Form Of Nicotinamide Adenine Dinucleotide Research Articles

Crosstalk Between the Mitochondrial Dynamics and Oxidative Stress in Zinc-induced Cytotoxicity.

Zinc is an essential trace element, which plays an important role in multiple biological activities. However, excessive exposure to zinc can cause toxic damage to living organism. Here, we investigated the relationship between oxidative stress and mitochondrial dynamics in the zinc-induced cytotoxicity. Results showed that excess exposure to zinc could significantly reduce cell viability and induce cell vacuolation in PK-15 cells. Additionally, zinc exposure caused mitochondrial dynamics disorder, manifested as mitochondrial fission, and the elevated mRNA level of Drp1 and downregulated mRNA levels of OPA1, Mfn1, and Mfn2. Meanwhile, zinc could induce oxidative damage, evidenced by the increasing levels of hydrogen peroxide, malondialdehyde, lipid peroxidation, oxidized form of nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide phosphate, oxidized glutathione/glutathione, superoxide dismutase activity, and the mRNA expression of SOD-1 and NOQ1, and decreasing levels of catalase activity, glutathione peroxidase activity, glutathione reductase activity, and the mRNA expression of CAT, and GPX1. Interestingly, N-acetyl-L-cysteine, an inhibitor for oxidative stress, could reduce the mitochondrial fission under zinc treatment. Besides, Mdivi-1, a mitochondrial fission inhibitor, could relieve oxidative stress caused by excess zinc. Ingeneral, these results suggested that mitochondrial fission and oxidative stress induced by zinc were interrelated in PK-15 cells, which is conducive to explore the new mechanism of zinc toxicity and proposes a theoretical foundation for selecting effective drugs to alleviate the toxic effects caused by zinc.

Genetic Associations of Visfatin Polymorphisms with EGFR Status and Clinicopathologic Characteristics in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common histologic type of lung cancer. Mutations of the epidermal growth factor receptor (EGFR) gene are among the most common genetic alterations in LUAD and are the targets of EGFR tyrosine kinase inhibitors. The enzyme visfatin is involved in the generation of the oxidized form of nicotinamide adenine dinucleotide (NAD+) and regulation of intracellular adenosine triphosphate (ATP), critical processes in cancer cell survival and growth. This study explored the relationship between visfatin single nucleotide polymorphisms (SNPs) with EGFR status and the clinicopathologic development of LUAD in a cohort of 277 Taiwanese men and women with LUAD. Allelic discrimination of four visfatin SNPs rs11977021, rs61330082, rs2110385 and rs4730153 was determined using a TaqMan Allelic Discrimination assay. We observed higher prevalence rates of advanced (T3/T4) tumors and distant metastases in EGFR wild-type patients carrying the rs11977021 CT + TT and rs61330082 GA + AA genotypes, respectively, compared with patients carrying the CC and GG genotypes. EGFR wild-type patients carrying the rs11977021 CT + TT genotypes were also more likely to develop severe (stage III/IV) malignancy compared with patients carrying the CC genotype. An analysis that included all patients found that the association persisted between the rs11977021 CT + TT and rs61330082 GA + AA genotypes and the development of T3/T4 tumors compared with patients carrying the rs11977021 CC and rs61330082 GG genotypes. In conclusion, these data indicate that visfatin SNPs may help to predict tumor staging in LUAD, especially in patients with EGFR wild-type status.

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5weeks of age and muscle atrophy by 11weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

Enzyme-embedded electrospun fiber sensor of hydrophilic polymer for fluorometric ethanol gas imaging in vapor phase

Non-invasive measurement of volatile organic compounds (VOCs) emitted from living organisms is a powerful technique for diagnosing health conditions or diseases in humans. Bio-based gas sensors are suitable for the sensitive and selective measurement of a target VOC from a complex mixture of VOCs. Conventional bio-based sensors are normally prepared as wet-type probes to maintain proteins such as enzymes in a stable state, resulting in limitations in the commercialization of sensors, their operating environment, and performance. In this study, we present an enzyme-based fluorometric electrospun fiber sensor (eFES) mesh as a gas-phase biosensor in dry form. The eFES mesh targeting ethanol was fabricated by simple one-step electrospinning of polyvinyl alcohol with an alcohol dehydrogenase and an oxidized form of nicotinamide adenine dinucleotide. The enzyme embedded in the eFES mesh worked actively in a dry state without pretreatment. Substrate specificity was also maintained, and the sensor responded well to ethanol with a sufficient dynamic range. Adjustment of the pH and coenzyme quantity in the eFES mesh also affected enzyme activity. The dry-form biosensor—eFES mesh—will open a new direction for gas-phase biosensors because of its remarkable performance and simple fabrication, which is advantageous for commercialization.

Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state.

.SignificanceThe optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability.AimDetermine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability.ApproachSW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h.ResultsChanges in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition.ConclusionsThe ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Genetic Ablation of Na,K-ATPase α4 Results in Sperm Energetic Defects.

The Na,K-ATPase alpha 4 isoform (NKAα4) is expressed specifically in the male germ cells of the testes and is particularly abundant in mature spermatozoa. Genetic deletion of NKAα4 in mice (NKAα4 KO mice) results in complete infertility of male, but not female mice. The reduced fecundity of NKAα4 KO male mice is due to a series of defects, including a severe impairment in total and hyperactive sperm motility. In this work, we show that deletion of NKAα4 also leads to major defects in sperm metabolism and energetics. Thus, compared to wild-type sperm, sperm from NKAα4 KO mice display a significant reduction in the extracellular acidification rate (ECAR), indicative of impaired glycolytic flux. In addition, mitochondrial function is disrupted in sperm lacking NKAα4, as indicated by a reduction in the mitochondrial membrane potential and lower oxygen consumption rate (OCR). Moreover, the ratio between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD/NADH) is increased in NKAα4 KO sperm, indicating a shift in the cellular redox state. These metabolic changes are associated with augmented reactive oxygen species (ROS) production and increased lipid peroxidation in NKAα4 KO sperm. Altogether, these findings reveal a novel link between NKAα4 activity and sperm energetics, highlighting the essential role of this ion transporter in sperm physiology.

Metabolism of Enantiomers of Rhododendrol in Human Skin Homogenate.

We reported that raspberry ketone (RK) is produced from rhododendrol (RD) in excised mouse skin. We confirmed that RK is also produced from RD in human skin homogenates. We also observed more conversion of RD to RK when the oxidized form of nicotinamide adenine dinucleotide (NAD+), a coenzyme of alcohol dehydrogenase (ADH), was added to human skin homogenates. Chiral column analysis of the consumption of RD enantiomers in human skin homogenates also showed that more of the R enantiomers of RD remained than the S enantiomers of RD. This suggests that the S-enantiomer of RD is more easily oxidized in human skin. We confirmed that RD is partially metabolized to RK in human skin, thus suggesting that ADH in the skin may be the main cause of the appearance of this oxidation product.

Label-Free Imaging to Track Reprogramming of Human Somatic Cells.

The process of reprogramming patient samples to human-induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient, leading to a heterogeneous population of cells. In this study, we track the reprogramming status of patient-derived erythroid progenitor cells (EPCs) at the single-cell level during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear morphometry to identify high-quality iPSCs. EPCs isolated from human peripheral blood of three donors were used for our proof-of-principle study. We found distinct patterns of autofluorescence lifetime for the reduced form of nicotinamide adenine dinucleotide (phosphate) and flavin adenine dinucleotide during reprogramming. Random forest models classified iPSCs with ∼95% accuracy, which enabled the successful isolation of iPSC lines from reprogramming cultures. Reprogramming trajectories resolved at the single-cell level indicated significant reprogramming heterogeneity along different branches of cell states. This combination of micropatterning, autofluorescence imaging, and machine learning provides a unique, real-time, and nondestructive method to assess the quality of iPSCs in a biomanufacturing process, which could have downstream impacts in regenerative medicine, cell/gene therapy, and disease modeling.

The Effect of Antihypertensive Drugs on NADH in Newly Diagnosed Primary Hypertension.

