The Urinary Excretory Ratio of Nicotinamide Catabolites Was Associated with the Conversion Ratio of Tryptophan to Nicotinamide in Growing Rats Fed a Niacin-Free 20% Casein Diet

Weaning rats were fed a niacin-free 20% casein diet. Twenty-four-h-urine samples were collected, and nicotinamide and its catabolites were measured. A correlation was found between the urinary excretory ratio of nicotinamide catabolites (N ¹-methyl-2-pyridone-5-carboxamide + N ¹-methyl-4-pyridone-3-carboxamide)/N ¹-methylnicotinamide and the tryptophan-nicotinamide conversion ratio during growing period of the rats. This indicates the possibility that the conversion ratio can be deduced from the excretory ratio.     

Simultaneous measurement of nicotinamide and its catabolites,nicotinamide N-oxide,N1-methyl-2-pyridone-5-carboxamide,and N1-methyl-4-pyridone-3-carboxamide,in mice urine

Nicotinamide N-oxide is a major nicotinamide catabolite in mice but not in humans and rats. A high-performance liquid chromatographic method for the simultaneous measurement of nicotinamide, nicotinamide N-oxide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone-3-carboxamide in mice urine was developed by modifying the mobile phase of a reported method for measurement of nicotinamide N-oxide.     

Effects of fatty liver induced by niacin-free diet with orotic acid on the metabolism of tryptophan to niacin in rats

The effects of dietary orotic acid on the metabolism of tryptophan to niacin in weaning rats was investigated. The rats were fed with a niacin-free, 20% casein diet containing 0% (control diet) or 1% orotic acid diet (test diet) for 29 d. Retardation of growth, development of fatty liver, and enlargement of liver were observed in the test group in comparison with the control group. The concentrations of NAD and NADP in liver significantly decreased, while these in blood did not decrease compared to the control group. The formation of the upper metabolites of tryptophan to niacin such as anthranilic acid, kynurenic acid, and 3-hydroxyanthranilic acid were not affected, but the quinolinic acid and beyond, such as nicotinamide, N1-methylnicotinamide, N1-methyl-2-pyridone-5-carboxamide, and N1-methyl-4-pyridone-3-carboxamide, were significantly reduced by the administration of orotic acid. Therefore, the conversion ratio of tryptophan to niacin significantly decreased in the test group in comparison with the control group.     

Effects of Vitamin B6 Deficiency on the Conversion Ratio of Tryptophan to Niacin

To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone3- carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.     

Effect of Adding Methionine and Threonine to a Protein-free Diet on the Metabolism of Nicotinamide

We have been attempting to confirm the hypothesis that the excretion ratio of nicotinamide metabolites, [N¹-methyl-2-pyridone-5-carboxamide (2-Py) + N¹-methyl-4-pyridone-3-carboxamide (4-Py)]/N¹-methylnicotinamide (MNA), reflects the adequacy of amino acid nutrition, but not of niacin nutrition. It is known that methionine and threonine supplementation to a protein-free diet reduced body weight loss. In this paper, we investigated whether rats fed with a protein free-diet supplemented with methionine and threonine would result in this excretion ratio being increased or not. The body weight loss was markedly reduced by the supplementation with both amino acids, as has been reported, and under the conditions, the excretion ratio significantly increased. The activity of 4-Py-forming MNA oxidase, which controls the change in excretion ratio, also increased significantly. From the present results and our previous results, it was proved that the excretion ratio of nicotinamide metabolites, (2-Py +4-Py)/MNA, reflects the adequacy of amino acid nutrition, but not of niacin nutrition.     

Effects of Peroxidation Products of Linoleic Acid on Tryptophan–nicotinamide Metabolism in Rats

