Nicotinamide Adenine Dinucleotide Phosphate Concentrations Research Articles

Butyrate ameliorates lithium-induced cardiometabolic disorders in male Wistar rats

BackgroundCardiometabolic disease is becoming a main cause of death worldwide with over ten million fatalities annually. Lithium (Li) has been reported to be one of the major causes of cardiometabolic disorders due to its narrow therapeutic index. Sodium butyrate (NaB) which is a short chain fatty acid is implicated to possess an amazing therapeutic effect against cardiometabolic disorders. As a result, the current study aims to evaluate sodium butyrate impact on lithium-induced cardiometabolic disorders. MethodsTwenty male Wistar rats weighing between 230 and 250 g were used for the experiment. They were grouped into control and treated groups respectively as (CTR; normal chow), Lithium only (Li; 5 mg/kg, po), Sodium butyrate only (NaB) (200 mg/kg, po) and NaB +Lithium (Li; 5 mg/kg +NaB; 200 mg/kg, po) for six weeks, after a week of acclimation; n=5. The animals were anesthetized using sodium pentobarbital at a dose of 50 mg/kg intraperitoneally, blood samples were collected by cardiac puncture and cardiac tissue homogenates were measured for plasma Fasting Blood Sugar (FBS), plasma Insulin, Phosphatidylinositol 3 Kinase (PI3K), Nitric oxide (NO), Glucose-6-phosphate dehydrogenase (G6PDH), Malonyladehyde (MDA), Catalase (CAT), Superoxide Dismutase (SOD), Nicotinamide adenine dinucleotide phosphate (NADPH), Glutathione Peroxidase (GPx), and Adenosine deaminase /Xanthine oxidase /uric acid (ADA/XO/UA) using respective ELISA kits. ResultsThe results showed that lithium significantly increased fasting blood sugar (FBS), plasma insulin, GPx, CAT, SOD, and MDA while causing a statistical reduction in plasma and cardiac concentration of NADPH, G6PDH, PI3K, endothelial nitric oxide synthase (eNOS), and Nitric Oxide (NO) in comparison with the control. However, butyrate attenuated plasma GPx, CAT, SOD, MDA, FBS, plasma insulin and significantly elevated plasma and cardiac PI3K, eNOS, NO, plasma NADPH and G6PDH levels in relation to control. Also no change was observed in the plasma and cardiac ADA/XO/UA following sodium butyrate. ConclusionTherefore, butyrate has a remarkable therapeutic potential in ameliorating lithium induced cardiometabolic disorders through glucometabolic control.

Nesfatin-1, Dopamine, and NADPH levels in Infertile Women with Polycystic Ovary Syndrome: Is There a Relationship Between Their Levels and Metabolic and Hormonal Variables.

Polycystic ovary syndrome (PCOS) is commonest endocrine disease occurring in women of reproductive age. This study conducted to clarify altered concentrations of Nesfatin-1, nicotinamide adenine dinucleotide phosphate (NADPH), and dopamine in PCOS women and controls. Also, to assess their role in PCOS pathophysiology and their correlation with measured biochemical parameters. In this observational study, 60 PCOS patients and 24 controls included. Medical history was recorded and full examinations were done. Serum concentrations of lipid profile, fasting blood glucose (FBG), fasting insulin (FSI), luteinizing hormone (LH), follicle stimulating hormone (FSH), prolactin, testosterone, progesterone, estradiol, Nesfatin-1, dopamine, and NADPH were measured by ELISA kits. Values were analyzed using unpaired t-test and Pearson Chi-square test. The p<0.05 was considered statistically significant. In this study, there was significantly elevated waist hip ratio (WHR) and body mass index (BMI) in PCOS patients versus controls (p<0.0001 and p=0.014). There was significant increase in FSH, LH, prolactin, estradiol, testosterone, Nesfatin-1, and dopamine (p=0.021, p=0.015, p<0.0001, p<0.0001, p=0.006, p=0.017, p< 0.0001) and decrease of NADPH (p<0.0001) in PCOS patients. There were significant positive correlations between Nesfatin-1, prolactin, and dopamine levels. Also, there was significant positive correlation between dopamine and BMI, FSI, FSH, LH, estradiol, and prolactin levels; however, significant negative correlations observed between NADPH and BMI, FSI, estradiol, and prolactin levels. Elevated serum Nesfatin-1 concentrations and their association with hyperprolactinemia indicate that they have a role in PCOS pathophysiology. Moreover, elevated dopamine and decreased NADPH concentrations could play role in PCOS pathogenesis.

