NADH Concentration Research Articles

Effect of tirapazamine on selected kidney parameters in rats treated with cisplatin

Abstract Hypoxic cancer cells are more aggressive and responsible for more efficient metastasis and recurrence. It seems worth-while, hence, to supplement current cytostatic drugs therapy (i.e. cisplatin) with hypoxia cytotoxic agents (i.e. tirapazamine), the toxicity of which is activated by hypoxia. Cisplatin and tirapazamine can change a redox equilibrium and consequently lead to changes in cell metabolism, fibrosis and apoptosis. The aim of this study was to evaluate the cisplatin/tirapazamine toxicological synergism. In doing so we tested selected kidney oxidative stress parameters, as well as nephrotoxicity markers, in plasma and urine. Once a week for 6 weeks, rats received intraperitoneally two doses of tirapazamine (5 or 10 mg/kg bw), 2 hours before cisplatin (2 mg/kg bw) was applied. Our results show that Tirapazamine (TP) had no significant adverse effect on the redox balance, oxidative stress and kidney function in rats receiving cisplatin (CP). However, TP significantly increased protein concentration in the kidneys of rats. In all tested groups, a significant decrease in NADH concentration in kidneys was recorded, which could indicate disorder in the cell metabolism. TP also was found to have prevented bacterial infection caused by CP. In summary, there was no nephrotoxic synergy of TP with CP at an unacceptable level.

Global analysis and Decay Associated Images (DAI) derived from Fluorescence Lifetime Imaging Microscopy (FLIM).

The extraction of fluorophore lifetimes in a biological sample provides useful information about the probe environment that is not readily available from fluorescence intensity alone. Cell membrane potential, pH, concentration of oxygen ([O2]), calcium ([Ca2+]), NADH and other ions and metabolites are all regularly measured by lifetime-based techniques. These measurements provide invaluable knowledge about cell homeostasis, metabolism and communication with the cell environment. Fluorescence lifetime imaging microscopy (FLIM) produces spatial maps with time-correlated single-photon counting (TCSPC) histograms collected and analyzed at each pixel, but traditional TCSPC analysis is often hampered by the low number of photons that can reasonably be collected while maintaining high spatial resolution. More important, traditional analysis fails to employ the spatial linkages within the image. Here, we present a different approach, where we work under the assumption that mixtures of a global set of lifetimes (often only 2 or 3) can describe the entire image. We determine these lifetime components by globally fitting precise decays aggregated over large spatial regions of interest, and then we perform a pixel-by-pixel calculation of decay amplitudes (via simple linear algebra applied to coarser time-windows). This yields accurate amplitude images (Decay Associate Images, DAI) that contain stoichiometric information about the underlying mixtures while retaining single pixel resolution. We collected FLIM data of dye mixtures and bacteria expressing fluorescent proteins with a two-photon microscope system equipped with a commercial single-photon counting card, and we used these data to benchmark the gDAI program.

Electrocatalytic oxidation of NADH in a new nanostructured interface with an entrapped butylpyrene nitroaromatic derivative

In a previous study, we revealed a new way to modify multi-walled carbon nanotubes (MWCNTs) with 3,5-dinitrobenzoic acid (35DNB). Furthermore, we proved that a 35DNB-MWCNTGC electrode efficiently catalysed the oxidation of NADH. In this paper, with the aim of obtaining a stronger bond of the electrocatalytic mediator 35DNB with the MWCNTs, we have synthesized a modified compound of 35DNB. The modification consisted of adding a 4-(piren-1-yl)butyl terminal to 35DNB to form 4-(pyren-1-yl)butyl 3,5-dinitrobenzoate (35DNBpy). The synthesis of 35DNBpy was obtained with a yield of 78.3% (mp=202–204°C). With this change, we improved the electrocatalytic activity by several oxidation cycles, conferring electrocatalytic stability.The preparation and modification of electrodes with carbon nanotubes and 35DNBpy is fast and simple. The electrochemical study was performed by cyclic voltammetry in 3mM NADH solutions. Oxidation of NADH occurs at 0.1 (V vs Ag/AgCl) in aqueous media at pH7 and the same electrode can be used for up to 50min.In this work, the electrocatalytic activity of new modified electrodes with MWCNTs and 35DNBpy is reported. The electrocatalytic activity was evaluated by cyclic voltammetry. The electrocatalytic capacity of the electrode and the heterogeneous electron transfer rate constant were calculated.Sensitivity of the modified electrode 35DNBpy/MWCNT/GCE is measured by voltammetric method which showed linear variation with NADH concentration with limit of detection (LOD) and sensitivity of 0.885μM (3S/N) and 0.33μA/μM, respectively.