The flavin and pyridine nucleotide coenzymes are involved in the detoxication of autoxidation products of lipids. In tryptophan-nicotinamide metabolism, kynurenine 3-hydroxylase and N¹-methylnicotinamide (MNA) oxidase contain FAD as a coenzyme. So, the effects of dietary autoxidation products of linoleic acid on the metabolism of tryptophan-nicotinamide were investigated using rats. The administration of linoleic acid hydroperoxides or secondary products reduced the urinary excretion of xanthurenic acid, nicotinamide and its metabolites such as MNA, N¹-methyl-2-pyridone-5-carboxamide (2-Py), and N¹-methyl-4-pyridone-3-carboxamide (4-Py) as compared with the group administered saline or linoleic acid. Among the enzyme activities involved in the tryptophan-nicotinamide metabolism, the activity of NAD⁺ synthetase was decreased by the administration of linoleic acid hydroperoxides or secondary products. The activities of tryptophan oxygenase and 4-Py-forming MNA oxidase were also decreased by the administration of secondary products. These results indicate that the conversion of tryptophan to nicotinamide would be lower in the groups administered the hydroperoxides and secondary products than in saline and linoleic acid groups.     

Evidence for an Ester Linkage between Lignin and Glucuronic Acid in Lignin–Carbohydrate Complexes by DDQ-Oxidation

The effects of ethanol feeding on the tryptophan-niacin metabolism were investigated in rats. Male rats of the Wistar strain (3 weeks old) were fed with a 20% casein diet and 15% ethanol ad libitum for 56 days. Urine samples were collected every week during the experimental period. Urinary excretion of N¹-methylnicotinamide (MNA) was always higher in the ethanol-fed group than in the control group, but urinary excretion of its oxidized metabolites N¹-methyl-2-pyridone-5-carboxamide (2-Py) and N¹-methyl-4-pyridone-3-carboxamide (4-Py) was always lower. Therefore, the ratio of (2-Py + 4-Py)/MNA excretion was lower in the ethanol-fed group than in the control group. The rats were killed on day 57 and liver enzyme activities involved in the tryptophan-niacin metabolism were also measured. Tryptophan oxygenase, kynureninase, nicotinamide mononucleotide adenylyltransferase, NAD⁺ synthetase, and nicotinamide methyltransferase activities were similar in both groups, but, 2-Py-forming MNA oxidase and 4-Py-forming MNA oxidase activities in the ethanol-fed group were 43% and 18% of the control, respectively. Therefore, the increase in MNA excretion and the decrease in the ratio of (2-Py + 4-Py)/MNA excretion might be attributed to the inhibition of the two MNA oxidase activities by ethanol feeding. Furthermore, it happened to be found that this excretion ratio also increased with growth up to nine weeks and this change was attributed to the increased reaction MNA → 4-Py with growth.     

Identification of Arsenobetaine,a Water Soluble Organo-arsenic Compound in Muscle and Liver of a Shark,Prionace glaucus

After male rats of the Sprague Dawley strain, 5 weeks old, were fed a 20% casein diet for 12 days, 70 mg of streptozotocin/kg body weight (STZ group) or 70 mg of streptozotocin and 500 mg of nicotinamide/kg body weight (STZ-Nam group) was injected intraperitoneally into the rats. The rats were kept for 21 more days on the 20% casein diet and killed by decapitation. Urine was collected for the last 2 days. The level of blood glucose was 2-fold higher in the STZ group than in the STZ-Nam group. Urinary excretion of large amounts of glucose was observed only in the STZ group. Extremely reduction of urinary excretion of nicotinamide was observed in the STZ group, but, urinary excretion of N¹-methylnicotinamide (MNA) and N-¹-methyl-2-pyridone-5-carboxamide (2-py) was about the same in the two groups and that of N¹-methyl-4-pyridone-3-carboxamide (4-py) was higher in the STZ group than in the STZ-Nam group. The sum of urinary excretion of nicotinamide, MNA, 2-py, and 4-py was higher in the STZ group than in the STZ-Nam group. The levels of NAD in liver, pancreas, and blood in the STZ group tended to be higher, or rather not to decrease compared to the STZ-Nam group. For enzyme activities concerned with the tryptophan-NAD metabolism, a marked increase was observed in the activities of aminocarboxymuconate-semialdehyde decarboxylase, 3-hydroxyanthranilic acid oxygenase, and nicotinamide methyltransferase, on the other hand, the activity of NAD⁺ synthetase decreased in the STZ group compared to the STZ-Nam group. The activities of tryptophan oxygenase, kynureninase, NMN adenylyltransferase, and MNA oxidase were about the same in the two groups. These changes in the above enzyme activities mean that the conversion ratio from tryptophan to NAD is lower in the streptozotocin diabetic rats than normal rats, but the tryptophan metabolites such as NAD and 4-py were higher in the STZ group than in the STZ-Nam group. This might be due to the higher food intake and the lower body weight gain in the STZ group than in the STZ-Nam group. Similar phenomena have reported in alloxan diabetic rats.     

