Nicotinamide N-oxide Research Articles

Pharmacokinetics and biochemistry studies on nicotinamide in the mouse

Nicotinamide sensitizes murine tumours to the effect of radiation, but the pharmacokinetics are not well characterized at doses that are achievable in humans. In the mouse, nicotinamide given i.p. at doses of 100-500 mg/kg showed biphasic elimination with dose-dependent changes in half-life. The initial half-life increased significantly (P < 0.05) from 0.8 to 2 h and the terminal half-life increased from 3.4 to 5.6 h over the dose range studied. Clearance, however, decreased significantly from 0.3 to 0.24 l kg-1 h-1 only at the highest dose. Peak concentrations increased in a dose-dependent manner from 1,000 to 4,800 nmol/ml. The main plasma metabolite in the mouse is nicotinamide N-oxide, the peak concentration of which increased only from 80 to 160 nmol/ml. The N-oxide, which is also a weak radiosensitizer, is subject to reduction to the parent nicotinamide following administration at a dose of 276 mg/kg; peak concentrations of the N-oxide of 1900 nmol/ml were reached in 10 min, whereas concentrations of nicotinamide produced by reduction reached a maximum of 144 nmol/ml at 1 h. Elimination of the N-oxide was also biphasic, with initial and terminal half-lives being 0.39 and 1.8 h, respectively. The bioavailability of both drugs given via the i.p. as compared with the i.v. route was close to 100%. Tumour concentrations of nicotinamide paralleled those in the plasma after a short lag. Tumour nicotinamide adenine dinucleotide (NAD) concentrations were elevated by factors of 1.5 and 1.8 following doses of 100 and 500 mg/kg nicotinamide, respectively. Maximal concentrations were seen after 3-6 h, but levels remained elevated for 16 h. No change in tumour energy charge or in plasma 5-hydroxytryptamine was detected following a dose of 500 mg/kg nicotinamide.

Reduction of N-oxides and sulfoxide by the same terminal reductase inProteus mirabilis

Proteus mirabilis can grow anaerobically on the fermentable substrate, glucose. When the glucose medium was supplemented with an electron acceptor, growth doubled. However, the organism failed to grow anaerobically on the oxidizable substrate glycerol unless the medium was supplemented with an external electron acceptor. Dimethyl sulfoxide (DMSO), trimethylamine N-oxide (TMAO), nicotinamide N-oxide (NAMO), and nitrate (NO3) can serve this function. Cell-free extracts ofP. mirabilis can reduce these compounds in the presence of various electron donors. In order to determine whether the same or different terminal reductase(s) are involved in the reduction of these compounds, we isolated mutants unable to grow on glycerol/DMSO medium. When these mutants were tested on glycerol medium containing TMAO, NAMO, and NO3 as electron acceptors, it was found that there were two groups. Group I mutants were unable to grow with DMSO, TMAO, and NAMO, while their growth was unaffected with NO3. Group II mutants were unable to grow on any electron acceptor including NO3. Enzyme assays using reduced benzyl viologen with both groups of mutants were in agreement with growth studies. On the basis of these results, we conclude that the same terminal reductase is involved in the reduction of DMSO, TMAO, and NAMO (group I) and that the additional loss of NO3 reductase in group II mutants is probably owing to a defect in the synthesis or insertion of molybdenum cofactor.

Nutritional Efficiency of Nicotinamide N-Oxide and N'-Methylnicotinamide as Niacin in Rats.

The nutritional efficiency of nicotinamide N-oxide and N’-methylnicotinamide as substitutes for nicotinic acid was investigated using weanling rats fed with a nicotinic acid-free and tryptophan-limited diet (basal diet; negative control), the basal diet containing 0.003% nicotinic acid (positive control), or basal diet containing various amounts of nicotinamide N-oxide or N’-methylnicotinamide. The relative niacin activity of nicotinamide N-oxide and N’-methylnicotinamide to nicotinic acid was about 1/5 and 1/2 on a weight basis, respectively, judged from the following comparisons: body weight gain, food intake, food efficiency ratio, the NAD contents in liver and blood, the total nicotinamide content in liver, and the urinary excretion of nicotinamide and its metabolites such as N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide.

