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.     

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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

Potential urinary and plasma biomarkers of peroxisome proliferation in the rat: identification of N-methylnicotinamide and N-methyl-4-pyridone-3-carboxamide by 1H nuclear magnetic resonance and high performance liquid chromatography

This study identified two potential novel biomarkers of peroxisome proliferation in the rat. Three peroxisome proliferator-activated receptor (PPAR) ligands, chosen for their high selectivity towards the PPARα, -δ and -γ subtypes, were given to rats twice daily for 7 days at doses known to cause a pharmacological effect or peroxisome proliferation. Fenofibrate was used as a positive control. Daily treatment with the PPARα and -δ agonists produced peroxisome proliferation and liver hypertrophy. ¹H nuclear magnetic resonance spectroscopy and multivariate statistical data analysis of urinary spectra from animals given the PPARα and -δ agonists identified two new potential biomarkers of peroxisome proliferation - N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY) - both endproducts of the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway. After 7 days, excretion of NMN and 4PY increased 24- and three-fold, respectively, following high doses of fenofibrate. The correlation between total NMN excretion over 7 days and the peroxisome count was r=0.87 (r²=0.76). Plasma NMN, measured using a sensitive high performance liquid chromatography method, was increased up to 61-fold after 7 days' treatment with high doses of fenofibrate. Hepatic gene expression of aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) was downregulated following treatment with the PPARα and -δ agonists. The decrease was up to 11-fold compared with controls in the groups treated with high doses of fenofibrate. This supports the link between increased NMN and 4PY excretion and regulation of the tryptophan-NAD⁺ pathway in the liver. In conclusion, NMN, and possibly other metabolites in the pathway, are potential non-invasive surrogate biomarkers of peroxisome proliferation in the rat.     

Tryptophan Metabolism of Rats Fed on an Amino Acid Diet Devoid of One Branched Chain Amino Acid

In an attempt to study on metabolic changes in rats fed on an amino acid diet devoid of one branched chain amino acid and of niacin, rats were force-fed a leucine-free, isoleucine-free, valine-free or complete amino acid diet for 3 or 4 days and killed 3 hr after the feeding on day 4 or 5 to observe the body weight changes, the urinary nitrogen and N¹-methylnicotinamide (MNA), and liver tryptophanpyrrolase (TPase) and tyrosine-α-keto-glutarate transaminase (TKase) activities.

The excretion of the urinary nitrogen and MNA, TPase and TKase activities, and fat content of livers of rats force-fed these amino acid deficient diets were higher than those fed the complete amino acid diet. It was further confirmed in the present study that changes in TPase activity of rats given diets devoid of one essential amino acid were in the same direction with changes in urinary MNA which was observed in the previous studies on rats given threonine-free, tryptophan-free, methionine-free, lysine-free and complete amino acid diets. However, such metabolic changes in rats fed the leucine-free diet were not so remarkable, compared with those of rats fed the other amino acid deficient diets.     

Increased plasma concentration of 4-pyridone-3-carboxamide-1-ß-D-ribonucleoside (4PYR) in lung cancer. Preliminary studies

Abstract

4-pyridone-3-carboxamide-1-β-D-ribonucleoside (4PYR) is a new nicotinamide derivative, which is potentially toxic to the endothelium. Dysfunction of the endothelium promotes cancer cell proliferation, invasiveness, and inflammatory signaling. The aim of this study was to analyze 4PYR concentration in the plasma of lung cancer patients and its relationship to other known biochemical parameters associated with the endothelium function.

The concentration of 4PYR, nicotinamide, 1-methylnicotinamide (MNA), amino acids, and their derivatives were measured in samples obtained from patients with primary squamous cell carcinoma (n?=?48) and control group (n?=?100).

The concentration of 4PYR and 4PYR/MNA ratio were significantly higher in lung cancer patients as compared to controls (0.099?±?0.009 vs. 0.066?±?0.006?µmol/L and 1.10?±?0.08 vs. 1.97?±?0.15, respectively). The plasma arginine/asymmetric dimethylarginine (Arg/ADMA) ratio was considerably lower in lung cancer patients (253?±?17 vs. 369?±?19) as well as plasma MNA (0.057?±?0.004 vs. 0.069?±?0.003?µmol/L). There was no difference in the plasma concentrations of nicotinamide and nicotinamide riboside in both groups (0.116?±?0.019 vs. 0.131?±?0.014 and 0.102?±?0.006 vs. 0.113?±?0.011, respectively).

In this study, a higher 4PYR concentration was observed for the first time in patients with squamous cell carcinoma. This change may be related to the endothelial dysfunction that promote cancer progression since 4PYR and its derivatives are known to disrupt glycolytic pathway.     