Urine metabolomics reveals novel physiologic functions of human aldehyde oxidase and provides biomarkers for typing xanthinuria

Classical xanthinuria is a rare inherited metabolic disorder caused by either isolated xanthine dehydrogenase (XDH) deficiency (type I) or combined XDH and aldehyde oxidase (AO) deficiency (type II). XDH and AO are evolutionary related enzymes that share a sulfurated molybdopterin cofactor. While the role of XDH in purine metabolism is well established, the physiologic functions of AO are mostly unknown. XDH and AO are important drug metabolizing enzymes. Urine metabolomic analysis by high pressure liquid chromatography and mass spectrometry of xanthinuric patients was performed to unveil physiologic functions of XDH and AO and provide biomarkers for typing xanthinuria. Novel endogenous products of AO, hydantoin propionic acid, N1-methyl-8-oxoguanine and N-(3-acetamidopropyl) pyrrolidin-2-one formed in the histidine, nucleic acid and spermidine metabolic pathways, respectively, were identified as being lowered in type II xanthinuria. Also lowered were the known AO products, N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-5-carboxamide in the nicotinamide degradation pathway. In contrast to the KEGG annotations, the results suggest minor role of human AO in the conversion of pyridoxal to pyridoxate and gentisaldehyde to gentisate in the vitamin B6 and tyrosine metabolic pathways, respectively. The perturbations in purine degradation due to XDH deficiency radiated further from the previously known metabolites, uric acid, xanthine and hypoxanthine to guanine, methyl guanine, xanthosine and inosine. Possible pathophysiological implications of the observed metabolic perturbations are discussed. The identified biomarkers have the potential to replace the allopurinol-loading test used in the past to type xanthinuria, thus facilitating appropriate pharmacogenetic counseling and gene directed search for causative mutations.     

Changes in Nicotinamide Metabolism by One Amino Acid Deficiency. (I) Threonine-, Tryptophan-, Aspartic Acid-, Lysine-, Leucine-, or Methionine-free Diet

Effects of a threonine-, tryptophan-, aspartic acid-, lysine-, leucine-, or methionine-free diet fed to rats on the metabolism of nicotinamide were investigated. The body weights of rats and food intakes were greatly decreased by feeding of the diet excluding any of the above essential amino acids compared to the control group, however, not by feeding of an aspartic acid-free diet. The sum of the urinary excretion of nicotinamide, N¹-methylnicotinamide (MNA), N¹ -methyl-2-pyridone-5-carboxamide (2-Py), and N¹ -methyl-4-pyridone-3-carboxamide (4-Py) changed roughly in proportion to food intake. In the groups fed with the threonine- and lysine-free diets, the urinary excretion of MNA greatly increased compared with the control group during the whole experimental period and in the groups fed with the leucine- and methionine-free diets, increased excretion of MNA was observed on day o–day 1. Whenever the increase in MNA excretion was observed, a decrease in 4-Py excretion was observed. This was attributed to the activity of 4-Py-forming MNA oxidase decreasing when rats were fed with the diet excluding one of the essential amino acid except for tryptophan. Therefore, the (2-Py +4-Py)/MNA excretion was greatly decreased by feeding of the diet excluding one of the essential amino acids except for the tryptophan-free diet. These results strengthened our hypothesis that the (2-Py +4-Py)/MNA excretion reflects the adequacy of amino acid nutrition.     

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-methyl-4-pyridone-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.     

