Oxidized Nicotinamide Adenine Dinucleotide Research Articles

Electrochemical Oxidation of NADH at Highly Boron-Doped Diamond Electrodes

Conductive boron-doped chemical vapor-deposited diamond thin films, already known to have superior properties for general electroanalysis, including low background current and a wide potential window, are here shown to have additional advantages with respect to electrochemical oxidation of nicotinamide adenine dinucleotide (NADH), including high resistance to deactivation and insensitivity to dissolved oxygen. Cyclic voltammetry, amperometry, and the rotating disk electrode technique were used to study the reaction in neutral pH solution. Highly reproducible cyclic voltammograms for NADH oxidation were obtained at as-deposited diamond electrodes. The response was stable over several months of storage in ambient air, in contrast to glassy carbon electrodes, which deactivated within 1 h. The diamond electrode exhibited very high sensitivity for NADH, with an amperometric detection limit of 10 nM (S/N = 7). The response remained stable, even in the very low concentration range, for several months. In addition, interference effects due to ascorbic acid were minimal when the concentrations of NADH and ascorbic acid were comparable. An NADH-mediated dehydrogenese-based ethanol biosensor incorporating an unmodified diamond electrode is demonstrated. The present results indicate that diamond is a useful electrode material for the analytical detection of NADH, making it attractive for use in sensors based on enzyme-catalyzed reactions involving NADH as a cofactor.

Nitric oxide and its congeners in mitochondria: implications for apoptosis.

Apoptosis is an evolutionarily conserved form of physiologic cell death important for tissue development and homeostasis. The causes and execution mechanisms of apoptosis are not completely understood. Nitric oxide (NO) and its congeners, oxidative stress, Ca2+, proteases, nucleases, and mitochondria are considered mediators of apoptosis. Recent findings strongly suggest that mitochondria contain a factor or factors that upon release from the destabilized organelles, induce apoptosis. We have found that oxidative stress-induced release of Ca2+ from mitochondria followed by Ca2+ reuptake (Ca2+ cycling) causes destabilization of mitochondria and apoptosis. The protein product of the protooncogene bcl-2 protects mitochondria and thereby prevents apoptosis. We have also found that NO and its congeners can induce Ca2+ release from mitochondria. Thus, nitrogen monoxide (.NO) binds to cytochrome oxidase, blocks respiration, and thereby causes mitochondrial deenergization and Ca2+ release. Peroxynitrite (ONOO-), on the other hand, causes Ca2+ release from mitochondria by stimulating a specific Ca2+ release pathway. This pathway requires oxidized nicotinamide adenine dinucleotide (NAD+) hydrolysis to adenosine diphosphate ribose and nicotinamide. NAD+ hydrolysis is only possible when some vicinal thiols are cross-linked. ONOO- is able to oxidize them. Our findings suggest that NO and its congeners can induce apoptosis by destabilizing mitochondria via deenergization and/or by inducing a specific Ca2+ release followed by Ca2+ cycling.

Poly(ADP-ribose) synthetase activation mediates increased permeability induced by peroxynitrite in Caco-2BBe cells

Background & Aims: Peroxynitrite induces cytotoxicity by generating DNA single-strand breaks and activating poly(ADP-ribose) synthetase (PARS), a nuclear enzyme that consumes oxidized nicotinamide adenine dinucleotide (NAD +) and depletes cellular adenosine triphosphate (ATP). The aim of this study was to examine this mechanism of injury in an intestinal epithelial cell model after exposure to exogenous peroxynitrite (ONOO −) and nitric oxide (NO). Methods: Caco-2BBe cell monolayers exposed to donors of peroxynitrite (3-morpholino-sydnonimine [SIN-1], 3 mmol/L) or NO ( S-nitroso- N-acetyl penicillamine [SNAP]; 3 mmol/L) were analyzed for DNA strand breaks, [NAD +], [ATP], and transepithelial flux of fluorescein sulfonic acid. Results: SIN-1 but not SNAP induced DNA single-strand breakage. Both SIN-1 and SNAP reduced [ATP], but only SIN-1 reduced [NAD +]. Inhibition of PARS activity by the PARS inhibitors 5-iodo-6-amino 1,2- benzopyrone or 3-aminobenzamide prevented the SIN-1–induced reduction in [NAD +] and [ATP] but had no effect on the SNAP-induced reduction in [ATP]. PARS inhibition reduced SIN-1–but not SNAP-induced hyperpermeability. Conclusions: Peroxynitrite but not NO increases transepithelial permeability by inducing DNA strand breaks that activate the PARS pathway and cause the depletion of intracellular energy stores. Inhibition of PARS activity may represent a novel strategy in ameliorating peroxynitrite-mediated epithelial injury during intestinal inflammation. GASTROENTEROLOGY 1998;114:510-518

Kinetic isotope effect characterization of the transition state for oxidized nicotinamide adenine dinucleotide hydrolysis by pertussis toxin.