Intracellular Analysis of Individual Cells and Organelles for Both Oxygen Concentration/Consumption and NADH Free/Bound Redox State using Fluorescence Lifetime Imaging

Measuring the oxygen consumption and linked metabolic pathways in cancer cells, especially their covariant and/or compensatory interplay, is important for understanding transformation mechanisms, mechanisms of malignant growth, cancer cell drug resistance, and for discovering potential drugs specific for cancer cells. Fluorescence lifetime imaging (FLIM) already offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH and FAD. FLIM can also be used in combination with our novel Förster resonance energy transfer (FRET)-based oxygen sensing probe, Myoglobin-mCherry (Myo-mCherry). This allows real-time, non-invasive monitoring of the intracellular (or even organelle-specific) oxygen concentration ([O2]). Herein, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration ([NADH]). These contemporaneous intracellular measurements of both [O2] and the changes in metabolic redox ratio ([NADH]/[FAD]), via NADH conformation and environment, provides a foundation for quantitative functional imaging of cancer energy metabolism and can shed light on the mechanisms of adaptive responses to hypoxia - or some common physiological and disease states.

Age- and AD-related redox state of NADH in subcellular compartments by fluorescence lifetime imaging microscopy.

Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 to 41% more reducing NADH redox state than cytoplasm for each age, genotype, and sex. Aging diminished the mitochondrial free NADH concentration in NTg neurons by 43% and in 3xTg-AD by 50%. The lower free NADH with age suggests a decline in capacity to regenerate free NADH for energetic supply to power oxidative phosphorylation which further worsens in AD. Applying this non-invasive approach, we showed the most explicit measures yet of bioenergetic deficits in free NADH with aging at the subcellular level in live neurons from in-bred mice and an AD model.

62 - Generation of superoxide radical by human cytochrome b5 reductase is modulated by cytochrome b5

Cytochrome b5 reductase (CYB5R) plays a central role in many diverse metabolic reactions by coupling NAD(P)H oxidation to reduction of proteins, such as cytochrome b5 (CYB5). Together, CYB5R and CYB5 transfer electrons to terminal oxidases, such as the mitochondrial amidoxime reducing component (mARC) or hemoglobin. Previous studies have demonstrated that nitric oxide (NO) production by the mARC/CYB5/CYB5R system is quenched by molecular oxygen (O2) and suggested that superoxide radical production was involved, as the addition of Cu,Zn-superoxide(SOD1) dismutase partially recovered NO production. Flavin proteins are known to generate superoxide, suggesting that the CYB5R was the likely site of superoxide radical production. However, the direct formation of superoxide by human CYB5R had not been studied. Here, studies were performed to understand the related mechanisms of radical generation by CYB5R in the absence and presence of CYB5. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with CYB5R and NADH. An EPR spectrum corresponding to the superoxide radical adduct of DMPO (DMPO-OOH) was obtained. This EPR signal was quenched by the addition of the SOD1. The amount of DMPO-OOH formed from the oxidation of NADH by the CYB5R increased with NADH concentration. Importantly, the formation of DMPO-OOH was abrogated in the presence of CYB5. Spectral kinetics demonstrate that NADH consumption is O2-dependent, as no NADH consumption was observed in the absence of O2. From these results, we propose a mechanism in which NADH-reduced CYB5R catalyzes superoxide radical formation. Our results demonstrate that human CYB5R catalyzes the O2-dependent oxidation of NADH to generate superoxide radical. Additionally, CYB5 may protect biological targets from oxidative damage, by inhibiting CYB5R superoxide radical production. The ability of CYB5R to catalyze superoxide radical production in response to O2 has potential pathophysiological implications for the ischemia/reperfusion induced damages, endothelial dysfunction, and cardiovascular and neurological diseases.