Study of the electrochemical reduction of nicotinamide N-oxide in aqueous solutions

Nicotinamide N 1-oxide was studied by dc and DP polarography and voltammetry from strongly acidic solutions (5 M H 2SO 4) to solutions of pH 12. Tafel slopes and reaction orders were obtained at potentials corresponding to the foot of the polarographic waves. The effect of pH, reactant concentration, proton donors and drop time on the polarographic and kinetic parameters is studied. The results indicate that the protonated form of the N-oxide group is reduced in acidic media, whereas the unprotonated form is reduced at pH > 9. In both cases the reduction product is nicotinamide. At pH < 9, the rate-determining step, rds, consists of the elimination of an OH − group, this reaction being placed between the two one-electron transfers. At pH > 9, the protonation of the species formed after the first one-electron transfer is the rds. In the latter case, the proton donors present in the solution are involved in the reaction.

Fates of N'-methylnicotinamide and nicotinamide N-oxide in mice.

This experiment was performed to investigate the possibility that N'-methylnicotinamide (N'-methyl-3-pyridinecarboxamide) and nicotinamide N-oxide have niacin activity or not in animals. When 20 mg N'-methylnicotinamide per mouse was administered, urinary excretion of nicotinamide, N1-methylnicotinamide (MNA), N1-methyl-2-pyridone-5-carboxamide (2-Py), and N1-methyMpyridone-3-carboxamide (4-Py) increased 24-, 3-, 3-, and 3-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were almost the same as those when 20 mg nicotinamide was administered. Therefore, the relative activity of N'-methylnicotinamide to nicotinamide as niacin was considered to be about 1. When 20 mg nicotinamide N-oxide per mouse was administered, urinary excretion of nicotinamide, MNA, 2-Py, and 4-Py increased 6.4-, 1.8-, 1.6-, and 1.7-fold, respectively, compared with the control values. The increased ratios of MNA, 2-Py, and 4-Py were about 1/2 of those when 20 mg nicotinamide was administered, so the relative activity of nicotinamide N-oxide to nicotinamide as niacin is considered to be about 1/2. In conclusion, it was found the possibility that the reactions N'-methylnicotinamide→nicotinamide and nicotinamide N-oxide→nicotinamide occur, at least in mice, and that therefore N'-methylnicotinamide and nicotinamide N-oxide have niacin activity.

Niacin catabolism in rodents.

Urinary excretion of nicotinamide and its metabolites in mouse, guinea pig, and hamster with a pharmacological amount of nicotinamide or N1-methylnicotinamide (MNA) was investigated to compare them with those of rat. In mouse, nicotinamide N-oxide was the most abundant metabolite, accounting for 35% of urinary excretion of nicotinamide and its metabolites, and followed by N1-methyl-2-pyridone-5-carboxamide (2-Pyr), 20%; nicotinamide, 18; MNA, 16%; and N1-methyl-4-pyridone-3-carboxamide (4-Pyr), 11%. In guinea pig, 2-Pyr was the most abundant metabolite, accounting for 80% of urinary excretion, and was followed by MNA, 11%; nicotinamide, 3%; 4-Pyr, 3%; and nicotinamide N-oxide, 3%. In hamster, nicotinamide was the most abundant metabolite, accounting for 44%, and followed by 2-Pyr, 21%; nicotinamide N-oxide, 15%; MNA, 10%; and 4-Pyr, 10%. Urinary excretion of nicotinic acid and nicotinuric acid was not detected in mouse, guinea pig, and hamster. When a pharmacological amount of nicotinamide was intraperitoneally injected into mouse, the excretion of nicotinamide N-oxide increased to 79.7% of the nicotinamide metabolites, while those of MNA (4.9%), 2-Pyr (11.2%), and 4-Pyr (6.3%) decreased. Nicotinic acid and nicotinuric acid were again not detected. When a pharmacological amount of nicotinamide was intraperitoneally injected into guinea pig and hamster, the deamidated metabolites nicotinic acid and nicotinuric acid occupied significant percentages of the nicotinamide metabolites: 50.4% and 26.3%, respectively, in guinea pig; and 7.7% and 79.5%, respectively, in hamster. The ratio of 2-Pyr to 4-Pyr excretion did not change when nicotinamide or MNA was located into mouse, guinea pig, and hamster. From these results, the catabolism of nicotinamide in rodent is discussed.