Pertussis toxin from Bordatella pertussis catalyzes the ADP ribosylation of several G-proteins, using NAD+ as a substrate. In the absence of an acceptor protein, the toxin acts as a NAD+ glycohydrolase. Pertussis toxin is one of the virulent factors for whooping cough and therefore a target for site-specific inhibitors based on the transition state structure. A family of kinetic isotope effects was determined for the hydrolysis reaction, using NAD+ labeled with 3H, 14C, and 15N as substrates. Primary isotope effects were 1.021 +/- 0.001 for [1'N-14C]NAD+ and 1.021 +/- 0.004 for [1N-15N]NAD+, and the double-primary effect of [1'N-14C,1N-15N]NAD+ was 1.049 +/- 0.004. Secondary kinetic isotope effects were 1.207 +/- 0.010 for the [1'N-3H]-, 1.144 +/- 0.005 for the [2'N-3H]-, 0.989 +/- 0.001 for the [4'N-3H]-, and 1.019 +/- 0.004 for the [5'N-3H]NAD+, respectively. Commitment to catalysis was excluded by isotope trapping experiments, and the experimental kinetic isotope effects were independent of pH. The measured isotope effects are therefore intrinsic. The isotope effects are remarkable because they indicate an oxocarbenium-like ribose ring at the transition state but a stiffer than expected vibrational environment for C1' at the reaction center. On the basis of these isotope effects, a bond order vibrational analysis was performed to locate a transition state structure consistent with the isotope effects. The kinetic isotope effects predict a residual bond order to the nicotinamide leaving group of 0.11, corresponding to a distance of 2.14 A. Participation of the water nucleophile is weak, consistent either with an S(N)1-like transition state with no water interaction or with the water oxygen no closer than 3.5 A from the reaction center. The positive charge of the ribose oxocarbenium is stabilized by delocalization between the C1'-O4' and C1'-C2' bonds. The enzyme contacts restrict the vibrational environment of the reaction coordinate requiring increased bonding force constants for the enzyme-stabilized transition state. NAD+ analogues with the nicotinamide ribose replaced by an iminoribitol ring, mimicking the flattened ribose ring of the transition state, are expected to be transition state inhibitors.

Transition State Analysis of NAD+Hydrolysis by the Cholera Toxin Catalytic Subunit

The transition state for NAD+ (oxidized nicotinamide adenine dinucleotide) hydrolysis by the cholera toxin A1 polypeptide (CTA) has been characterized by multiple V/K kinetic isotope effects (KIEs) using labeled NAD+ as the substrate. CTA causes cholera by catalyzing the ADP-ribosylation of the signal-transducing Gsα protein. In vitro, CTA catalyzes the ADP-ribosylation of several simple guanidino compounds as well as the slow hydrolysis of NAD+ (kcat = 8 min-1, Km = 14 mM) to form ADP−ribose and nicotinamide. KIEs for NAD+ hydrolysis are the following: primary 14C = 1.030 ± 0.005, primary 15N = 1.029 ± 0.004, α-secondary 3H = 1.186 ± 0.004, β-secondary 3H = 1.108 ± 0.004, γ-secondary 3H = 0.986 ± 0.003, δ-secondary 3H = 1.020 ± 0.003, and primary double = 1.052 ± 0.004. On the basis of steady-state kinetic parameters for CTA-catalyzed NAD+ hydrolysis, as well as a comparison with KIEs measured for NAD+ solvolysis, the enzymatic KIEs are near-intrinsic and describe a transition state that is relatively d...