Simultaneous determination of nicotinic acid and its four metabolites in rat plasma using high performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS)

A sensitive and specific liquid chromatography electrospray ionization–tandem mass spectrometry method for the simultaneous quantitation of nicotinic acid (NicA) and its metabolites nicotinamide (NA), 1-methylnicotinamide (MNA), 1-methyl-2-pyridone-5-carboxamide (M2PY) and 1-methyl-4-pyridone-5-carboxamide (M4PY) in rat plasma has been developed and validated. As an internal standard, 6-chloronicotinamide was used. The samples (100 μL) were subjected to deproteinization with acetonitrile (200 μL) and then, after centrifugation, 150 μL of the supernatant was transferred into conical vial and evaporated. Dry residue was reconstituted in 100 μL of the ACN/water (10:90, v/v) mixture. Chromatography was performed on a Waters Spherisorb^® 5 μm CNRP 4.6 × 150 mm analytical column with gradient elution using a mobile phase containing acetonitrile and water with 0.1% of formic acid. The full separation of all compounds was achieved within 15 min of analysis. Detection was performed by an Applied Biosystems MDS Sciex API 2000 triple quadrupole mass spectrometer set at unit resolution. The mass spectrometer was operated in the selected reactions monitoring mode (SRM), monitoring the transition of the protonated molecular ions m/z 153–110 for M2PY, 153–136 for M4PY, 124–80 for NicA, 123–80 for NA and 137–94 for MNA. The mass spectrometric conditions were optimized for each compound by continuously infusing the standard solution at the rate of 5 μL/min using a Harvard infusion pump. Electrospray ionization (ESI) was used for ion production. The instrument was coupled to an Agilent 1100 LC system. The precision and accuracy for both intra- and inter-day determination of all analytes ranged from 1.3% to 13.3% and from 94.43% to 110.88%. No significant matrix effect (ME) was observed. Stability of compounds was established in a battery of stability studies, i.e. bench-top, autosampler and long-term storage stability as well as freeze/thaw cycles. The method proved to be suitable for various applications. In particular using this method we detected increased concentration of MNA and its metabolites in rat plasma after treatment with exogenous MNA (100 mg/kg), as well as increased concentration of endogenous NA and MNA in rat plasma in the early phase of hypertriglyceridemia development in rats fed high-fructose diet.     

Development of a multivariate statistical model to predict peroxisome proliferation in the rat,based on urinary 1H-NMR spectral patterns

A previous report of this work (Ringeissen et al. 2003) described the use of nuclear magnetic resonance (NMR) spectroscopy coupled with multivariate statistical data analysis (MVDA) to identify novel biomarkers of peroxisome proliferation (PP) in Wistar Han rats. Two potential biomarkers of peroxisome proliferation in the rat were described, N-methylnicotinamide (NMN) and N-methyl-4-pyridone-3-carboxamide (4PY). The inference from these results was that the tryptophan-nicotinamide adenine dinucleotide (NAD⁺) pathway was altered in correlation with peroxisome proliferation, a hypothesis subsequently confirmed by TaqMan® analysis of the relevant genes encoding two key enzymes in the pathway, aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) and quinolinate phosphoribosyltransferase (EC 2.4.2.19). The objective of the present study was to investigate these data further and identify other metabolites in the NMR spectrum correlating equally with PP. MVDA Partial Least Squares (PLS) models were constructed that provided a better prediction of PP in Wistar Han rats than levels of 4PY and NMN alone. The resulting Wistar Han rat predictive models were then used to predict PP in a test group of Sprague Dawley rats following administration of fenofibrate. The models predicted the presence or absence of PP (above on arbitrary threshold of >2-fold mean control) in all Sprague Dawley rats in the test group.     

Nicotinamide N-methyltransferase in endothelium protects against oxidant stress-induced endothelial injury

Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1.) plays an important role in the growth of many different tumours and is also involved in various non-neoplastic disorders. However, the presence and role of NNMT in the endothelium has yet to be specifically explored. Here, we characterized the functional activity of NNMT in the endothelium and tested whether NNMT regulates endothelial cell viability. NNMT in endothelial cells (HAEC, HMEC-1 and EA.hy926) was inhibited using two approaches: pharmacological inhibition of the enzyme by NNMT inhibitors (5-amino-1-methylquinoline – 5MQ and 6-methoxynicotinamide – JBSF-88) or by shRNA-mediated silencing. Functional inhibition of NNMT was confirmed by LC/MS/MS-based analysis of impaired MNA production. The effects of NNMT inhibition on cellular viability were analyzed in both the absence and presence of menadione.Our results revealed that all studied endothelial lines express relatively high levels of functionally active NNMT compared with cancer cells (MDA-MB-231). Although the aldehyde oxidase 1 enzyme was also expressed in the endothelium, the further metabolites of N1-methylnicotinamide (N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide) generated by this enzyme were not detected, suggesting that endothelial NNMT-derived MNA was not subsequently metabolized in the endothelium by aldehyde oxidase 1. Menadione induced a concentration-dependent decrease in endothelial viability as evidenced by a decrease in cell number that was associated with the upregulation of NNMT and SIRT1 expression in the nucleus in viable cells. The suppression of the NNMT activity either by NNMT inhibitors or shRNA-based silencing significantly decreased the endothelial cell viability in response to menadione. Furthermore, NNMT inhibition resulted in nuclear SIRT1 expression downregulation and upregulation of the phosphorylated form of SIRT1 on Ser47. In conclusion, our results suggest that the endothelial nuclear NNMT/SIRT1 pathway exerts a cytoprotective role that safeguards endothelial cell viability under oxidant stress insult.     

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.     

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.