Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem‐mass spectrometry

Nicotinamide adenine dinucleotide (NAD) is a major co‐factor that mediates multiple biological processes including redox reaction and gene expression. Recently, NAD metabolism has received considerable attention because administration of NAD precursors exhibited beneficial effects against aging‐related metabolic disorders in animals. Although numerous studies have reported that NAD levels decline with aging in multiple animal tissues, the pathway and kinetics of NAD metabolism in aged organs are not completely understood. To determine the NAD metabolism upon aging, we developed targeted metabolomics based on an LC/MS/MS system. Our method is simple and applicable to crude biological samples, including culture cells and animal tissues. Unlike a conventional enzymatic cycling assay, our approach can determine NAD and NADH (reduced form of NAD) by performing a single sample preparation. Further, we validated our method using biological samples and investigated the alteration of the NAD metabolome during aging. Consistent with previous reports, the NAD levels in the liver and skeletal muscle decreased with aging. Further, we detected a significant increase in nicotinamide mononucleotide and nicotinamide riboside in the kidney upon aging. The LC/MS/MS‐based NAD metabolomics that we have developed is extensively applicable to biomedical studies, and the results will present innovative ideas for the aging studies, especially for that of NAD metabolism.     

Noniterative biexponential fluorescence lifetime imaging in the investigation of cellular metabolism by means of NAD(P)H autofluorescence.

The cofactors NADH and NADPH, hereafter NAD(P)H [NAD(P)= nicotinamide adenine dinucleotide (phosphate)], belong to the principal endogenous indicators of energetic cellular metabolism. Since the metabolic activity of cells is given by the ratio between the concentrations of free and protein-bound NAD(P)H, the development of autofluorescence techniques which accurately measure the modifications to this ratio is particularly significant. Hitherto the methods applied in the monitoring of cellular metabolism have provided either imprecise results, due to interference of the NAD(P)H signal by perturbing factors, or they have required a complicated internal calibration. We employ biexponential fluorescence lifetime imaging (FLIM) in order to discriminate between the free and protein-bound NAD(P)H without any previous calibration. Thus, we have obtained directly, and for the first time, a high-resolution map of cellular metabolism, that is, an image of the contribution of the protein-bound NAD(P)H to the cumulative NAD(P)H fluorescence signal. Moreover, we demonstrate that protein-NAD(P)H complexes characterised by different fluorescence lifetimes are not uniformly distributed all over the cell, as assumed until now, but are concentrated in certain cellular regions. The different fluorescence lifetimes indicate either different protein-NAD(P)H complexes or different bond strengths between NAD(P)H and the protein in these complexes. Since an important aspect in biological applications is to monitor the dynamics of the relevant processes (such as cellular metabolism), rapid dynamical techniques, for example, rapid biexponential fluorescence lifetime imaging, are needed. Furthermore, it is necessary to reduce the evaluation effort as much as possible. Most of the evaluation techniques in multiexponential FLIM are time-expensive iterative methods. The few exceptions are connected with a loss of information, for example, global analysis; or a loss in accuracy, for example, the rapid evaluation technique (RLD). We implement for the first time in FLIM a noniterative, nonrestrictive method originally developed by Prony for approximations of multiexponential decays. The accuracy of this method is verified in biexponential FLIM experiments in time-domain on mixtures of two chromophores both in homogenous and in heterogeneous media. The resulting fluorescence lifetimes agree (within error margins) with the lifetimes of the pure substances determined in monoexponential FLIM experiments. The rapidity of our evaluation method as compared to iterative pixel-by-pixel methods is evidenced by a reduction of the evaluation time by more than one order of magnitude. Furthermore, the applicability of this method for the biosciences is demonstrated in the investigation of cellular metabolism by means of NAD(P)H endogenous fluorescence.     

Pyridine metabolism and trigonelline synthesis in leaves of the mangrove legume trees Derris indica (Millettia pinnata) and Caesalpinia crista

The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 microg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.     

Ubiquinone‐Rhodol (UQ‐Rh) for Fluorescence Imaging of NAD(P)H through Intracellular Activation

The nicotinamide adenine dinucleotide (NAD) derivatives NADH and NADPH are critical components of cellular energy metabolism and operate as electron carriers. A novel fluorescent ubiquinone‐rhodol derivative (UQ‐Rh) was developed as a probe for NAD(P)H. By using the artificial promoter [(η⁵‐C₅Me₅)Ir(phen)(H₂O)]²⁺, intracellular activation and imaging of NAD(P)H were successfully demonstrated. In contrast to bioorthogonal chemistry, this “bioparallel chemistry” approach involves interactions with native biological processes and could potentially be used to control or investigate cellular systems.     