Redox polymers for electrocatalytic oxidation of NADH – Cationic styrene and ethylenimine polymers

AbstractThe electrocatalytic oxidation of nicotinamide adenine dinucleotide (NADH) by two redox polymers was evaluated from steady‐state measurements at drop‐coated solid graphite electrodes in 0.25 M phosphate buffer, pH 7.0, at 0 mV (vs. SCE). The redox polymers studied comprised a styrene polymer incorporating positive charges through quaternary amines and Toluidine Blue 0 (TBO) moieties (ST) and a branched polyethylenimine with part of its primary amine groups loaded with TBO (PE). The catalytic efficiencyjudged from the maximum NADH sensitivity for the PE polymer was estimated to be 230 μa cm.−2 mM−1 at a coverage of 40 μgcm−2 and for the ST polymer 300 μAcm−2 cmM−1 at about 100 μg cm−2. Reagentless biosensors sensing D‐glucose and L‐malate were constructed from bulk modified carbon paste containing the PE redox polymer, NAD+, polyethylenimine (PEI) and D‐glucose dehydrogenase or L‐malate dehydrogenase. The D‐glucose and L‐malate sensors were characterized in a flow injection system by apparent j′max− and K′M‐values of 230 μA cm−2 and 62mM and 3.3 μAcm−2 and 1.7mM, respectively. It was shown for glucose dehydrogenase that the presence of PEI in the paste in addition to the PE polymer, drastically improved the enzymatic conversion of glucose to gluconic acid.

Protective effects of glucose-6-phosphate and NADP against α-chaconine-induced developmental toxicity in xenopus embryos

In previous studies a metabolic activation system (MAS) composed of Aroclor 1254-induced rat liver microsomes led to an apparent reduction of potato glycoalkaloid developmental toxicity in the frog embryo teratogenesis assay-Xenopus (FETAX). The reasons for this reduction were investigated in this study. The effect of the exogenous MAS on glycoalkaloid developmental toxicity was examined in two experiments in which a concentration series of α-chaconine was tested with a MAS with and without a reduced nicotinamide adenine dinucleotide (NADPH) generator system consisting of NADPH, oxidized nicotinamide adenine dinucleotide (NADP), glucose-6-phosphate (G6P) and glucose-6-phosphate dehydrogenase. The NADPH generator system and each of its individual components were tested at a single high concentration of α-chaconine to evaluate their potential effects on toxicity. The findings indicated that the protective effect of the MAS was not the result of detoxification by microsomal enzyme systems, but was caused by two components of the NADPH generator system, namely NADP and G6P. G6P was more protective of α-chaconine-induced toxicity than NADP at the concentrations tested. Thus, FETAX with a MAS must be performed with appropriate controls that take into account the possible interactions with individual components of the system.

Crystal structure of Pseudomonas mevalonii HMG-CoA reductase at 3.0 angstrom resolution.

The rate-limiting step in cholesterol biosynthesis in mammals is catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a four-electron oxidoreductase that converts HMG-CoA to mevalonate. The crystal structure of HMG-CoA reductase from Pseudomonas mevalonii was determined at 3.0 angstrom resolution by multiple isomorphous replacement. The structure reveals a tightly bound dimer that brings together at the subunit interface the conserved residues implicated in substrate binding and catalysis. These dimers are packed about a threefold crystallographic axis, forming a hexamer with 23 point group symmetry. Difference Fourier studies reveal the binding sites for the substrates HMG-CoA and reduced or oxidized nicotinamide adenine dinucleotide [NAD(H)] and demonstrate that the active sites are at the dimer interfaces. The HMG-CoA is bound by a domain with an unusual fold, consisting of a central alpha helix surrounded by a triangular set of walls of beta sheets and alpha helices. The NAD(H) is bound by a domain characterized by an antiparallel beta structure that defines a class of dinucleotide-binding domains.

Immobilization of alcohol dehydrogenase enzyme in a Langmuir-Blodgett film of stearic acid: its application as an ethanol sensor

A stable monolayer of alcohol dehydrogenase (ADH) enzyme has been prepared by spreading an aqueous solution of ADH on a water subphase containing stearic acid monolayer. This ADH-stearic acid monolayer has been successfully transferred onto a conducting polypyrrole-coated glass electrode by the Langmuir-Blodgett (LB) technique. ADH catalyses the reaction between the coenzyme ß-NAD + (β form of oxidized nicotinamide adenine dinucleotide) and ethanol to produce NADH and acetaldehyde. Conducting polypyrrole acts as a mediator for transferring electrons produced on NADH oxidation. This LB-immobilized polypyrrole-mediated enzyme electrode can be used as an ethanol sensor. It shows better sensitivity than sensors consisting of ADH immobilized by chemical cross-linking with glutaraldehyde. This LB sensor can be used for low ethanol concentration (in the micro-molar range).