Enzymatic CO2 reduction to formate by formate dehydrogenase from Candida boidinii coupling with direct electrochemical regeneration of NADH

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Redox control and autoxidation of class 1, 2 and 3 phytoglobins from Arabidopsis thaliana

Despite a recent increase in interest towards phytoglobins and their importance in plants, much is still unknown regarding their biochemical/biophysical properties and physiological roles. The present study presents data on three recombinant Arabidopsis phytoglobins in terms of their UV-vis and Raman spectroscopic characteristics, redox state control, redox potentials and autoxidation rates. The latter are strongly influenced by pH for all three hemoglobins – (with a fundamental involvement of the distal histidine), as well as by added anion concentrations – suggesting either a process dominated by nucleophilic displacement of superoxide for AtHb2 or an inhibitory effect for AtHb1 and AtHb3. Reducing agents, such as ascorbate and glutathione, are found to either enhance– (presumably via direct electron transfer or via allosteric regulation) or prevent autoxidation. HbFe3+ reduction was possible in the presence of high (presumably not physiologically relevant) concentrations of NADH, glutathione and ascorbate, with differing behaviors for the three globins. The iron coordination sphere is found to affect the autoxidation, redox state interconversion and redox potentials in these three phytoglobins.

The soluble transhydrogenase UdhA affecting the glutamate-dependent acid resistance system of Escherichia coli under acetate stress.

ABSTRACTThe soluble transhydrogenase (UdhA) is one of two transhydrogenases that play a role in maintaining the balance between NAD(H) pools and NADP(H) pools in Escherichia coli. Although UdhA has been extensively used in metabolic engineering and biocatalysis for cofactor regeneration, its role in acid resistance has not been reported. Here we used DNA microarray to explore the impact of UdhA on transcript levels. We demonstrated that during growth on acetate, the expression of genes involved in the respiratory chain and Gad acid resistance system was inhibited in the udhA-knockout strain. The deletion of udhA significantly repressed the expression of six genes (gadA, gadB, gadC, gadE, hdeA and hdeB) which are involved in Gad acid resistance and resulted in low survival of the bacterium at a low pH of 4.9. Moreover, UdhA was essential for NADH production which is important for the adaptive growth of E. coli on acetate, while NADH concentration in the udhA-knockout strain was quite low and supplemental NADH significantly increased the expression of acid resistance genes and survival of the udhA-knockout strain. These results demonstrated that UdhA is an important source of NADH of E. coli growth on acetate and affects Gad acid resistance system under acetate stress.

Optical Biopsy for Prostate Cancer Diagnosis Using Fluorescence Spectroscopy

Native fluorescence spectra are acquired from fresh normal and cancerous human prostate tissues. The fluorescence data are analyzed using an unsupervised machine learning algorithm such as non-negative matrix factorization. The nonnegative spectral components are retrieved and attributed to the native fluorophores such as collagen, reduced nicotinamide adenine dinucleotide (NADH), and flavin adenine dinucleotide (FAD) in tissue. The retrieved scores of the components are used to estimate the relative concentrations of the native fluorophores such as NADH and FAD and the redox ratio. A supervised machine learning algorithm such as support vector machine (SVM) is used to classify normal and cancerous tissue samples based on either the relative concentrations of NADH and FAD or the redox ratio alone. Various statistical measures such as sensitivity, specificity, and accuracy, along with the area under receiver operating characteristic (ROC) curve are used to show the classification performance. A cross validation method such as leave-one-out is used to further evaluate the predictive performance of the SVM classifier to avoid bias due to overfitting, and the accuracy was found to be 93.3%.