Optimizing the energy status of skin cells during solar radiation.

Ionizing- and ultraviolet-radiation cause cell damage or death by directly altering DNA and protein structures and by production of reactive oxygen species (ROS) and reactive carbonyl species (RCS). These processes disrupt cellular energy metabolism at multiple levels. The formation of DNA strand breaks activates signaling pathways that consume NAD, which can lead to the depletion of cellular ATP. Poly(ADP)-ribose polymerase (PARP-1) is the enzyme responsible for much of the NAD degradation following DNA damage, although numerous other PARPs have been discovered recently that await functional characterization. Studies on mouse epidermis in vivo and on human cells in culture have shown that UV-B radiation provokes the transient degradation of NAD and the synthesis of ADP-ribose polymers by PARP-1. This enzyme functions as a component of a DNA damage surveillance network in eukaryotic cells to determine the fate of cells following genotoxic stress. Additionally, the activation of PARP-1 results in the activation of a nuclear proteasome that degrades damaged nuclear proteins including histones. Identifying approaches to optimize these responses while maintaining the energy status of cells is likely to be very important in minimizing the deleterious effects of solar radiation on skin.     

Reduction in DNA synthesis during two-photon microscopy of intrinsic reduced nicotinamide adenine dinucleotide fluorescence

Two-photon laser scanning microscopy (TPLSM) of endogenous reduced nicotinamide adenine dinucleotide (NAD(P)H) provides important information regarding the cellular metabolic state. When imaging the punctate mitochondrial fluorescence originating from NAD(P)H in a rat basophilic leukemia (RBL) cell at low laser powers, no morphological changes are evident, and photobleaching is not observed when many images are taken. At higher powers, mitochondrial NAD(P)H fluorescence bleaches rapidly. To assess the limitations of this technique and to quantify the extent of photodamage, we have measured the effect of TPLSM on DNA synthesis. Although previous reports have indicated a threshold power for "safe" two-photon imaging, we find the laser power to be an insufficient indicator of photodamage. A more meaningful metric is a two-photon-absorbed dose that is proportional to the number of absorbed photon pairs. A temporary reduction of DNA synthesis in RBL cells occurs whenever a threshold dose of approximately 2 x 10(53) photon2 cm-4 s-1 is exceeded. This threshold is independent of laser intensity when imaging with average powers ranging from 5 to 17 mW at 740 nm. Beyond this threshold, the extent of the reduction is intensity dependent. DNA synthesis returns to control levels after a recovery period of several hours.     

Influence of Dietary Chitosan Feeding Duration on Glucose and Lipid Metabolism in a Diabetic Rat Model

This study was designed to investigate the influence of dietary chitosan feeding-duration on glucose and lipid metabolism in diabetic rats induced by streptozotocin and nicotinamide [a non-insulin-dependent diabetes mellitus (NIDDM) model]. Male Sprague-Dawley rats were used as experimental animals and divided into short-term (6 weeks) and long-term (11 weeks) feeding durations, and each duration contained five groups: (1) control, (2) control + 5% chitosan, (3) diabetes, (4) diabetes + 0.8 mg/kg rosiglitazone (a positive control), and (5) diabetes + 5% chitosan. Whether the chitosan feeding was for 6 or 11 weeks, the chitosan supplementation decreased blood glucose and lipids levels and liver lipid accumulation. However, chitosan supplementation decreased plasma tumor necrosis factor (TNF)-α, insulin levels, alanine aminotransferase (ALT) activity, insulin resistance (HOMA-IR), and adipose tissue lipoprotein lipase activity. Meanwhile, it increased plasma high-density lipoproteins (HDL)-cholesterol level, plasma angiopoietin-like-4 protein expression, and plasma triglyceride levels (at 11-week feeding duration only). Taken together, 11-week (long-term) chitosan feeding may help to ameliorate the glucose and lipid metabolism in a NIDDM diabetic rat model.     

Studies of inhibitor binding to the [4Fe-4S] cluster of quinolinate synthase

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de?novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in?vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster.     