Niacin Status Indicators and Their Relationship with Metabolic Parameters in Dairy Cows during Early Lactation.

Simple SummaryThe active forms of niacin that represent niacin status are nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP) and the NAD:NADP ratio. Previous studies have shown metabolic changes in the function of niacin form and dose, but it has not been determined whether there are changes in the function of active form of niacin that indicate the vitamin status in the body. In this study, we examined differences in NAD, NADP and NAD:NADP concentration in blood and their relationship with metabolic parameters in cows receiving and not receiving additional niacin in food. We concluded that NAD and NADP are good indicators of the ability of an additional niacin source to create functional cofactors due to their concentration changes, while the NAD:NADP ratio is a good indicator of the biological effects of additional niacin due to correlation with many metabolites.Previous experimental models on cows have examined the difference in the metabolic adaptation in cows after niacin administration, without identifying the most important mediators between niacin administration and its biological effects, namely active forms of niacin. All tissues in the body convert absorbed niacin into its main metabolically active form, the coenzyme nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP). The aim of this study was to determine the influence of niacin administration in periparturient period on NAD, NADP and the NAD:NADP ratio and to determine relationship between these indicators of an active form of niacin with metabolic parameters in cow blood. The study included 90 healthy cows: 45 cows receiving niacin and 45 cows were negative control. The niacin group was treated with nicotinic acid for two weeks before, as well as two weeks after parturition. Nicotinic acid was applied per os with feed. In cows receiving niacin, there was a significantly higher concentration of NAD and NADP, but the NAD:NADP ratio did not differ compared with control. All three indicators were able to separate cows who received and who did not receive additional niacin. NAD and NADP are good indicators of the availability of niacin from additional sources. The NAD:NADP ratio is a good indicator of the biological effect of applied niacin on metabolites in cows due to its correlation with a number of metabolites: positive correlation with glucose, insulin, glucose to insulin ratio and the revised quantitative insulin sensitivity check index (RQUICKIBHB) of insulin resistance, triglycerides and cholesterol, and a negative correlation with nonesterified fatty acid (NEFA), beta hydroxybutyrate (BHB), gamma-glutamyltranspherase (GGT) and urea in cows receiving niacin. The same amount of added niacin in feed can produce different concentrations of NAD, NADP and NAD:NADP in the blood, and this was not related to their concentration before the addition of niacin. The change in the concentration of the active form of niacin (NAD, NADP and NAD:NADP) further correlates with the concentration of metabolic parameters, which indicates that the intensity of the biological effect of additional niacin can be accurately determined only if we know the concentrations of its active forms in blood. Under basal conditions (without additional niacin), active forms of niacin that already exist in the blood do not show significant correlations with metabolic parameters.

Foliar-Applied Glutathione Mitigates Cadmium-Induced Oxidative Stress by Modulating Antioxidant-Scavenging, Redox-Regulating, and Hormone-Balancing Systems in Brassica napus.

The antioxidant glutathione (GSH) mitigates adverse physio-metabolic effects and defends against abiotic types of stress, such as cadmium (Cd) stress. However, its function and role in resisting Cd phytotoxicity by leveraging plant antioxidant-scavenging, redox-regulating, and hormone-balancing systems have not been comprehensively and systematically demonstrated in the Cd-hyperaccumulating plant Brassica napus L. cv. Tammi (oilseed rape). In this study, the effects of exogenously applied GSH to the leaves of B. napus seedlings exposed to Cd (10 μM) were investigated. As a result, Cd stress alone significantly inhibited growth and increased the levels of reactive oxygen species (ROS) and the bioaccumulation of Cd in the seedlings compared with those in unstressed controls. Furthermore, Cd stress induced an imbalance in plant stress hormone levels and decreases in endogenous GSH levels and GSH redox ratios, which were correlated with reductions in ascorbate (AsA) and/or nicotinamide adenine dinucleotide phosphate (NADPH) redox states. However, the exogenous application of GSH to Cd-stressed B. napus seedlings reduced Cd-induced ROS levels and enhanced antioxidant-scavenging defenses and redox regulation by both increasing seedling AsA, GSH, and NADPH concentrations and rebalancing stress hormones, thereby enhancing Cd uptake and accumulation. These results demonstrate that GSH improved plant redox status by upregulating the AsA-GSH-NADPH cycle and reestablishing normal hormonal balance. This indicates that exogenously applied GSH can mitigate Cd phytotoxicity in B. napus and possibly other plants. Therefore, GSH can potentially be applied to Cd-polluted soil for plant remediation.