Background Some antihypertensive medications alter cellular energy production, presumably by modification of the mitochondrial function. In vivo studies of such effects are challenging in humans. We applied a noninvasive forearm skin measurement of the 460-nm fluorescence specific for the reduced form of nicotinamide adenine dinucleotide (NADH) to study the 6-week effects of four different antihypertensive medications on mitochondrial function using the Flow-Mediated Skin Fluorescence (FMSF). Methods In a prospective open-label study, we compared the long-term effects of a 6-week treatment with either amlodipine (5 mg), perindopril (5 mg), nebivolol (5 mg), or metoprolol (50 mg) on the dynamic flow-mediated changes in the skin NADH content in 76 patients (29 women) with untreated primary arterial hypertension (HA). Patients underwent 24-hour ambulatory blood pressure monitoring. To study mitochondrial function, the FMSF was measured at rest, during 100-second ischemia and postischemic reperfusion. The control group consisted of 18 healthy people (7 women). Results There were no significant differences in the FMSF parameters between the control and the study group before medication. After the 6-week treatment, all drugs similarly reduced blood pressure. Neither amlodipine, perindopril, nor nebivolol changed the flow-mediated 460-nm skin fluorescence significantly. However, metoprolol raised this fluorescence at rest, during ischemia and reperfusion (P at most <0.05), indicating an increase in the total NADH skin content. Conclusion Amlodipine, perindopril, and nebivolol appear neutral for the skin NADH content during the 6-week antihypertensive treatment. Similar treatment with metoprolol increased skin NADH at rest, during ischemia and reperfusion, probably due to an effect on microcirculation and altered mitochondrial function. Explanation of the potential mechanisms behind metoprolol influence on the skin NADH metabolism requires further investigation.

Electrochemical Immunosensor for Lactate Dehydrogenase Detection Through Analyte‐driven Catalytic Reaction on Multi‐walled Carbon Nanotubes and Gold Nanoparticle Modified Carbon Electrode

AbstractA novel electrochemical immunosensor for lactate dehydrogenase (LDH) detection was proposed based on analyte‐driven catalytic reaction by attaching LDH antibodies on multi‐walled carbon nanotubes (MWCNTs) and gold nanoparticles (AuNPs) modified glassy carbon electrodes (GCE). As LDH was captured by the antibodies on electrode surface, it catalyzed the formation of pyruvate and the reduced form of nicotinamide adenine dinucleotide (NADH), thus a sensitive electrochemical signal obtained from the above redox reaction was recorded by differential pulse voltammetry (DPV). Under optimum conditions, the developed immunosensor exhibits high sensitivity for LDH quantification ranging from 0.001 μg/mL to 0.5 μg/mL with a low detection limit at 0.39 ng/mL. This developed immunosensor reveals ideal accuracy and feasibility for LDH detection in Streptococcus uberis (S. uberis) induced bovine mammary epithelial cells (MECs) samples by comparison with conventional commercial kit, which shows remarkably application potential in diseases diagnosis.

Nitrobenzoic acid-functionalized gold nanoparticles: DET promoter of multicopper oxidases and electrocatalyst for NAD-dependent glucose dehydrogenase

We report the synthesis of coated gold nanoparticles with nitrobenzoic moieties (AuNPs-TNB). They displayed small and narrow size distribution (2 nm) as shown by TEM imaging. These functionalized AuNPs-TNB were physically adsorbed on multi-walled carbon nanotubes (MWCNTs) electrodes. Their stable adsorption was monitored by electrochemical methods. The stability of the activated hydroxylamine layer was enhanced compared to the adsorption of the dinitro precursor onto the CNTs layer. Unactivated AuNPs-TNB enhanced the oxygen reduction reaction catalyzed by multicopper oxidases through increased aromatic and electrostatic interactions with limiting current densities higher than 500 µA cm−2. Next, the electrocatalytic properties of the active form toward the oxidation of the reduced form of nicotinamide adenine dinucleotide (NADH) with low overpotential was shown at near neutral pH. Associated with a NAD-dependent glucose dehydrogenase, the bioelectrodes were assessed for glucose electrooxidation. A complete glucose/O2 enzymatic fuel cell was developed based on unactived and activated AuNPs as cathodic DET promoter and anodic electrocatalyst respectively. The nanostructured assembly exhibited efficient glucose oxidation with current densities of 450 µA cm−2 in high glucose concentration and O2 saturated conditions. In concentration close to those found in physiological fluids (0.04 to 4.00 mmol L−1), the enzymatic biofuel cell displayed a linear range for glucose concentration and a low limit of detection (2.5 ± 0.9 µmol L−1).

NAD(P)H Drives the Ascorbate-Glutathione Cycle and Abundance of Catalase in Developing Beech Seeds Differently in Embryonic Axes and Cotyledons.