Design, synthesis and biological characterization of novel inhibitors of CD38

Human CD38 is a novel multi-functional protein that acts not only as an antigen for B-lymphocyte activation, but also as an enzyme catalyzing the synthesis of a Ca(2+) messenger molecule, cyclic ADP-ribose, from NAD(+). It is well established that this novel Ca(2+) signaling enzyme is responsible for regulating a wide range of physiological functions. Based on the crystal structure of the CD38/NAD(+) complex, we synthesized a series of simplified N-substituted nicotinamide derivatives (Compound 1-14). A number of these compounds exhibited moderate inhibition of the NAD(+) utilizing activity of CD38, with Compound 4 showing the highest potency. The crystal structure of CD38/Compound 4 complex and computer simulation of Compound 7 docking to CD38 show a significant role of the nicotinamide moiety and the distal aromatic group of the compounds for substrate recognition by the active site of CD38. Biologically, we showed that both Compounds 4 and 7 effectively relaxed the agonist-induced contraction of muscle preparations from rats and guinea pigs. This study is a rational design of inhibitors for CD38 that exhibit important physiological effects, and can serve as a model for future drug development.     

Indirect electrochemical reduction of nicotinamide coenzymes

Nicotinamide coenzymes nicotinamide adenine dinucleotide (NAD(+)) and nicotinamide adenine dinucleotide phosphate (NADP(+)) were electrochemically reduced to NADH and NADPH, respectively. As direct reduction of nicotinamide coenzymes leads to inactive by-products, an indirect method using (pentamethylcyclopentadienyl-2,2'-bipyridine aqua) rhodium (III) as the mediator, was applied. A phosphate buffer solution, pH 8, with 1-10 mM NAD(P)(+) and 2.5-200 microM mediator, was pumped through a glassy carbon packed bed cathode. Virtually all the NAD(P)(+) was reduced to NAD(P)H in the cell. No sign of mediator loss due to side-reactions was detected though the mediator molecules shuttled hundreds of times between the oxidised and the reduced form. Adsorption of mediator molecules on the surface of the carbon cathode was found to be important for the reduction process. Due to strong adsorption, only minute amounts of mediator were consumed.     

Unusual enzymatic hydrolysis of NAD by solubilized form of NAD(+) glycohydrolase

Using solubilized form (sNADase) of membrane-bound porcine brain NAD(+) glycohydrolase (pNADase), the NADase-catalyzed hydrolysis and transglycosidation reactions of NAD (1) were examined. Unexpectedly, products in the reactions were found to be nicotinamide (5'-O-diphosphono)-beta-D-ribofuranoside (4) and adenosine (5). Adenosine 5'-diphosphate (ADP)-ribose (2) and nicotinamide (3) as well as a transglycosylated product, which are formed in a usual NAD/pNADase reaction system, were scarcely produced in the NAD/sNADase system. Setting aside the mechanical aspects of this unusual cleaving, it is quite interesting that the sNADase-catalyzed hydrolytic reaction of NAD resulted in the selective cleavage of the P-O bond of the adenosine side without the appreciable hydrolysis of the labile quaternary nicotinamide-ribose pyridinium linkage.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiae

A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH(3)CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV-Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures.     

Synthesis of Carba-NAD and the Structures of Its Ternary Complexes with SIRT3 and SIRT5

Carba-NAD is a synthetic compound identical to NAD except for one substitution, where an oxygen atom adjacent to the anomeric linkage bearing nicotinamide is replaced with a methylene group. Because it is inert in nicotinamide displacement reactions, carba-NAD is an unreactive substrate analogue for NAD-consuming enzymes. SIRT3 and SIRT5 are NAD-consuming enzymes that are potential therapeutic targets for the treatment of metabolic diseases and cancers. We report an improved carba-NAD synthesis, including a pyrophosphate coupling method that proceeds in approximately 60% yield. We also disclose the X-ray crystal structures of the ternary complexes of SIRT3 and SIRT5 bound to a peptide substrate and carba-NAD. These X-ray crystal structures provide critical snapshots of the mechanism by which human sirtuins function as protein deacylation catalysts.     

Mechanism of action of choleragen.