The effects of aspartate and 2-oxoglutarate upon glycolytic energy metabolites and mechanical recovery following global ischaemia in isolated rat hearts

The hypothesis that aspartate and 2-oxoglutarate might help to relieve the inhibition of glycolysis during global myocardial ischaemia and improve post-ischaemic mechanical recovery was tested in isolated rat hearts. The hearts were attached to a lumped parameter model of the rat vascular impedance and initially perfused in the working mode with buffered Krebs-Henseleit solution containing 10.1 mmol/l glucose, with or without 3.3 mmol/l of aspartate and 3.3 mmol/l of 2-oxoglutarate, prior to imposing global ischaemia for 5, 10 or 15 mins. The addition of aspartate and 2-oxoglutarate improved the preservation of tissue CP after 5 mins of ischaemia and of ATP after 10 mins of ischaemia. The total adenine nucleotide pool was higher in the supplemented hearts after all three periods of ischaemia. Dihydroxyacetone phosphate, alanine, succinate and lactate accumulated during ischaemia, but the dihydroxyacetone phosphate accumulation was reduced while the alanine and succinate concentrations were increased by the addition of aspartate and 2-oxoglutarate to the perfusate. These observations lend support to the hypothesis that ischaemia arrests glycolysis at the glyceraldehyde phosphate dehydrogenase step due to a lack of oxidised nicotinamide adenine dinucleotide. Increasing the substrate concentrations of aspartate and 2-oxoglutarate may permit glycolysis to proceed for longer into the period of ischaemia by stimulating an additional pathway for nicotinamide adenine dinucleotide reoxidation. Small improvements in mechanical recovery were associated with the metabolic changes.

The inhibitory effect of extracts of cigarette tar on electron transport of mitochondria and submitochondrial particles

Acetonitrile extracts of cigarette tar inhibit state 3 and state 4 respiration of intact mitochondria. Exposure of respiring submitochondrial particles to acetonitrile extracts of cigarette tar results in a dose-dependent inhibition of oxygen consumption and reduced nicotinamide adenine dinucleotide (NADH) oxidation. This inhibition was not due to a solvent effect since acetonitrile alone did not alter oxygen consumption or NADH oxidation. Intact mitochondria are less sensitive to extracts of tar than submitochondrial particles. The NADH-ubiquinone (Q) reductase complex is more sensitive to inhibition by tar extract than the succinate-Q reductase and cytochrome complexes. Nicotine or catechol did not inhibit respiration of intact mitochondria. Treatment of submitochondrial particles with cigarette tar results in the formation of hydroxyl radicals, detected by electron spin resonance (ESR) spin trapping. The ESR signal attributable to the hydroxyl radical spin adduct requires the presence of NADH and is completely abolished by catalase and to a lesser extent superoxide dismutase (SOD). Catalase and SOD did not protect the mitochondrial respiratory chain from inhibition by tar extract, indicating that the radicals detected by ESR spin trapping are not responsible for the inhibition of the electron transport. We propose that tar causes at least two effects: (1) Tar components interact with the electron transport chain and inhibit electron flow, and (2) tar components interact with the electron transport chain, ultimately to form hydroxyl radicals.

Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Depression of force by phosphate in skinned skeletal muscle fibers of the frog

The relation between isometric force and phosphate concentration in skinned skeletal muscle fibers of the frog is found to depend on fiber size. Force decreased with increasing phosphate concentration, but depression of force in thick fibers was smaller than in thin segments. When the external phosphate concentration was abruptly altered during a sustained contracture, force changed. The half-time of the force change was proportional to the cross-sectional area of the preparation. From this relation, a value for the diffusion constant of phosphate in skinned fibers of 0.9 x 10(-10) m2/s was derived. The rate of phosphate production was determined photometrically via the enzymatic coupling of the resynthesis of ATP to the oxidation of nicotinamide adenine dinucleotide. The average value (+/- SE) of the rate of ATP hydrolysis (at 4 degrees C) was 2.7 +/- 0.3 mumol.s-1.g dry wt-1, which corresponds to 0.34 mmol.l-1.s-1. From a calculation based on the diffusion constant and the rate of phosphate production determined, it follows that the dependency of the force-phosphate relation on fiber diameter is due to phosphate accumulation inside the fiber.