European beech is an important component of European lowland forests in terms of ecology, and produces irregular seeds categorized as intermediate due to their limited longevity. Removal of the excess of reactive oxygen species is crucial for redox homeostasis in growing plant tissues. Hydrogen peroxide (H2O2) is detoxified via the plant-specific ascorbate-glutathione cycle, and enzymatically, mainly by catalase (CAT). The reduced and oxidized (redox) forms of ascorbate (AsA, DHA) and glutathione (GSH, GSSG) decreased during maturation as the content of redox forms of nicotinamide adenine dinucleotide (NADH, NAD+) phosphate (NADPH, NADP+), cofactors of ascorbate–glutathione enzymes, declined and limited this cycle. The degree of oxidation of glutathione peaked at approximately 80%, at the exact time when the NADP content was the lowest and the NADPH/NADP+ ratio reached the highest values. The glutathione pool was reflected in changes in the NADP pool, both in embryonic axes (R2 = 0.61) and in cotyledons (R2 = 0.98). A large excess of NADPH was reported in embryonic axes, whereas cotyledons displayed more unified levels of NADP redox forms. As a result, anabolic redox charge and reducing power were higher in embryonic axes. CAT was recognized as two proteins, and the abundance of the 55 kDa protein was correlated with all redox forms of ascorbate, glutathione, NAD, and NADP, whereas the 37 kDa protein was oppositely regulated in embryonic axes and cotyledons. Here, we discuss the role of NAD(P) in the regulation of the ascorbate–glutathione cycle, catalase, and seed longevity concerning a putative role of NAD(P)H as a redox biomarker involved in predefining seed quality, because NAD(P)H-derived redox homeostasis was found to be better controlled in embryonic axes than cotyledons.

Nicotinamide effects on the metabolism of human fibroblasts and keratinocytes assessed by quantitative, label-free fluorescence imaging.

Alterations in metabolism are central to the aging process. Therefore, understanding the subcellular functional and structural changes associated with metabolic aging is critical. Current established methods for exploring cell metabolism either require the use of exogenous agents or are destructive to the tissue or cells. Two-photon excited fluorescence (TPEF) imaging has emerged as a method for monitoring subtle metabolic changes non-invasively. In this study, we use TPEF imaging to acquire high-resolution fluorescence images from two coenzymes, NAD(P)H (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), within human fibroblasts and keratinocytes in response to B3 (a nicotinamide precursor) supplementation and/or UV irradiation, without addition of exogenous labels. In addition, multi-parametric analysis methods are used to extract functional information of cellular metabolism, including cellular redox state, NAD(P)H fluorescence lifetime, and mitochondrial organization. Our results demonstrate that such optical metabolic assessments can serve as sensitive, label-free, non-destructive reporters of known effects of B3 to maintain and in some cases even enhance the respiratory function of mitochondria, while lowering oxidative damage. Thus, TPEF imaging, supported by highly-quantitative analysis, can serve as a tool to understand aging-dependent metabolic changes as well as the effect of actives on human epidermal and dermal cells.

NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence.

In many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD+ ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae, also known as group B streptococcus (GBS). We present structures of Rex bound to NAD+ and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD+ complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD+ stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD+ levels (or growth rate) rather than to the NADH/NAD+ ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host.

Effect of Dietary Coenzyme Q10 Plus NADH Supplementation on Fatigue Perception and Health-Related Quality of Life in Individuals with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Prospective, Randomized, Double-Blind, Placebo-Controlled Trial.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, multisystem, and profoundly debilitating neuroimmune disease, probably of post-viral multifactorial etiology. Unfortunately, no accurate diagnostic or laboratory tests have been established, nor are any universally effective approved drugs currently available for its treatment. This study aimed to examine whether oral coenzyme Q10 and NADH (reduced form of nicotinamide adenine dinucleotide) co-supplementation could improve perceived fatigue, unrefreshing sleep, and health-related quality of life in ME/CFS patients. A 12-week prospective, randomized, double-blind, placebo-controlled trial was conducted in 207 patients with ME/CFS, who were randomly allocated to one of two groups to receive either 200 mg of CoQ10 and 20 mg of NADH (n = 104) or matching placebo (n = 103) once daily. Endpoints were simultaneously evaluated at baseline, and then reassessed at 4- and 8-week treatment visits and four weeks after treatment cessation, using validated patient-reported outcome measures. A significant reduction in cognitive fatigue perception and overall FIS-40 score (p < 0.001 and p = 0.022, respectively) and an improvement in HRQoL (health-related quality of life (SF-36)) (p < 0.05) from baseline were observed within the experimental group over time. Statistically significant differences were also shown for sleep duration at 4 weeks and habitual sleep efficiency at 8 weeks in follow-up visits from baseline within the experimental group (p = 0.018 and p = 0.038, respectively). Overall, these findings support the use of CoQ10 plus NADH supplementation as a potentially safe therapeutic option for reducing perceived cognitive fatigue and improving the health-related quality of life in ME/CFS patients. Future interventions are needed to corroborate these clinical benefits and also explore the underlying pathomechanisms of CoQ10 and NADH administration in ME/CFS.