Choleragen exerts its effect on cells through activation of adenylate cyclase. Choleragen initially interacts with cells through binding of the B subunit of the toxin to the ganglioside GM1 on the cell surface. Subsequent events are less clear. Patching or capping of toxin on the cell surface may be an obligatory step in choleragen action. Studies in cell-free systems have demonstrated that activation of adenylate cyclase by choleragen requires NAD. In addition to NAD, requirements have been observed for ATP, GTP, and calcium-dependent regulatory protein. GTP also is required for the expression of choleragen-activated adenylate cyclase. In preparations from turkey erythrocytes, choleragen appears to inhibit an isoproterenol-stimulated GTPase. It has been postulated that by decreasing the activity of a specific GTPase, choleragen would stabilize a GTP-adenylate cyclase complex and maintain the cyclase in an activated state. Although the holotoxin is most effective in intact cells, with the A subunit having 1/20th of its activity and the B subunit (choleragenoid) being inactive, in cell-free systems the A subunit, specifically the A1 fragment, is required for adenylate cyclase activation. The B protomer is inactive. Choleragen, the A subunit, or A1 fragment under suitable conditions hydrolyzes NAD to ADP-ribose and nicotinamide (NAD glycohydrolase activity) and catalyzes the transfer of the ADP-ribose moiety of NAD to the guandino group of arginine (ADP-ribosyltransferase activity). The NAD glycohydrolase activity is similar to that exhibited by other NAD-dependent bacterial toxins (diphtheria toxin, Pseudomonas exotoxin A), which act by catalyzing the ADP-ribosylation of a specific acceptor protein. If the ADP-ribosylation of arginine is a model for the reaction catalyzed by choleragen in vivo, then arginine is presumably an analog of the amino acid which is ADP-ribosylated in the acceptor protein. It is postulated that choleragen exerts its effects on cells through the NAD-dependent ADP-ribosylation of an arginine or similar amino acid in either the cyclase itself or a regulatory protein of the cyclase system.     

1H and 13C NMR characterization of hemiamidal isoniazid-NAD(H) adducts as possible inhibitors of InhA reductase of Mycobacterium tuberculosis

Isoniazid (INH) is easily oxidized with manganese(III) pyrophosphate, a chemical model of the KatG protein involved in activation of INH inside the bacteria Mycobacterium tuberculosis. Performed in the presence of NAD(+), this oxidation generates a family of isomeric INH-NAD(H) adducts, which have been shown to be effective inhibitors of InhA, an enzyme essential in mycolic acid biosynthesis. In this work, we fully characterized by (1)H and (13)C NMR spectroscopy four main species of INH-NAD(H) adducts that coexist in solution. Two of them are open diastereoisomers consisting of the covalent attachment of the isonicotinoyl radical at position four of the nicotinamide coenzyme. The other two result from a cyclization involving the amide group from the nicotinamide and the carbonyl group from the isonicotinoyl radical to give diastereoisomeric hemiamidals. Although an INH-NAD(H) adduct with a 4S configuration has been characterized within the active site of InhA from Xray crystallography and this bound adduct interpreted as an open form (Rozwarski et al., Science 1998, 279, 98-102), it is legitimate to raise the question about the effective active form(s), open or cyclic, of INH-NAD(H) adduct(s). Is there a single active form or are several forms able to inhibit the InhA activity with different levels of inhibitory potency?     

脱氢酶电化学生物传感器的研究进展

自然界中超过400种脱氢酶使用辅酶-烟酰胺腺嘌呤二核苷酸(NAD+)或烟酰胺腺嘌呤二核苷酸磷酸(NADP+)作为生物催化反应中氢和电子的传递体,因此烟酰胺型辅酶的电化学氧化对构筑此类脱氢酶电化学生物传感器具有重要的意义.本文介绍了还原型辅酶在人工电子媒介体存在下的电化学氧化,以及脱氢酶电化学生物传感器的设计和应用.     

光催化NAD（P）H再生的研究进展

辅酶与酶催化反应紧密相关,是酶催化氧化还原反应过程中不可缺失的重要组成,其中,烟酰胺类辅酶NAD和NADP参与了大多数的酶催化氧化还原反应,是辅酶中最重要的一类。然而,辅酶的高成本限制了其实际应用。因此,烟酰胺辅酶的高效和低成本再生具有特别重要的意义。本文总结了还原型烟酰胺辅酶光催化再生方法的相关研究进展以及各种光敏剂的优缺点,提出了光催化NAD(P)H再生仍需要解决的问题。