Quantitation of the niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in random spot urine samples, by ion-pairing reverse-phase HPLC with UV detection, and the implications for the use of spot urine samples in the assessment of niacin status

A simple ion-pairing reverse-phase HPLC method, with UV diode array detection, was developed and validated for quantitation of the urinary niacin metabolites 1-methylnicotinamide and l-methyl-2-pyridone-5-carboxamide in a single run. Urine samples were purified using a polymer-based mixed mode anion exchange reverse-phase cartridge. Analysis was performed on a reverse-phase C18 column, using a methanol gradient elution system, containing phosphate buffer pH 7.0, 1-heptanesulphonic acid as the ion-pairing agent and trimethylamine as a modifier. The assay was applied to the measurement of the niacin status of two subjects using spot urine samples. The samples were collected over 4 consecutive days and at four time points during 1 day. Status, expressed as the concentration ratios (2-PYR or 1-MN)/creatinine and 2-PYR/l-MN, varied within and between days and was least for fasting samples. This work illustrates the potential of spot urine sampling for niacin status assessment, but highlights the need for further validation prior to its use in field nutritional surveys.     

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.     

Niacin and its metabolites as master regulators of macrophage activation

Niacin is a broad-spectrum lipid-regulating drug used for clinical therapy of chronic high-grade inflammatory diseases. However, the mechanisms by which either niacin or the byproducts of its catabolism ameliorate these inflammatory diseases are not clear yet. Human circulating monocytes and mature macrophages were used to analyze the effects of niacin and its metabolites (NAM, NUA and 2-Pyr) on oxidative stress, plasticity and inflammatory response by using biochemical, flow cytometry, quantitative real-time PCR and Western blot technologies. Niacin, NAM and 2-Pyr significantly decreased ROS, NO and NOS2 expression in LPS-treated human mature macrophages. Niacin and NAM skewed macrophage polarization toward antiinflammatory M2 macrophage whereas a trend toward proinflammatory M1 macrophage was noted following treatment with NUA. Niacin and NAM also reduced the inflammatory competence of LPS-treated human mature macrophages and promoted bias toward antiinflammatory CD14⁺CD16⁺⁺ nonclassical human primary monocytes. This study reveals for the first time that niacin and its metabolites possess antioxidant, reprogramming and antiinflammatory properties on human primary monocytes and monocyte-derived macrophages. Our findings imply a new understanding of the mechanisms by which niacin and its metabolites favor a continuous and gradual plasticity process in the human monocyte/macrophage system.     

Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells

Niacin is a widely used lipid-regulating agent in dyslipidemic patients. Previously, we have shown that niacin inhibits triacylglycerol synthesis. In this report, using HepG2 cells, we have examined the effect of niacin on the mRNA expression and microsomal activity of diacylglycerol acyltransferase 1 and 2 (DGAT1 and DGAT2), the last committed but distinctly different enzymes for triglyceride synthesis. Addition of niacin to the DGAT assay reaction mixture dose-dependently (0-3 mM) inhibited DGAT activity by 35-50%, and the IC(50) was found to be 0.1 mM. Enzyme kinetic studies showed apparent K(m) values of 8.3 microM and 100 microM using [(14)C]oleoyl-CoA and sn-1,2-dioleoylglycerol as substrates, respectively. A decrease in apparent V(max) was observed with niacin, whereas the apparent K(m) remained constant. A Lineweaver-Burk plot of DGAT inhibition by niacin showed a noncompetitive type of inhibition. Niacin selectively inhibited DGAT2 but not DGAT1 activity. Niacin inhibited overt DGAT activity. Niacin had no effect on the expression of DGAT1 and DGAT2 mRNA. These data suggest that niacin directly and noncompetitively inhibits DGAT2 but not DGAT1, resulting in decreased triglyceride synthesis and hepatic atherogenic lipoprotein secretion, thus indicating a major target site for its mechanism of action.     

Elucidation of the toxic mechanism of the plasticizers,phthalic acid esters,putative endocrine disrupters: effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats

We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan-niacin metabolism.     

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.     