Ionic Liquids as Bifunctional Cosolvents Enhanced CO2 Conversion Catalysed by NADH-Dependent Formate Dehydrogenase

Efficient CO2 conversion by formate dehydrogenase is limited by the low CO2 concentrations that can be reached in traditional buffers. The use of ionic liquids was proposed as a manner to increase CO2 concentration in the reaction system. It has been found, however, that the required cofactor (NADH) heavily degraded during the enzymatic reaction and that acidity was the main reason. Acidity, indeed, resulted in reduction of the conversion of CO2 into formic acid and contributed to overestimate the amount of formic acid produced when the progression of the reaction was followed by a decrease in NADH absorbance (method N). Stability of NADH and the mechanism of NADH degradation was investigated by UV, NMR and by DFT calculations. It was found that by selecting neutral–basic ionic liquids and by adjusting the concentration of the ionic liquid in the buffer, the concentration of NADH can be maintained in the reaction system with little loss. Conversion of CO2 to methanol in BmimBF4 (67.1%) was more than twice as compared with the conversion attained by the enzymatic reaction in phosphate buffer (24.3%).

Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM

Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and phasor analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.

Tuned Amperometric Detection of Reduced β-Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the p-Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. p-hydroxy-phenylacetic acid ( p-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with p-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes.

Asymmetric Nanopore Electrode-Based Amplification for Electron Transfer Imaging in Live Cells.

Capturing real-time electron transfer, enzyme activity, molecular dynamics, and biochemical messengers in living cells is essential for understanding the signaling pathways and cellular communications. However, there is no generalizable method for characterizing a broad range of redox-active species in a single living cell at the resolution of cellular compartments. Although nanoelectrodes have been applied in the intracellular detection of redox-active species, the fabrication of nanoelectrodes to maximize the signal-to-noise ratio of the probe remains challenging because of the stringent requirements of 3D fabrication. Here, we report an asymmetric nanopore electrode-based amplification mechanism for the real-time monitoring of NADH in a living cell. We used a two-step 3D fabrication process to develop a modified asymmetric nanopore electrode with a diameter down to 90 nm, which allowed for the detection of redox metabolism in living cells. Taking advantage of the asymmetric geometry, the above 90% potential drop at the two terminals of the nanopore electrode converts the faradaic current response into an easily distinguishable bubble-induced transient ionic current pattern. Therefore, the current signal was amplified by at least 3 orders of magnitude, which was dynamically linked to the presence of trace redox-active species. Compared to traditional wire electrodes, this wireless asymmetric nanopore electrode exhibits a high signal-to-noise ratio by increasing the current resolution from nanoamperes to picoamperes. The asymmetric nanopore electrode achieves the highly sensitive and selective probing of NADH concentrations as low as 1 pM. Moreover, it enables the real-time nanopore monitoring of the respiration chain (i.e., NADH) in a living cell and the evaluation of the effects of anticancer drugs in an MCF-7 cell. We believe that this integrated wireless asymmetric nanopore electrode provides promising building blocks for the future imaging of electron transfer dynamics in live cells.

Spatial Characterization of NADH Concentration and Diffusion in Cells and Tissue

Spatial Characterization of NADH Concentration and Diffusion in Cells and Tissue

The effect of dehydrogenase enzyme activity in glycolysis on the colour stability of mutton during postmortem storage

This study investigated the influence of activities of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase-B (LDH-B) on the colour stability of mutton. From 60 sheep (Bayannur mutton sheep), 15 longissimus dorsi (LD) muscles were selected on the basis of colour stability (R630/580 and a* value) during the storage and classified into three groups (5 for each group) as high colour stability (HCS), intermediate colour stability (ICS) and low colour stability (LCS). The activities of GAPDH and LDH-B, muscle colour attributes, nicotinamide adenine dinucleuotide (NADH) concentration and lactate concentration were measured. The samples in HCS had higher activities of GAPDH and LDH-B than the samples in the LCS, and the samples in the HCS group also possessed higher NADH and lower lactate concentration. The higher activity of dehydrogenase enzyme may result in higher NADH concentrations and colour stability in muscle tissue. The results suggest that the activity of GAPDH and LDH-B may also play a role in maintaining colour stability.