Effects of dietary pyrazinamide, an antituberculosis agent, on the metabolism of tryptophan to niacin and of tryptophan to serotonin in rats

The effects of pyrazinamide on the metabolism of tryptophan to niacin and of tryptophan to serotonin were investigated to elucidate the mechanism for pyrazinamide action against tuberculosis. Weanling rats were fed with a diet with or without 0.25% pyrazinamide for 61 days. Urine samples were periodically collected for measuring the tryptophan metabolites. The administration of pyrazinamide significantly increased the metabolites, 3-hydroxyanthranilic acid and beyond, especially quinolinic acid, nicotinamide, N'-methylnicotinamide, and N1-methyl-4-pyridone-3-carboxamide, and therefore significantly increased the conversion ratio of tryptophan to niacin and the blood NAD level . However, no difference in the upper metabolites of the tryptophan to niacin pathway such as anthranilic acid, kynurenic acid and xanthurenic acid was apparent between the two groups. No difference in the concentrations of trytptophan and serotonin in the blood were apparent either. It is suggested from these results that the action of pyrazinamide against tuberculosis is linked to the increase in turnover of NAD and to the increased content of NAD in the host cells.     

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.     

Effects of 1-methylnicotinamide and its metabolite N-methyl-2-pyridone-5-carboxamide on streptozotocin-induced toxicity in murine insulinoma MIN6 cell line

1-methylnicotinamide (MNA) is a primary metabolite of nicotinamide. In recent years several activities of MNA have been described, such as anti-inflammatory activity in skin diseases, induction of prostacyclin synthesis via COX-2, aortal endothelium protection in diabetes and hypertriglyceridaemia and increased survival rate of diabetic rats. 1-methylnicotinamide was also suggested to protect pancreatic cells from streptozotocin in vivo. Streptozotocin toxicity is known to be mediated by poly-ADP-ribose polymerase. Nicotinamide and its derivatives have been shown to ameliorate poly-ADP-ribose polymerase-dependent nucleotide pool reduction. We aimed to verify if 1-methylnicotinamide and its metabolite, N-methyl-2-pyridone-5-carboxamide, can protect insulinoma cells from streptozotocin-induced toxicity. We found that N-methyl-2-pyridone-5-carboxamide, but not 1-methylnicotinamide, restores the pool of ATP and NAD+ in streptozotocin-treated cells, but neither compound improved the cell viability. We conclude that inhibition of poly-ADP-ribose polymerase-dependent nucleotide pool reduction may not be sufficient to protect cells from streptozotocin toxicity.     

Increased urinary excretion of pyrimidine and nicotinamide derivatives in rats treated with methyl methanesulphonate

Rats treated with methyl methanesulphonate (MMS) excreted significantly higher quantities of deoxycytidine, thymidine, uracil, 1-methylnicotinamide (1-meNmd) and 1-methyl-6-pyridone-3-carboxylamide (6-pyr-1-meNmd) in their urine 0–24 h after MMS injection (100 $\text{mg}\text{kg}$). Excretion of thymidine, which was not detectable in untreated rats, was dose-dependent. No increase in urinary 7-methylguanine was found, and creatinine excretion was decreased by MMS treatment. Experiments with methyl-¹⁴C-labelled MMS showed transfer of ¹⁴C-label to 7-methylguanine and 1-meNmd. X-Irradiation (500 rad) caused increased excretion of pyrimidines, like MMS, but did not increase excretion of the nicotinamide derivatives.     

Lunatoic Acid A,a Morphogenic Substance Inducing Chlamydospore-like Cells in Some Fungi

The effect of the addition of 0.26 % free tryptophan (Trp) to a 20 % casein diet containing 6 mg of nicotinic acid per 100 g of diet on the ratio of N¹-methyl-2-pyridone-5-carboxamide (2-py) plus N¹-methyl-4-pyridone-3-carboxamide (4-py) to -methylnicotinamide (MNA) excretion was investigated in rats. The urinary excretion of MNA, 2-py and 4-py, respectively, increased statistically significantly with the feeding of a 0.26% Trp (the same as the content of the 20% casein diet) supplemented 20% casein diet, although it did not increase with the feeding of a 40% casein diet, compared with in the case of the 20 % casein diet [Agric. Biol. Chem., 52, 1765 (1988)]. So, the total urinary excretion of Nam and its metabolites was 1.8 times higher in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet. However, the ratio of 2-py plus 4-py to MNA excretion was much lower in the group fed the Trp supplemented diet than in the group fed the 20 % casein diet (13.16 ± 3.75→5.49 ± 2.25). This decreased ratio was considered to be partially due to a decrease in the 4-py forming MNA oxidase, which decreased significantly with the feeding of the Trp supplemented diet. Furthermore, the metabolic fate of Trp was greatly affected by the form of Trp, free or bound.     