不同浓度有机物对雷公藤不定根生长和次生代谢产物含量的影响

以NT为基本培养基,雷公藤(Tripterygium wilfordii)不定根为材料,研究了肌醇、VB1、烟酸、VB6、甘氨酸、叶酸、生物素等有机物质对雷公藤不定根生长及其次生代谢产物雷公藤甲素和总生物碱含量的影响。结果表明:NT培养基中原有浓度的肌醇、VB1含量即可使雷公藤不定根生长量、雷公藤甲素含量、总生物碱含量及产量达到最大值。在添加的其他有机物中,添加1 mg/L烟酸、1 mg/L VB6和5 mg/L甘氨酸适合不定根的生长;添加0.5 mg/L烟酸、0.5 mg/L生物素、1 mg/L VB6、1 mg/L甘氨酸和1 mg/L叶酸适合雷公藤甲素的积累;添加0.5 mg/L甘氨酸、1 mg/L VB6、1 mg/L叶酸和1 mg/L生物素则适合不定根中雷公藤总生物碱的合成。     

Triglyceride modulation by acifran analogs: activity towards the niacin high and low affinity G protein-coupled receptors HM74A and HM74

Niacin is known to exert profound beneficial effects on cholesterol levels in humans, although its use is somewhat hampered by the gram quantities necessary to exert effects and the prevalence of compliance-limiting skin flushing side effects that occur. Recently, two G protein-coupled receptors (GPCRs) for niacin were identified and characterized as high (HM74A; GPR109A) and low (HM74; GPR109B) affinity receptors based on the binding affinities of niacin. These receptors also bind acifran (AY-25,712), which is known to modulate lipid levels like niacin, with similar affinities. Twelve analogs of acifran were chemically synthesized. One analogue demonstrated a dose-dependent decrease in serum triglycerides in rats within 3h of oral administration. Next, the acifran analogs were assessed for their activity towards the high and low affinity niacin receptors expressed in CHO-K1 cells. Constructs expressing HM74A or HM74 were stably transfected into CHO-K1 cells and shown to elicit phosphorylation of p42 and p44 mitogen-activated protein kinase (ERK1/ERK2) phosphorylation upon addition of niacin or acifran. The presence of functionally coupled GPCRs was further confirmed using Pertussis toxin, which completely inhibited the ability of either niacin or acifran to elicit phospho-ERK1/ERK2. The EC(50) of p-ERK1/ERK2 for niacin for the high and low affinity receptors was 47nM and indeterminate (i.e., >100microM), respectively, while the EC(50) for acifran was 160 and 316nM, respectively. Two chemical analogs of acifran demonstrated robust phosphorylation of ERK1/ERK2. Collectively, these data suggest that the synthesis of acifran analogs may be a suitable path for developing improved HM74A agonists.     

Water-soluble vitamin homeostasis in fasting northern elephant seals (Mirounga angustirostris) measured by metabolomics analysis and standard methods

Despite the importance of water-soluble vitamins to metabolism, there is limited knowledge of their serum availability in fasting wildlife. We evaluated changes in water-soluble vitamins in northern elephant seals, a species with an exceptional ability to withstand nutrient deprivation. We used a metabolomics approach to measure vitamins and associated metabolites under extended natural fasts for up to 7 weeks in free-ranging lactating or developing seals. Water-soluble vitamins were not detected with this metabolomics platform, but could be measured with standard assays. Concentrations of measured vitamins varied independently, but all were maintained at detectable levels over extended fasts, suggesting that defense of vitamin levels is a component of fasting adaptation in the seals. Metabolomics was not ideal for generating complete vitamin profiles in this species, but gave novel insights into vitamin metabolism by detecting key related metabolites. For example, niacin level reductions in lactating females were associated with significant reductions in precursors suggesting downregulation of the niacin synthetic pathway. The ability to detect individual vitamins using metabolomics may be impacted by the large number of novel compounds detected. Modifications to the analysis platforms and compound detection algorithms used in this study may be required for improving water-soluble vitamin detection in this and other novel wildlife systems.     

Comparison of the Activity of the Tryptophan-NAD Pathway between Rats Fed a Fat-free Diet and a Fat Diet

The effect of dietary fat on tryptophan-NAD metabolism was investigated. Weanling male rats of the Sprague Dawley strain were fed a 40% casein diet (nicotinic acid-free) with or without 20% fat for 13 days. Although the food intake in 13 days was significantly higher in the fat-free group than in the fat group, the gains in body weight in the two groups were almost the same, because of the same energy intakes. The urinary excretion of tryptophan metabolites such as quinolinic acid, niacin and N¹-methylnicotinamide was greatly increased in the fat group in comparison with that in the fat-free group. The urinary excretion of xanthurenic acid was almost the same in the two groups. The blood NAD level of the fat group was significantly increased. The activities of liver amino-carboxymuconate-semialdehyde decarboxylase and liver nicotinamide methyltransferase in the fat group were significantly reduced, and that of liver NMN adenylyltransferase was significantly increased. The changes of these three enzymes could be advantageous for the increased formation of NAD from tryptophan. As a result, the feeding of a high fat diet to rats increased the formation of niacin and niacin-related compounds.     