Oxygen-atom transfer catalyzed by an oxo-bridged molybdenum(V) compound

Mo 2 O 3 (dtc) 2 I 2 (THF) 2 can be used to reduce a variety of oxygen atom donating heterocyclic amine oxides and sulfoxides, including pyridine N-oxide, nicotinamide N-oxide, dimethyl sulfoxide, diphenyl sulfoxide, and biotin S-oxide, in either CH 2 Cl 2 or THF. In these reactions oxygen atom transfer results in the appropriate heterocyclic amine or sulfide and an MoO 2 2+ compound

High-performance liquid chromatographic measurement of nicotinamide N-oxide in urine after extracting with chloroform.

The measurement of nicotinamide N-oxide by high-performance liquid chromatography is described. The method used a Chemcosorb 5-ODS-H (150 × 4.5 mm i.d., particle size 5 jim) column eluted with 10 mM potassium dihydrogenphosphate (pH was adjusted to 3.0 by adding concentrated phosphoric acid) containing 0.1 g/l of sodium 1-octanesulfonate-methanol (100:4, v/v) at a flow-rate of l.Oml/min. The UV detector was set at 260nm. The detection limit for nicotinamide N-oxide was 2pmol (276 pg) at a signal-to-noise ratio of 5:1. Under these conditions, nicotinamide N-oxide was eluted at about 3.2 min. This technique was used to analyze rat urine following effective clean-up steps with chloroform, the total analysis time being about 13 min.

Purification of aldehyde oxidase from bovine ciliary body.

Ocular aldehyde oxidase was purified for the first time from bovine ciliary body cytosol by ammonium sulfate fractionation and successive HPLC using DEAE anion-exchange and hydroxyapatite columns. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be about 150,000 by electrophoresis and to be about 300,000 by gel filtration HPLC on a TSK gel G3000SWXL column, indicating that the enzyme consists of two subunits with the same molecular weight. On the other hand, nicotinamide N-oxide reductase activity was associated with aldehyde oxidase activity throughout the purification steps of the latter enzyme. This fact indicated that nicotinamide N-oxide reductase activity of the ciliary body cytosol is due to aldehyde oxidase present in the tissue preparation.

High-performance Liquid Chromatographic Measurement of Nicotinamide N-oxide in Urine after Extracting with Chloroform

High-performance Liquid Chromatographic Measurement of Nicotinamide N-oxide in Urine after Extracting with Chloroform

Metabolism of drugs in the eye. Drug-reducing activity of preparations from bovine ciliary body.

Drug-metabolizing activities, especially the reductase activities towards N-oxide, hydroxamic acid, sulfoxide and nitro compounds were comparatively examined with bovine ciliary body. As described previously, the cytosol from the ocular tissue exhibits the nicotinamide N-oxide reductase activity when supplemented with 2-hydroxypyrimidine, an electron donor of aldehyde oxidase. When the cytosol was fractionated with ammonium sulfate, followed by assays of aldehyde oxidase and nicotinamide N-oxide reductase activities in each fraction, the distribution of aldehyde oxidase activity in the resultant ammonium sulfate fractions was nearly parallel to that of nicotinamide N-oxide reductase activity. Furthermore, reductase activities towards drugs such as sulfoxide, hydroxamic acid and nitro compounds were observed with the cytosol in the presence of 2-hydroxypyrimidine or N1-methylnicotinamide. In general, these reductase activities of the fraction were markedly inhibited by menadione, an inhibitor of aldehyde oxidase. These results suggest that aldehyde oxidase present in ciliary body plays an important role in the reduction of a variety of xenobiotics in mammalian eyes. However, in the case of imipramine N-oxide, its reduction in the ocular tissue appears to be more readily catalyzed by a menadione-linked enzyme different from aldehyde oxidase.