Attenuated SIRT1 Activity Leads to PER2 Cytoplasmic Localization and Dampens the Amplitude of Bmal1 Promoter-Driven Circadian Oscillation.

The circadian clock possesses robust systems to maintain the rhythm approximately 24 h, from cellular to organismal levels, whereas aging is known to be one of the risk factors linked to the alternation of circadian physiology and behavior. The amount of many metabolites in the cells/body is altered with the aging process, and the most prominent metabolite among them is the oxidized form of nicotinamide adenine dinucleotide (NAD+), which is associated with posttranslational modifications of acetylation and poly-ADP-ribosylation status of circadian clock proteins and decreases with aging. However, how low NAD+ condition in cells, which mimics aged or pathophysiological conditions, affects the circadian clock is largely unknown. Here, we show that low NAD+ in cultured cells promotes PER2 to be retained in the cytoplasm through the NAD+/SIRT1 axis, which leads to the attenuated amplitude of Bmal1 promoter-driven luciferase oscillation. We found that, among the core clock proteins, PER2 is mainly affected in its subcellular localization by NAD+ amount, and a higher cytoplasmic PER2 localization was observed under low NAD+ condition. We further found that NAD+-dependent deacetylase SIRT1 is the regulator of PER2 subcellular localization. Thus, we anticipate that the altered PER2 subcellular localization by low NAD+ is one of the complex changes that occurs in the aged circadian clock.

Non-invasive in vivo human model of post-ischaemic skin preconditioning by measurement of flow-mediated 460-nm autofluorescence.

Transient ischaemia and reperfusion (TIAR) induce early ischaemic preconditioning (IPC) in different tissues and organs, including the skin. IPC protects tissues by modifying the mitochondrial function and decreasing the amount of the reduced form of nicotinamide adenine dinucleotide (NADH). Skin 460-nm autofluorescence is proportional to the NADH content and can be non-invasively measured during TIAR. We propose a non-invasive in vivo human model of skin IPC for studying the effects of repeated TIARs on the NADH content. Fifty-one apparently healthy volunteers (36 women) underwent three 100-second forearm ischaemia episodes induced by inflation of brachial pressure cuff to the pressure of 60 mmHg above systolic blood pressure, followed by 500-second long reperfusion episodes. Changes in skin NADH content were measured using 460-nm fluorescence before and during each of the three TIARs. The first two TIARs caused a significant reduction in the skin NADH content before (P = .0065) and during the third ischaemia (P = .0011) and reperfusion (P = .0003) up to 3.0%. During the third TIAR, the increase in skin NADH was 20% lower than during the first ischaemia (P = .0474). The measurement of the 460-nm fluorescence during repeated TIARs allows for a non-invasive in vivo investigation of human skin IPC. Although IPC reduces the overall NADH skin content, the most noticeable NADH reduction appears during ischaemia after earlier TIARs. Studying the skin model of IPC may provide new avenues for in vivo physiological, clinical and pharmacological research on mitochondrial metabolism.