Regeneration of Nicotinamide Adenine Dinucleotide Phosphate by a Chlorophyll a-Coated TiO2 Film Electrode.

A TiO2 electrode was coated with chlorophyll a to regenerate nicotinamide adenine dinucleotide phosphate (NADPH), which can enhance the photovoltages of the electrodes for photoelectrochemical capacitors. The photovoltage of an uncoated TiO2 electrode was high during the first cycle but then steadily reduced owing to the oxidization of NADPH in the electrolyte during the photo-charge-discharge cycling. By contrast, a chlorophyll a-coated TiO2 electrode maintained high photovoltages for 100 cycles. Residual NADPH concentrations after 100 cycles increased from 73% to 90% because of the coating, demonstrating that NADPH was regenerated by photoexcited chlorophyll a similar to a photosynthetic reaction in nature.

Nicotinamide adenine dinucleotides are associated with distinct redox control of germination in Acer seeds with contrasting physiology.

Seed germination is a complex process enabling plant reproduction. Germination was found to be regulated at the proteome, metabolome and hormonal levels as well as via discrete post-translational modification of proteins including phosphorylation and carbonylation. Redox balance is also involved but less studied. Acer seeds displaying orthodox and recalcitrant characteristics were investigated to determine the levels of redox couples of nicotinamide adenine dinucleotide (NAD) phosphate (NADP) and integrated with the levels of ascorbate and glutathione. NAD and NADP concentrations were higher in Norway maple seeds and exceptionally high at the germinated stage, being the most contrasting parameter between germinating Acer seeds. In contrast, NAD(P)H/NAD(P)+ ratios were higher in sycamore seeds, thus exhibiting higher reducing power. Despite distinct concentrations of ascorbate and glutathione, both seed types attained in embryonic axes and cotyledons had similar ratios of reduced/oxidized forms of ascorbate and half-cell reduction potential of glutathione at the germinated stage. Both species accomplished germination displaying different strategies to modulate redox status. Sycamore produced higher amounts of ascorbate and maintained pyridine nucleotides in reduced forms. Interestingly, lower NAD(P) concentrations limited the regeneration of ascorbate and glutathione but dynamically drove metabolic reactions, particularly in this species, and contributed to faster germination. We suggest that NAD(P) is an important player in regulating redox status during germination in a distinct manner in Norway maple and sycamore seeds.

Exogenous NADPH ameliorates myocardial ischemia-reperfusion injury in rats through activating AMPK/mTOR pathway.

Our previous study shows that nicotinamide adenine dinucleotide phosphate (NADPH) plays an important role in protecting against cerebral ischemia injury. In this study we investigated whether NADPH exerted cardioprotection against myocardial ischemia/reperfusion (I/R) injury. To induce myocardial I/R injury, rats were subjected to ligation of the left anterior descending branch of coronary artery for 30 min followed by reperfusion for 2 h. At the onset of reperfusion, NADPH (4, 8, 16 mg· kg−1· d−1, iv) was administered to the rats. We found that NADPH concentrations in plasma and heart were significantly increased at 4 h after intravenous administration. Exogenous NADPH (8−16 mg/kg) significantly decreased myocardial infarct size and reduced serum levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTn-I). Exogenous NADPH significantly decreased the apoptotic rate of cardiomyocytes, and reduced the cleavage of PARP and caspase-3. In addition, exogenous NADPH reduced mitochondrial vacuolation and increased mitochondrial membrane protein COXIV and TOM20, decreased BNIP3L and increased Bcl-2 to protect mitochondrial function. We conducted in vitro experiments in neonatal rat cardiomyocytes (NRCM) subjected to oxygen–glucose deprivation/restoration (OGD/R). Pretreatment with NADPH (60, 500 nM) significantly rescued the cell viability and inhibited OGD/R-induced apoptosis. Pretreatment with NADPH significantly increased the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in OGD/R-treated NRCM. Compound C, an AMPK inhibitor, abolished NADPH-induced AMPK phosphorylation and cardioprotection in OGD/R-treated NRCM. In conclusion, exogenous NADPH exerts cardioprotection against myocardial I/R injury through the activation of AMPK/mTOR pathway and inhibiting mitochondrial damage and cardiomyocyte apoptosis. NADPH may be a potential candidate for the prevention and treatment of myocardial ischemic diseases.