Impairment of NADH dehydrogenase and regulation of anaerobic metabolism by the small RNA RyhB and NadE for improved biohydrogen production in Enterobacter aerogenes

BackgroundEnterobacter aerogenes is a facultative anaerobe and is one of the most widely studied bacterial strains because of its ability to use a variety of substrates, to produce hydrogen at a high rate, and its high growth rate during dark fermentation. However, the rate of hydrogen production has not been optimized. In this present study, three strategies to improve hydrogen production in E. aerogenes, namely the disruption of nuoCDE, overexpression of the small RNA RyhB and of NadE to regulate global anaerobic metabolism, and the redistribution of metabolic flux. The goal of this study was to clarify the effect of nuoCDE, RyhB, and NadE on hydrogen production and how the perturbation of NADH influences the yield of hydrogen gas from E. aerogenes.ResultsNADH dehydrogenase activity was impaired by knocking out nuoCD or nuoCDE in E. aerogenes IAM1183 using the CRISPR-Cas9 system to explore the consequent effect on hydrogen production. The hydrogen yields from IAM1183-CD(∆nuoC/∆nuoD) and IAM1183-CDE (∆nuoC/∆nuoD/∆nuoE) increased, respectively, by 24.5 and 45.6% in batch culture (100 mL serum bottles). The hydrogen produced via the NADH pathway increased significantly in IAM1183-CDE, suggesting that nuoE plays an important role in regulating NADH concentration in E. aerogenes. Batch-cultivating experiments showed that by the overexpression of NadE (N), the hydrogen yields of IAM1183/N, IAM1183-CD/N, and IAM1183-CDE/N increased 1.06-, 1.35-, and 1.55-folds, respectively, compared with IAM1183. Particularly worth mentioning is that the strain IAM118-CDE/N reached 2.28 mol in H2 yield, per mole of glucose consumed. IAN1183/R, IAM1183-CD/R, and IAM1183-CDE/R showed increasing H2 yields in batch culture. Metabolic flux analysis indicated that increased expression of RyhB led to a significant shift in metabolic patterns. We further investigated IAM1183-CDE/N, which had the best hydrogen-producing traits, as a potential candidate for industry applications using a 5-L fermenter; hydrogen production reached up to 1.95 times greater than that measured for IAM1183.ConclusionsKnockout of nuoCD or nuoCDE and the overexpression of nadE in E. aerogenes resulted in a redistribution of metabolic flux and improved the hydrogen yield. Overexpression of RyhB had an significant change on the hydrogen production via NADH pathway. A combination of strategies would be a novel approach for developing a more economic and efficient bioprocess for hydrogen production in E. aerogenes. Finally, the latest CRISPR-Cas9 technology was successful for editing genes in E. aerogenes to develop our engineered strain for hydrogen production.

Acetone Biosensor Based on Fluorometry of Reduced Nicotinamide Adenine Dinucleotide Consumption in Reversible Reaction by Secondary Alcohol Dehydrogenase

A concentration of acetone in blood and urine can be used as a potential biomarker for clinical diagnosis such as diabetes mellitus type 1. In this paper, a fiber-optic biosensor exploiting consumption of nicotinamide adenine dinucleotide (NADH) was developed for determination of acetone concentration in an aqueous solution. An optical system for the biosensor was constructed with an ultraviolet-light emitting diode excitation system, a photomultiplier tube and an optical fiber probe. The biosensor was fabricated by attaching a membrane, where NADH-dependent secondary alcohol dehydrogenase (S-ADH) was immobilized to the fiber probe. Measurement of the acetone concentration was carried out by immersing the sensor probe in phosphate buffer solution, which contained a coenzyme, NADH. NADH was consumed by the enzymatic reaction with S-ADH, and the consumption was proportional to the acetone concentration. Fluorescence light emitted from NADH was then guided to the photomultiplier tube. Effect of the buffer pH and NADH concentration to the sensor performance, and the selectivity to acetone were also evaluated. As a result, the fluorescence intensity decreased as the acetone concentration increased. A relation between the change of fluorescence intensity and the acetone concentration was found from 0.2 to 50 μmol/L using decrease phenomena of NADH fluorescence for determination of the acetone concentration. The highly sensitive and selective acetone biosensor is the potential tool for diagnosis of diabetes in near future.