Photosynthetic reaction centers in bacteria

No abstract available.

Enzymic method for the determination of formaldehyde

This paper describes a sensitive method for the determination of formaldehyde using two sequential reactions catalyzed by two enzymes, formaldehyde dehydrogenase and diaphorase. The principle of this method is based on the quantitative oxidation of formaldehyde, in the presence of formaldehyde dehydrogenase and oxidized nicotinamide adenine dinucleotide (NAD{sup +}), to produce formic acid and the reduced dinucleotide (NADH). The NADH produced then reacts with oxidized 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium (INT), in the presence of diaphorase, to produce formazan, a highly chromogenic compound. The formazan formed is measured spectrophotometrically at 500 nm and will be directly proportional to the amount of formaldehyde in the assay solution. As low as 0.05 {mu}g/mL (1.6 {times} 10{sup {minus}6} M) formaldehyde concentration can be determined, and the linear range was up to 3 {mu}g/mL (1.0 {times} 10{sup {minus}4} M). This enzymatic method is simple, sensitive, selective and can be used for the determination of formaldehyde in air, particularly in indoor environments.

Changes in Mean Concentration, Phase Shifts, and Dissipation in a Forced Oscillatory Reaction

Experiments are presented that confirm earlier predictions that the mode of supply of reactants to a nonlinear (bio)chemical reaction determines or controls concentrations at steady states far from equilibrium. The oxidation of nicotinamide adenine dinucleotide (NADH) catalyzed by the enzyme horseradish peroxidase with continuous input of oxygen was studied; NAD+ is continuously recycled to NADH through a glucose-6-phosphate dehydrogenase system. A comparison of steady-state concentrations is made with an oscillatory oxygen input and a constant input at the same average oxygen input for both modes. By varying the frequency and amplitude of the perturbation (O2 influx), the following may be changed: the average concentration of NADH; the Gibbs free energy difference delta G of the reactants and products at steady state; the average rate of the reaction; the phase relation between the oscillatory rate and delta G; and the dissipation. These results confirm the possibility of an "alternating current chemistry," of control and optimization of thermodynamic efficiency and dissipation by means of external variation of constraints in classes of nonlinear reactions and biological pumps, and of improvements of the yield in such reactions (heterogeneous catalysis, for example).

Inhibition of NAD glycohydrolase and ADP-ribosyl transferases by carbocyclic analogs of oxidized nicotinamide adenine dinucleotide

Analogues of oxidized nicotinamide adenine dinucleotide (NAD+) in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ have recently been synthesized [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183]. Carbocyclic NAD+ analogues have been shown to inhibit NAD glycohydrolases and ADP-ribosyl transferases such as cholera toxin A subunit. In this study, the diastereomeric mixture of dinucleotides was separated, and the inhibitory capacity of each of the purified diastereomers was defined. The NAD+ analogue in which the D-dihydroxycyclopentane is substituted for the D-ribose is designated carba-NAD and was demonstrated to be a poor inhibitor of the Bungarus fasciatus venom NAD glycohydrolase. The diastereomeric dinucleotide pseudo-carbocyclic-NAD (psi-carba-NAD), containing L-dihydroxycyclopentane in place of the D-ribose of NAD+, was shown, however, to be a potent competitive inhibitor of the venom NAD glycohydrolase with an inhibitor dissociation constant (Ki) of 35 microM. This was surprising since psi-carba-NAD contains the carbocyclic analogue of the unnatural L-ribotide and was therefore expected to be a biologically inactive diastereomer. psi-Carba-NAD also competitively inhibited the insoluble brain NAD glycohydrolase from cow (Ki = 6.7 microM) and sheep (Ki = 31 microM) enzyme against which carba-NAD is ineffective. Sensitivity to psi-carba-NAD was found to parallel sensitivity to inhibition by isonicotinic acid hydrazide, another NADase inhibitor. psi-Carba-NAD is neither a substrate for nor an inhibitor of alcohol dehydrogenase, whereas carba-NAD is an efficient dehydrogenase substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical evaluation of naphthalene intoxication in the tropical acrid blood clam Anadara granosa