METOPROLOL COMPARED TO AMLODIPINE IMPAIRS MITOCHONDRIAL FUNCTION IN FOREARM SKIN IN HYPERTENSIVE PATIENTS

Objective: If oxygen is available, then mitochondria oxidase the reduced form of nicotinamide adenine dinucleotide (NADH) and produce energy. Oxygen delivery to mitochondria in skin cells depends on the function of microvascular circulation. Antihypertensive medications can modify both energy metabolism (e.g. gain of body weight) and microvascular function (cold fingers and toes). In this prospective study, we compared the effects of amlodipine vs metoprolol on the skin content of NADH in hypertensive patients. Design and method: Patients with newly diagnosed hypertension were randomly allocated to the six-week treatment with either 5 mg of amlodipine (20 patients) or 25 mg of metoprolol (17 patients) once a day. The 460-nm forearm skin fluorescence measured the skin mitochondrial content of NADH at rest, during transient 100-second ischaemia (by occlusion of the brachial artery with the inflated arm blood pressure cuff), and the following reperfusion. This measurement was made before and after the treatment initiation. Results: Hypotensive effects of both treatment were comparable after the six-week therapy. For NADH, amlodipine showed no significant change in the 460-nm fluorescence at rest (791.6 ± 281.1 to 733.7 ± 261.9 [kU] (p = 0.119), during ischemia (850.2 ± 292.2 to 805.6 ± 294.8 [kU] (p = 0.307), and reperfusion (662.3 ± 228.4 to 624.1 ± 225.0 [kU] (p = 0.210). In contrast, treatment with metoprolol significantly increased the 460-nm fluorescence at rest (678.2 ± 284.0 to 797.3 ± 247.0 [kU] (p &lt; 0.01), during ischemia (736.5 ± 302.1 to 859.7 ± 264.9 [kU] (p &lt; 0.01), and reperfusion (571.5 ± 243.2 to 662.2 ± 204.7 [kU] (p &lt; 0.01). Conclusions: Metoprolol significantly impairs the function of skin mitochondria, causing a significant increase in the NADH content. In contrast, amlodipine shows neutral effects on this function.

Single-Cell Oxidative Stress Events Revealed by a Renewable SERS Nanotip.

A nanotip sensitive to reactive oxygen species (ROS) and NAD+/NADH (oxidized/reduced forms of nicotinamide adenine dinucleotide) was designed and prepared to identify the redox events in a single living cell by surface-enhanced Raman scattering (SERS) spectroscopy. The nanotips were prepared by the one-step laser-induced Ag growth and deposition. A redox-reversible Raman reporter, 4-mercaptophenol (4-MP), was employed for the nanotip decoration along with the Ag deposition. 4-MP can be converted to SERS-inactive 4-mercaptocyclohexa-2,5-dienone (4-MC) by Fe3+ ions to complete signal rezeroing for multiple oxidative stress event loops. The SERS signal conversion from 4-MC to 4-MP provides a cue for the reduction process that is NADH-dependent. In contrast, by the conversion from 4-MP to 4-MC, the oxidative stress events and the signal transduction mechanism of cells stimulated by drugs (phorbol 12-myristate 13-acetate and H2O2) can be explored by SERS. This sensor is easy to fabricate and can be recycled. This tip-typed SERS nanosensor can be extendedly available for tracing other key markers in other NAD+/NADH-mediated respiratory chain and glycolysis, e.g., lactic acid, pyruvic acid, adenosine triphosphate, and antioxidants. It will be useful for investigating the diseases of abnormal oxidative stress and mitochondrial metabolism at the single-cell level.

ZnO Materials as Effective Anodes for the Photoelectrochemical Regeneration of Enzymatically Active NAD.

This work reports the study of ZnO-based anodes for the photoelectrochemical regeneration of the oxidized form of nicotinamide adenine dinucleotide (NAD+). The latter is the most important coenzyme for dehydrogenases. However, the high costs of NAD+ limit the use of such enzymes at the industrial level. The influence of the ZnO morphologies (flower-like, porous film, and nanowires), showing different surface area and crystallinity, was studied. The detection of diluted solutions (0.1 mM) of the reduced form of the coenzyme (NADH) was accomplished by the flower-like and the porous films, whereas concentrations greater than 20 mM were needed for the detection of NADH with nanowire-shaped ZnO-based electrodes. The photocatalytic activity of ZnO was reduced at increasing concentrations of NAD+ because part of the ultraviolet irradiation was absorbed by the coenzyme, reducing the photons available for the ZnO material. The higher electrochemical surface area of the flower-like film makes it suitable for the regeneration reaction. The illumination of the electrodes led to a significant increase on the NAD+ regeneration with respect to both the electrochemical oxidation in dark and the only photochemical reaction. The tests with formate dehydrogenase demonstrated that 94% of the regenerated NAD+ was enzymatically active.