mBFP to NADPH and the immediate fluorescence enhancement upon the addition of mBFP to a solution of interest containing NADPH. We can apply this detection scheme to directly quantitative assessment of NADP(H)-dependent enzyme activities in-vitro, and further accessed to quantitative assay of other nicotine amide cofactors, such as NAD+ and NADH, by coupling assay using NAD(H) kinase. Thus, our method enabled us to quantitatively assess the activity of nicotinamide cofactor-associated enzymes in both bacterial and human cell lysates.
... mM (Michelet et al., 1995;Zhang et al., 2014), blood: 0.85-1.8 mM (Michelet et al., 1995;Ogasawara, Funakoshi, & Ishii, 2009) (Mohri et al., 2005;Solaas et al., 2000) 0.4 mM ATP G-and T-amidation 1-10 mM (Chijiiwa et al., 2002) Plasma: 0.000028 mM (Gorman, Feigl, & Buffington, 2007) 5-7 mM (Mohri et al., 2005;Solaas et al., 2000) 1 mM NADPH Oxidation 0.2-0.3 mM (Slater, 1967) Red blood cells 0.02 mM (Ogasawara et al., 2009) 1 mM Wang et al., 2008;Yu, Cui, & Davis, 1999) 1 mM ATP, adenosine triphosphate; CoA, coenzyme A; G, glycine; GSH, glutathione; NADPH, nicotinamide adenine dinucleotide phosphate; PAPS, D-Saccharic acid 1,4-lactone, 3′-phosphoadenosine-5′-phosphosulfate; T, taurine; UDPGA, uridine diphosphate glucuronic acid. ...
... mM (Michelet et al., 1995;Ogasawara, Funakoshi, & Ishii, 2009) (Mohri et al., 2005;Solaas et al., 2000) 0.4 mM ATP G-and T-amidation 1-10 mM (Chijiiwa et al., 2002) Plasma: 0.000028 mM (Gorman, Feigl, & Buffington, 2007) 5-7 mM (Mohri et al., 2005;Solaas et al., 2000) 1 mM NADPH Oxidation 0.2-0.3 mM (Slater, 1967) Red blood cells 0.02 mM (Ogasawara et al., 2009) 1 mM Wang et al., 2008;Yu, Cui, & Davis, 1999) 1 mM ATP, adenosine triphosphate; CoA, coenzyme A; G, glycine; GSH, glutathione; NADPH, nicotinamide adenine dinucleotide phosphate; PAPS, D-Saccharic acid 1,4-lactone, 3′-phosphoadenosine-5′-phosphosulfate; T, taurine; UDPGA, uridine diphosphate glucuronic acid. ...
One of the mechanisms of drug‐induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High‐ and low‐resolution liquid chromatography–tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3‐O‐sulfates was a major pathway in human and chimpanzee (4.8%–52%) and it was a minor pathway in all other species (0.02%–14%). Amidation was primarily with glycine (62%–95%) in minipig and rabbit and it was primarily with taurine (43%–81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%–80%) in rat and mice followed by hamster, while it was lowest (1.6%–22%) in human, chimpanzee and minipig. C6‐β hydroxylation was predominant (65%–95%) in rat and mice, while it was at C6‐α position in minipig (36%–97%). Glucuronidation was highest in dog (10%–56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human. Species differences in bile acid (BA) metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably drug induced liver injury during the early stages of drug discovery and development. We developed an in vitro cofactor‐fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs between humans and several animal species. Major species differences were found in the metabolism of BAs and the relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo. In general, BA metabolism in the chimpanzee is most similar to human, while BA metabolism in rat and mice is most dissimilar from human.