Reduced nicotinamide adenine dinucleotide phosphate inhibits rat platelet aggregation and p38 phosphorylation

Previous studies found that reduced nicotinamide adenine dinucleotide phosphate (NADPH) protected neurons against ischemia/reperfusion-induced injury. In addition to ROS reduction and ATP increment, preliminary data suggested that NADPH inhibited ADP and thrombin-induced platelet aggregation. As the effect of NADPH on platelet function was not reported by other investigators, the actions of NADPH on platelet function and mechanisms of actions were investigated in the present study. In vitro studies, the effects of different concentrations of NADPH on platelet aggregation induced by ADP (10 μM), thrombin (0.05 U/mL) or AA (50 μM) were determined. The results showed that NADPH could inhibit platelet aggregation induced by ADP, thrombin or AA in a concentration dependent manner. When the inhibitory effects of NAD+, NADH, NADP+ and NADPH on platelet aggregation were compared, NADPH demonstrated the relatively best effect on platelet aggregation. In vivo studies, the effects of NADPH on platelet aggregation, tail bleeding time, coagulation response and ferric chloride-induced thrombosis were determined in mice or rats. The maximum aggregation rate of platelets of rats injected with NADPH (5 mg/kg) was lower than platelets from control rats. NADPH transiently prolonged tail bleeding time in mice at 30 min after the injection of NADPH (7.5 mg/kg), while aspirin (15 mg/kg) significantly prolonged the tail bleeding time in mice at all time points examined. NADPH (5 mg/kg), as well as aspirin (10 mg/kg), had no effect on coagulation response in rats. Using a FeCl3-induced abdominal aorta injury thrombosis model, administration of NADPH (5 mg/kg) significantly delayed the onset of vessel occlusion, while aspirin (10 mg/kg) almost completely prevented the vessel occlusion. With microscopic examination the thrombi in injured vessel sections of rats received NADPH were much smaller and less dense than that of rats received vehicle treatment. ADP induced an increase in phosphorylation of p38 and the effect was markedly inhibited by the p38 inhibitor SB203580. Similarly, NADPH also inhibited ADP-induced phosphorylation of p38. Similar to NADPH, SB203580 robustly inhibited ADP- and thrombin-induced platelet aggregation. In addition, NADPH also reduced ADP-induced increases in ROS in platelets. The current results demonstrated that NADPH inhibited platelet aggregation, oxidative stress and p38 phosphorylation, suggesting that NADPH might be a novel compound for management of high risk of cardiovascular disease.

Role of Ad4-binding protein/steroidogenic factor 1 in regulating NADPH production in adrenocortical Y-1 cells.

Ad4-binding protein/steroidogenic factor 1 (Ad4BP/SF-1), a member of the nuclear receptor superfamily, is expressed in steroidogenic cells and regulates all steroidogenic gene expression. We recently employed mRNA and chromatin immunoprecipitation sequence (ChIP-seq) to demonstrate that Ad4BP/SF-1 directly regulates the expression of nearly all glycolytic genes. The pentose phosphate pathway (PPP) contributes to the production of nicotinamide adenine dinucleotide phosphate (NADPH). Although the expression of PPP genes and intracellular NADPH were decreased by Ad4BP/SF-1 knockdown, these genes were not the direct targets of Ad4BP/SF-1. This study therefore investigates whether Ad4BP/SF-1 directly regulates genes implicated in NADPH production. Examination of previously published data sets of mRNA sequence (mRNA-seq) and ChIP-seq strongly suggested a possibility that other NADPH-producing genes, such as malic enzyme 1 (Me1) and methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), are the direct targets of Ad4BP/SF-1. Reporter gene assays and determination of intracellular NADPH concentration supported the notion that Ad4BP/SF-1 regulates NADPH production by regulating these genes. NADPH is required for macromolecule synthesis of compounds such as steroids, and for detoxification of reactive oxygen species. When synthesizing steroid hormones, steroidogenic cells consume NADPH through enzymatic reactions mediated by steroidogenic P450s. NADPH is also consumed through elimination of reactive oxygen species produced as the byproducts of the P450 reactions. Overall, Ad4BP/SF-1 potentially maintains the intracellular NADPH level through cooperative regulation of genes involved in the biological processes for consumption and supply.