NAD (P) H-dependent reduction of nicotinamide N-oxide by an unique enzyme system consisting of liver microsomal NADPH-cytochrome c reductase and cytosolic aldehyde oxidase

NAD(P) H-dependent reduction of nicotinamide N-oxide was investigated with rabbit liver preparations. Microsomes, microsomal NADPH-cytochrome c reductase or cytosolic aldehyde oxidase alone exhibited no nicotinamide N-oxide reductase activity in the presence of NADPH or NADH. However, when the microsomal preparations were combined with the cytosolic enzyme, a significant N-oxide reductase activity was observed in the presence of the reduced pyridine nucleotide. The activity was enhanced by FAD or methyl viologen. Cytosol alone supplemented with NADPH or NADH exhibited only a slight, but when combined with microsomes, a significant N-oxide reductase activity. Based on these facts, we propose a new electron transfer system consisting of NADPH-cytochrome c reductase and aldehyde oxidase, which exhibits nicotinamide N-oxide reductase activity in the presence of the reduced pyridine nucleotide.

Involvement of liver aldehyde oxidase in the reduction of nicotinamide N-oxide

The present paper describes that mammalian liver aldehyde oxidase is involved in the reduction of nicotinamide N-oxide to nicotinamide. Rabbit liver aldehyde oxidase supplemented with its electron donor exhibited a significant nicotinamide N-oxide reductase activity under anaerobic conditions. Liver cytosols from rabbits, hogs, guinea pigs, hamsters, rats and mice, all of them, similarly exhibited the N-oxide reductase activity in the presence of an electron donor of aldehyde oxidase, but not xanthine oxidase. The cytosolic N-oxide reductase activity was almost completely inhibited by menadione, an inhibitor of aldehyde oxidase.

Nicotinamide N-oxide formation by rat liver microsomes

Nicotinamide N-oxide formation by rat liver microsomes

Determination of nicotinamide N-oxide—a human excretory product

A method for the determination of nicotinamide N-oxide has been developed. It is based on the ability of the N-oxide to function as an electron acceptor in the xanthine oxidase catalyzed oxidation of xanthine. In simple mixtures the N-oxide can be converted quantitatively to nicotinamide and the latter determined by the cyanogen bromide method. The conversion is not always quantitative in complex mixtures, such as urine; an isotope dilution variation on the basic method permits the determination of the N-oxide in such situations. The basic method is applicable over the range 0.02–0.3 μmole of nicotinamide N-oxide. The new method has been used to verify the prominent excretory role of nicotinamide N-oxide in rodents. Application of the method to a study of human urines has permitted the detection of the N-oxide as an excretory metabolite in man. Only vanishingly small quantities of the N-oxide are excreted under normal conditions. However after the ingestion of 200 mg of nicotinamide, significant quantities of the N-oxide are detectable in human urine. Urine samples obtained from a number of other mammalian species contained little or no detectable nicotinamide N-oxide.

Catalysis of the Direct Transfer of Oxygen from Nicotinamide N-Oxide to Xanthine by Xanthine Oxidase

The role of the oxidizing agent in the xanthine oxidase reaction has been evaluated. The long standing notion that oxygen normally functions only as an electron acceptor in the xanthine oxidase reaction has been confirmed. When molecular oxygen was the oxidizing agent and the reaction was carried out at pH 7.5, 18O was incorporated into uric acid from 18O-labeled water; no 18O was found in uric acid when xanthine was oxidized in an atmosphere of 18O2. However, at pH 8.9 there was a small amount of direct oxygen transfer from 18O2 into uric acid. In experiments in which nicotinamide N-oxide was used in place of O2, the N-oxide appeared to function to a large extent by direct transfer of oxygen. It acted as an electron acceptor as well. Xanthine oxidase catalyzed the transfer of 18O from nicotinamide 18ON-oxide to xanthine in the course of the formation of uric acid. 18O from 18O-labeled water was also incorporated into uric acid when nicotinamide N-oxide was used as the oxidant in the xanthine oxidase reaction. Essentially identical results were obtained with both milk and liver xanthine oxidases. The possibility was raised that heterocyclic N-oxides may be capable of acting as general biological oxygenating agents.