An unusual pyridine nucleotide accumulating in erythrocytes: its identity and positive correlation with degree of renal failure

We have investigated an unusual nucleotide that accumulates, with precursors, in the erythrocytes of patients in uraemia. This nucleotide is related chemically to the NAD breakdown product, N1-methyl-2-pyridone-5-carboxamide (Me2Py), found in high concentrations in the plasma of uraemic patients. Both Me2Py and the nucleotide accumulate to high concentrations in the blood during uraemia: our investigations of samples from renal out-patients have provided information on a plausible link between the two compounds.     

Moderate food restriction suppresses the conversion of l-tryptophan to nicotinamide in weaning rats

Calorie restriction leads to a change in the metabolism of nutrients. Nicotinamide is biosynthesized from l-tryptophan. We attempted to determine the effects of food restriction on the biosynthesis of nicotinamide from l-tryptophan. Weaning male rats were fed a conventional chemically defined diet without preformed niacin for 63?d. However, the food intake was restricted to 80 and 65% of the intake of the ad libitum-fed control group of rats. The 24-h urine samples were periodically collected, and the urinary excretion of nicotinamide and its catabolites was measured. The conversion percentages were lower in both restricted groups than in the ad libitum-fed control group during the experimental period (control group, 1.37?±?0.24%; 80%-restricted group, 0.20?±?0.04%; 65%-restricted group, 0.15?±?0.02%; control vs. restricted groups, p?<?0.01). Food restriction, even at mild level, suppressed the conversion of l-tryptophan to nicotinamide when compared to the ad libitum-fed control group.     

The effect of N-methyl-2-pyridone-5-carboxamide--A nicotinamide catabolite on poly ADP-rybosylation and oxidative stress injury in endothelial cells

This study evaluated the effect of nicotinamide (NA) and its endogenous metabolite 2PY (N-methyl-2-pyridone-5-carboxamide) on the activity of poly (ADP-ribose) polymerase (PARP) and on peroxynitrite-induced injury in endothelial cells. 2PY and NA inhibited isolated PARP with half-maximal constants of 0.53 mM and 0.025 mM, respectively. Exposure to peroxynitrite caused a decrease of the NAD pool in cultured endothelial cells to below 10% of initial level. Addition of 2PY or NA provided partial protection from peroxynitrite-induced NAD depletion, with NA being more effective. 2PY and NA also provide protection from ATP depletion. We conclude that NA as well as 2PY protect from oxidative stress injury in endothelial cells by inhibition of PARP and protection from NAD depletion. This, in turn, protects energetics, allowing maintaining cellular ATP.     

Isolation and Restriction Mapping of a Pseudomonas Plasmid

The effects of the injection of a large amount of N¹ -methylnicotinamide (MNA) (500 mg per kg body weight) on the ratio of N¹ -methyl-4-pyridone-3-carboxamide (4-py) to N¹ -methyl-2-pyridone-5- carboxamide (2-py) excretion, and the activities of 2-py and 4-py forming MNA oxidases were investigated in rats. The injected MN A was excreted very rapidly into the urine; 46 % of the dose was excreted from 0~3hr post-injection, 15% from 3~6hr, 6% from 6~9hr and 1.5% from 9~ 12hr. The ratio of 4-py to 2-py also decreased rapidly; the ratio being about 0.6, 0.4, 0.4 and 0.6 from 0~3hr, 3~6hr, 6~9hr and 9~ 12hr post-injection, respectively. This ratio then recovered rapidly; being about 2, 5.5, 8.5 and 9.7 from 12~24 hr, 24 ~48 hr, 48~72 hr and 72 ~96 hr post-injection, respectively. The normal range of 4-py to 2-py excretion ratio is 8~14. So, this ratio returned to a normal level by day 3 post-injection. The rats were killed 5 hr after the MNA injection. At this time (the lowest ratio was observed around this time), the activities of 2-py and 4-py forming MNA oxidases in the injected group were 59 % and 11 % of the normal levels, respectively. Therefore, it was found that the decreased ratio of 4-py to 2-py excretion with the MNA injection was mainly due to the higher inhibition of the 4-py forming MNA oxidase than of the 2-py forming MNA oxidase by the MNA injection.