Native fluorescence spectroscopy of blood plasma of rats with experimental diabetes: identifying fingerprints of glucose-related metabolic pathways.

We present the results of a native fluorescence spectroscopy study of blood plasma of rats with experimental diabetes. It was shown that the fluorescence emission band shape at 320 nm excitation is the most indicative of hyperglycemia in the blood plasma samples. We provide the interpretation of this fact based on the changes in reduced nicotinamide adenine dinucleotide phosphate concentration due to glucose-related metabolic pathways and protein fluorescent cross-linking formation following nonenzymatic glycation.

Effect of biogenic photochromic electron acceptors on chlorophyll fluorescence

It is shown that the photophysical properties of chlorophyll a (Chl) depend on the nature and relative amounts of 2-methyl-1,4-naphthoquinone (MNQ) and nicotinamide adenine dinucleotide phosphate (NADP). Photoinduced charge separation occurs in aqueous ethanol solutions of Chl (1 × 10−5 M) and NADP (5 × 10−6–5 × 10−4 M), resulting in the dynamic quenching of Chl fluorescence. Coordination interaction between Chl and NADP is established at an NADP concentration of ≥5 × 10−4 M. The nonlinear Stern-Volmer dependence in this range is due to the input from static quenching. It is shown that the quenching of Chl fluorescence in an MNQ solution at Chl and MNQ concentrations of 1 × 10−5 M and 6.7 × 10−5–1 × 10−4 M, respectively, is described by a linear dependence in the Stern-Volmer coordinates; no complex formation is observed for Chl and MNQ under these conditions, and electron transfer is of the dynamic type. Static or mixed-type energy transfer from MNQ to Chl dominates at elevated MNQ concentrations.

NADPH fluorescence as an indicator of hydrogen production in the green algae Chlamydomonas reinhardtii

This study investigated cellular Nicotinamide Adenine Dinucleotide Phosphate (NADPH) fluorescence as a potential indicator of biohydrogen production in Chlamydomonas reinhardtii and a β-NADPH standard. NADPH fluorescence profiles of cultures grown in TAP-S (Tris-acetate phosphate minus sulphur) media, TAP (Tris-acetate phosphate) media and TAP + 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were subsequently compared. Hydrogen production induced from sulphur depletion was found to correlate directly (r = 0.941) with NADPH over the ten day period. The addition of leachate was used to increase hydrogen yields, and subsequently increased the NADPH concentration by 50%–70%. A direct correlation was observed (r = 0.929) between NADPH and hydrogen when the leachate supplemented media was used. As NADPH is the terminal electron acceptor in the photosynthetic chain, results show that NADPH has a pivotal role in hydrogen production as a carrier molecule. Under sulphur depletion, cellular NADPH fluorescence can be used as an indicator of hydrogen production.

Characterisation of anthocyanidin reductase from Shuchazao green tea

Flavan-3-ols, which account for approximately 700-800 g kg(-1) of tea polyphenols, exert many health-promoting effects. Anthocyanidin reductase (ANR) is an important enzyme involved in the biosynthesis of flavan-3-ols in the tea plant. The purpose of this study was to establish a suitable method for the determination of ANR activity. Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses showed that cyanidin and delphinidin were converted into epicatechin and epigallocatechin respectively via ANR by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme in the tea plant. In order to measure ANR activity via NADPH concentration changes at 340 nm, several interference factors were studied. The interferences from the high background absorbance of substrate and coenzyme and the oxidation reaction of substrate and product were excluded by devising control experiments, decreasing substrate and coenzyme concentrations or adding antioxidants. The optimal pH and concentrations of substrate and NADPH were chosen such that the ANR assays were carried out at 45 °C for 25 min in a total volume of 1.5 mL of reaction mixture containing 0.1 mol L(-1) phosphate buffer (pH 6.5), 0.0667 mmol L(-1) cyanidin, 1 mmol L(-1) NADPH, 0.53 mmol L(-1) ascorbic acid and 150 µg total protein. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that the trends in ANR gene expression corresponded with the enzyme activity in leaves at different development stages. The proposed method is simple, rapid, sensitive and suitable for the determination of ANR activity in the tea plant.