Exposure of the tropical arcid blood clam Anadara granosa L., collected from Bombay waters in 1986, to sublethal concentrations of naphthalene (Nap) (5, 10 and 15μg Nap ml-1), induced various changes in vital biochemical systems. The condition index (CI) remained largely within the control range, although tissue water content after 96h exposure increased significantly (p<0.001). Glycogen levels appreciably decreased, both in muscles (p<0.001) and digestive glands (p<0.01). AMP activity remained unaltered irrespective of the ambient Nap concentration. ADP levels decreased by more than 20% and ATP levels also decreased significantly (p≪0.001) by more than 60% with increases in ambient Nap levels from 5 to 15 μg ml-1. Lysosomal marker enzymes-arylsulphatase and acid phosphatase-showed various changes. Levels of the former were 10 to 17 times higher than the latter. Total and free arylsulphatase activity in hepatic cells decreased on exposure of clams to 5 and 10μg Nap ml-1, but not significantly. However, it was significantly inhibited (p<0.001) towards the upper limits of exposure concentration. The same was also true of acid phosphatase activity. Nicotinamide adenine dinucleotide (NADH2) oxidation of lipochrome showed a dramatic decrease in activity in the range of 37 to 88% over controls with increasing Nap from 5 to 15 μg ml-1. Fatty acids also exhibited significant changes in composition with a decrease in polyunsaturated acids and an increase in certain saturated fatty acids. Protein and carbohydrate levels increased significantly (p<0.001) towards upper limits of exposure concentration. Pyruvic acid levels increased significantly by 2 to 6 times over controls on exposure to 5 to 15 μg Nap ml-1. Explanation of these changes is attempted in terms of the glycolysis pathway involving pyruvic acid oxidation and oxidative phosphorylation in the electron transport system.

Changes in Glutathione Peroxidase System and Pyridine Nucleotide Phosphate Levels in Kidneys of Cephaloridine-Administered Rats

To elucidate the nephrotoxic mechanisms of cephalondine (CER), changes in renal contents of glutathione (GSH), glutathione disulfide (GSSG), reduced and oxidized nicotinamide adenine dinucleotide phosphates (NADPH and NADP) and changes in renal activities of glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase were examined for 15 days in rats that received single intravenous injections of CER in doses of 0 (control), 100 and 1.000 mg/kg body weight. Significantly different changes from the control group were observed in the 1.000 mg/kg group. The 1.000 mg/kg group showed elevations in renal NADP and NADPH contents and decrements in renal GSH content in the period of the 1 st to 3rd hour after the CFR-administration. Thus, the fall in renal GSH content was considered to be a cause for renal injury due to the oxygen radicals observed in the early period. After the 6th hour, the 1.000 mg/kg group showed decreases of renal glutathione peroxidase and glutathione reductase activities and increases of renal glucose-6-phosphate dehydrogenase activity as well as GSH content. Although accumulation of GSH in the kidney was clearly observed in the late period, the more highly aggravated renal injury was speculated to be due to the decreased level in the utilization of GSH according to the fall of renal glutathione peroxidase activity.

Chemically induced parkinson's disease II: Intermediates in the oxidation and reduction reactions of the 1-methyl-4-phenyl-2,3-dihydropyridinium ion and its deprotonated form

The one-electron reduction product of 1-methyl-4-phenyl-2,3-dihydropyridinium ion has been generated by pulse radiolysis and its absorption spectrum recorded. This radical was found to decay by second-order kinetics (2k = 9.5 x 10(8) M-1 s-1) to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-2,3-dihydropyridinium ion. Reactions of the above radical species and that formed by one-electron reduction of 1-methyl-4-phenylpyridinium ion, which can also be generated by one-electron oxidation of 1-methyl-4-phenyl-1,2-dihydropyridine, with a number of molecules of biochemical interest have been studied. The one-electron reduction product of oxidised nicotinamide adenine dinucleotide efficiently reduced 1-methyl-4-phenyl-2,3-dihydropyridinium ion (k = 2.2 x 10(9) M-1 s-1). The relevance of these results in relation to redox cycling, a possible mechanism for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity, is discussed.