Hidden Antioxidative Functions of Reduced Nicotinamide Adenine Dinucleotide Coexisting with Hemoglobin.

Ferrous oxyhemoglobin (HbO2) in red blood cells (RBCs) invariably and slowly autoxidizes to form ferric methemoglobin (metHb). However, the level of metHb is always maintained below 0.5% by intracellular metHb reduction of enzymatic systems with coenzymes, such as reduced nicotinamide adenine dinucleotide (NADH), and by superoxide dismutase (SOD) and catalase (CAT), which eliminate reactive oxygen species. Unquestionably, NADH cannot reduce metHb without the corresponding enzymatic system. Our study, however, demonstrated that a high concentration of NADH (100-fold of normal level, equimolar to HbO2) retarded autoxidation of HbO2 in a highly purified Hb solution with no enzymatic system. Furthermore, an inhibitory effect of NADH on metHb formation was observed with additions of oxidants such as H2O2, NO, and NaNO2. Our mechanism assessment elucidated extremely high pseudo-CAT and pseudo-SOD activities of NADH with coexistence of HbO2, and reactivity of NADH with NO. We prepared a model of RBCs (Hb-vesicles, Hb-V) encapsulating purified HbO2 solution and NADH, but no enzymatic system within liposome. We confirmed the inhibitory effect of NADH on both autoxidation and oxidant-induced metHb formation. In addition, an intravenous administration of these Hb-Vs to rats caused significant retardation of metHb formation by approximately 50% compared to the case without NADH coencapsulation. Based on these results, we elucidated a new role of NADH, that is, antioxidative effect via interaction with Hb, in addition to its classical role as a coenzyme.

Fluorescent diagnostics of epithelial neoplasms of different colon parts.

Changes in the biochemical composition of the tissue during colon cancer progression usually precede morphological changes registered by light microscopy. These changes are very sensitive and may be used for diagnostics in difficult cases, when it is impossible to obtain sufficient amount of material during colonoscopy. The aim of the study is analysis of spectral characteristics of sporadic adenomas and tumors in different parts of colon for improving tumors diagnostics in disputable cases. The spectra of fluorescence excitation of histological sections from 78 patients with colon cancer (adenocarcinoma) and colonic adenomas of different localizations were measured. The spectra of fluorescence excitation of all types of adenomas as well as adenocarcinoma have two maxima at 260/270 nm and at 330/340 nm. The first maximum is primarily defined by tryptophan and phenylalanin containing peptides, one of them is glucagon. The second maximum is mainly defined by collagen in stroma. Progression of precancer lesions to advanced cancer leads to increase of NADH concentration impacting on the second maximum of spectra. However, spectra of all types of the investigated lesions have peculiarities depending on localization. At odds to the previous data about similarities between distal colon and rectum, our results demonstrate similar spectra for proximal colon and rectum due to some similarities in morphological and, as a consequence, biochemical composition. Tumor can be detected by spectral techniques on histological slides even if the specimen contains very few tumorous cells in stroma. Biochemical changes and their similarities for precancer lesions and advanced colon cancer have described. Peculiarities of spectral data for different parts of colon may change the previous opinion about similar mechanisms of cancerogenesis for distal colon and rectum. Moreover, investigation of tissue specimen obtained for histological examination and containing lack of malignant epithelial cells in massive stroma does not interfere with analysis due to specific disproportion of spectrum maxima. Lasers Surg. Med. 49:763-766, 2017. © 2017 Wiley Periodicals, Inc.