Metabolic constraints on the recovery of sensitivity after visual pigment bleaching in retinal rods

The shutoff of active intermediates in the phototransduction cascade and the reconstitution of the visual pigment play key roles in the recovery of sensitivity after the exposure to bright light in both rod and cone photoreceptors. Physiological evidence from bleached salamander rods suggests this recovery of sensitivity occurs faster at the outer segment base compared with the tip. Microfluorometric measurements of similarly bleached salamander rods demonstrate that the reduction of all-trans retinal to all-trans retinol also occurs more rapidly at the outer segment base than at the tip. The experiments reported here were designed to test the hypothesis that these two phenomena are linked, e.g., that slowed recovery of sensitivity at the tip of outer segments is rate limited by the reduction of all-trans retinal and results from a shortage of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH), the reducing agent for all-trans retinal reduction. Extracellular measurements of membrane current and sensitivity were made from isolated salamander rods under dark-adapted and bleached conditions while intracellular NADPH concentration was varied by dialysis from a micropipette attached to the inner segment. Sensitivity at the base and tip of the outer segment was assessed before and after bleaching. After exposure to a light that photoactivates 50% of the visual pigment, rods were completely insensitive for nearly 10 minutes, after which the base recovered sensitivity and responsiveness with a time constant of ∼200 seconds, but tip sensitivity recovered more slowly with a time constant of ∼680 seconds. Dialysis of 5 mM NADPH into the rod promoted an earlier recovery and eliminated the previously observed tip/base difference. Dialysis of 1.66 mM NADPH failed to eliminate the tip/base recovery difference, suggesting the steady-state NADPH concentration in rods is ∼1 mM. These results indicate the inner segment is the primary source of reducing equivalents after pigment bleaching, with the reduction of all-trans retinal to all-trans retinol playing a key step in the recovery of sensitivity.

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

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 oxidation, we developed an improved method for measurement of NADPH in human RBCs using high-performance liquid chromatography (HPLC). The present method with fluorescent detection is reproducible and selective than enzymatic cycling method and HPLC methods with spectrometric detection. The calibration curve is linear over the range of 0.1-5.0 muM with a correlation coefficient of 0.999. The within-run precision of the assays for total NADPH (NADPH+NADP(+)) and NADPH concentrations in human RBC were 2.4% and 8.6%, respectively (n=5). After the RBC suspension was exposed to tert-butyl hydroperoxide (t-BHP), which is scavenged by glutathione peroxidase (GPX) along with NADPH consumption, a significant decrease in the NADPH ratio [(NADPH/(NADPH+NADP(+))] was observed after a transient decrease and rapid recovery of the reduced form of glutathione. The marked decrease in the NADPH ratio induced by t-BHP exposure was observed in the absence of glucose. However, the NADPH ratio was not decreased by t-BHP exposure after pre-treatment with a glutathione reductase inhibitor. This method may be useful for the measurement of small amounts of NADPH from various biological sources.

Spectrofluorimetric determination of trace amount of coenzyme II using ciprofloxacin–terbium complex as a fluorescent probe

A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using terbium ion (Tb 3+)–ciprofloxacin (CIP) complex as a fluorescent probe, in the buffer solution of pH = 9.00 , NADP can remarkably enhance the fluorescence intensity of the Tb 3+–CIP complex at λ = 545 nm and the enhanced fluorescence intensity of Tb 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.9×10 −7−3.7×10 −6 mol L −1 with detection limit of 1.3×10 −7 mol L −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Tb 3+-CIP system and the Tb 3+–CIP–NADP system have been also discussed.