The Reduction of Nicotinamide N-Oxide by Xanthine Oxidase

The following evidence has led to the conclusion that xanthine oxidase, with its usual electron donors, is capable of reducing nicotinamide N-oxide to nicotinamide. 1. The purest enzyme fraction from hog liver which catalyzes the reduction has the spectrum of a metalloflavoprotein and has xanthine oxidase activity. 2. The enzyme responsible for the reduction cofractionates with hog liver xanthine oxidase. 3. Milk xanthine oxidase also catalyzes the reduction of nicotinamide N-oxide. 4. The electron donors for the reduction are known substrates of xanthine oxidase. 5. Both milk and liver xanthine oxidases exhibit separate pH optima for the reductions dependent on reduced diphosphopyridine nucleotide and xanthine. 6. Xanthine oxidase activity and nicotinamide N-oxide reduction activity are destroyed by heat at the same rate. 7. Each activity decreases to the same extent in the livers of rats maintained on protein-deficient diets. 8. Xanthine oxidase activity and xanthine-dependent nicotinamide N-oxide reduction exhibit parallel inhibition by cyanide. It is proposed that xanthine oxidase might be responsible for other N-oxide reductions.

The Enzymatic Reduction of Nicotinamide N-Oxide

Abstract An enzymatic system which reduces nicotinamide N-oxide to nicotinamide was isolated from hog liver and purified about 280-fold. The reduction is dependent on the presence of reduced diphosphopyridine nucleotide or a low molecular weight constituent found in boiled liver supernatant. The two cofactors appear to be involved in the expression of two distinct enzymatic activities. Evidence has been presented which indicates both activities may be associated with a single protein or enzymatic unit. The DPNH-dependent reaction is the following. H+ + DPNH + nicotinamide N-oxide → DPN+ + nicotinamide + H2O The nature of the supernatant-dependent reaction is not completely understood, since the identity of the compound which is oxidized in the course of reduction of the N-oxide is not known.

Spectral and biological changes induced in nicotinic acid and related compounds by ultraviolet light.

1. Irradiation of nicotinic acid, nicotinamide, nicotinamide N-oxide, N'-methyl-4-pyridone-3-carboxamide, reduced nicotinamide-adenine dinucleotide and pyridine with ultraviolet light at 253.7mmu leads to striking spectral changes. 2. Nicotinic acid and nicotinamide are broken down to photosensitive intermediates which in turn undergo photodecomposition. 3. A major photoproduct of [7-(14)C]nicotinic acid is radioactive and absorbs ultraviolet light, but is inactive as a growth factor for Candida pseudotropicalis. 4. Irradiation of nicotinamide gives rise to small quantities of a biologically active photoproduct having the same R(F) as nicotinic acid. A second photoproduct is also formed, but its identity has not yet been established. 5. Irradiation of nicotinamide N-oxide leads to the formation of several photoproducts, one of which has the same R(F) as nicotinamide, absorbs ultraviolet light, and is biologically active. 6. Evidence is presented that irradiation of ethanolic solutions of N'-methyl-4-pyridone-3-carboxamide gives rise to acetaldehyde. 7. Irradiation of reduced nicotinamide-adenine dinucleotide in the presence of acetaldehyde leads to the formation of oxidized nicotinamide-adenine dinucleotide, which in turn can break down to nucleotide and/or nucleoside (depending on the conditions of the reaction). 8. The quantum yields of photolysis and the molar photosensitivities have been determined for N'-methyl-4-pyridone-3-carboxamide and nicotinamide N-oxide. 9. The possible biological significance of these photoreactions is discussed in relation to photosynthesis, visual-pigment metabolism and ultraviolet-light-induced cell damage. 10. A four-step theory is presented for the biochemical evolution of oxidation-reduction systems, involving photoactivated transformations of pyridine derivatives.