Fluorimetric study of the interaction between nicotinamide adenine dinucleotide phosphate and tetracycline–europium complex and its application

A new spectrofluorimetric method was developed for the determination of trace amount of nicotinamide adenine dinucleotide phosphate (NADP). Using europium (Eu 3+)–tetracycline (TC) complex as a fluorescent probe, in the buffer solution of pH 7.60. NADP can remarkably enhance the fluorescence intensity of the Eu 3+–TC complex at λ = 612 nm and the enhanced fluorescence intensity of Eu 3+ ion is in proportion to the concentration of NADP. Optimum conditions for the determination of NADP were also investigated. The dynamic range for the determination of NADP is 4.4 × 10 −7 to 2.2 × 10 −6 mol l −1 with detection limit of 6.9 × 10 −8 mol l −1. This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of NADP in synthetic water samples and in serum samples. Moreover, the enhancement mechanisms of the fluorescence intensity in the Eu 3+–TC system and the Eu 3+–TC–NADP system have been also discussed.

Influence of oxygen tension on reactive oxygen species production and human sperm function.

Human spermatozoa were exposed to the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to stimulate endogenous production of reactive oxygen species (ROS). They were incubated under a gas phase of 5% CO2/90% N2/5% O2, or 5% CO2/95% air (20% O2) to investigate whether a lower than atmospheric oxygen tension in the gas phase of the incubator limited the endogenous production of ROS by human spermatozoa and thus was able to reduce the cytotoxic effects of ROS on sperm function. Exposure of human spermatozoa or exogenous NADPH induced an 8-fold higher production of superoxide anion under ambient vs. low oxygen tension. This marked difference in the stimulation of superoxide anion generation was associated with significantly different sperm motility parameters, according to the oxygen tension in the gas phase of the incubator. Whereas under 5% oxygen the percentage of motile spermatozoa was unaffected by the presence of NADPH, all of the motility parameters recorded under an atmosphere of 5% CO2 in air were dramatically affected, not only compared to their respective controls, but also compared to the motility parameters observed under low oxygen tension. The presence of superoxide dismutase plus catalase protected spermatozoa against the toxic effects of NADPH, confirming a cause/effect relationship between the increased superoxide production and reduced sperm function. Even at a concentration of NADPH which did not alter the percentage of motile spermatozoa, hyperactivated motility and acrosome reaction were significantly lower under an atmosphere of 5% CO2 in air compared to a gas phase of 5% CO2/90% N2/5% O2. These results suggest that there is an advantage in using 5% O2 rather than 20% O2 in the gas phase of the incubator to prevent the excessive production of ROS by spermatozoa and related alterations of sperm functions. This may be of clinical value in fertilization programmes.

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min −1 ml −1 of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min −1 ml −1 at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients ( K m) app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V max, for immobilized microsomes is calculated to be 322 nmoles min −1 ml −1 compared with 145 nmols min −1 ml −1 for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.

Purification and Partial Kinetic and Physical Characterization of Two Chloroplast-Localized NADP-Specific Glutamate Dehydrogenase Isoenzymes and Their Preferential Accumulation in Chlorella sorokiniana Cells Cultured at Low or High Ammonium Levels

Two ammonium-inducible, chloroplast-localized NADP-specific glutamate dehydrogenase isoenzymes were purified to homogeneity from Chlorella sorokiniana. These isoenzymes were homopolymers of either alpha- or beta-subunits with molecular weights of 55,500 or 53,000, respectively. The alpha-isoenzyme was preferentially induced at low ammonium concentrations (2 millimolar or lower), whereas only the beta-isoenzyme accumulated after cells were fully induced (120 minutes) at high ammonium concentrations (29 millimolar). Purification of isoenzymes was achieved by (NH(4))(2)SO(4) fractionation, gel-filtration, anion-exchange fast protein liquid chromatography, and affinity chromatography. The alpha- and beta-isoenzymes were separated by their differential binding to Type 4 nicotinamide adenine dinucleotide phosphate-Sepharose. Both isoenzymes bound to an antibody affinity column to which purified antibody (prepared against beta-isoenzyme) was covalently attached. Peptide mapping of the subunits showed them to have a high degree of sequence homology. Both subunits were synthesized in vitro from precursor protein(s) with a molecular weight of 58,500. Although the subunits have similar chemical, physical, and antigenic properties, their holoenzymes have strikingly different ammonium K(m) values. The ammonium K(m) of the beta-isoenzyme remained constant at approximately 75 millimolar, whereas this K(m) of the alpha-isoenzyme ranged from 0.02 to 3.5 millimolar, depending upon nicotinamide adenine dinucleotide phosphate concentration.