SIRT1 genetic variation and mortality in type 2 diabetes: interaction with smoking and dietary niacin

SIRT1 protects cells against oxidative stress and aging. Its activity may be modulated by dietary niacin (vitamin B3) intake. We studied the association of SIRT1 genetic variation with mortality in subjects with increased oxidative stress (type 2 diabetes and smokers) in relation to dietary niacin. In 4573 participants from the Rotterdam Study, including 413 subjects with prevalent and 378 with incident type 2 diabetes, three SIRT1 tagging SNPs were genotyped and all-cause mortality was studied (average follow-up12 years). We found no association between SIRT1 variation and mortality in the total population or in smokers. In subjects with prevalent type 2 diabetes, homozygous carriers of the most common SIRT1 haplotype, 1, had 1.5 times (95%CI 1.1–2.1) increased mortality risk compared to noncarriers. This risk further increased among smokers and those with low niacin intake. In the lowest tertile of niacin intake, mortality risk was increased 2.3 (95%CI 1.1–4.9) and 5.7 (95%CI 2.5–13.1) times for heterozygous and homozygous carriers of haplotype 1. Subjects with incident diabetes showed similar findings but only when they smoked. We conclude that in subjects with type 2 diabetes, SIRT1 genetic variation influences survival in interaction with dietary niacin and smoking. Correction of niacin deficiency and SIRT1 modulators may prolong the life span of patients with diabetes.     

Efficiency of D-tryptophan as Niacin in rats

L-tryptophan is a very important precursor of niacin in mammals. Food preparation in which proteins are exposed to an alkali and/or high temperature for a long period generate appreciable amounts of D-amino acids from racemization. The efficiency of D-tryptophan as niacin was thus investigated by using weanling rats. The availability of D-tryptophan was almost the same as that in L-tryptophan as the precursor of niacin and was 1/6 as active as niacin.     

Effects of l-carnitine and niacin supplied by drinking water on fattening performance,carcass quality and plasma l-carnitine concentration of broiler chicks

The present study was initiated to determine whether dietary supplemental L-carnitine and niacin affect growth performance, carcass yield, abdominal fat and plasma L-carnitine concentration of broiler chicks. One-day-old broiler chicks (COB500) were used in the experiment. A two by two factorial arrangement was employed with two levels (0 and 50 mg/l) of supplemental L-carnitine and two levels (0 or 50 mg/l) of supplemental niacin in drinking water as main effects. Body weight gain was significantly improved by L-carnitine, or L-carnitine + niacin supplementation during the first 3 weeks. However, supplemental L-carnitine and niacin did not change body weight gain during the last 3 weeks of the experimental period. Supplemental L-carnitine significantly improved feed intake during the first 3 weeks. Supplemental L-carnitine or niacin did not influence carcass weight, carcass yield and abdominal fat weight. L-carnitine content in the plasma was significantly higher in the groups receiving supplemental L-carnitine and L-carnitine + niacin. It is concluded that dietary supplemental L-carnitine or L-carnitine + niacin could have positive effects on body weight gain and feed intake during the early stages of growing. However, supplemental L-carnitine or L-carnitine + niacin were not of benefit regarding the complete growth period.     

Effects of niacin on milk yield,milk composition,and blood components of dairy cows in hot weather

Twenty multiparous Friesian cows, 90 ± 30 days postpartum, were allocated to two groups of ten cows according to calving date, lactation number and daily milk yield, and assigned randomly to one of two diets in a cross-over experiment. The experimental diet consisted of concentrate and maize silage in the proportions 1:1 (dry matter basis) containing either 0 or 10 g niacin per cow per day, which was handmixed into the concentrate. The diets were offered individually as total mixed rations in two equal proportions at 09:00 and 20:00 h in amounts sufficient to ensure 10% refusals. Dry matter, metabolisable energy and crude protein intakes, as well as milk yield, milk protein proportion and yield, milk lactose, total solids and solids-not-fat proportions were not affected by niacin supplementation. In contrast, niacin supplementation increased milk fat proportion and yield. No differences were observed in blood serum concentrations of glucose, total protein, triglycerides, cholesterol, Na, K, Ca, P or Mg. However, the serum concentration of urea was lower when the cows were supplemented with niacin.     

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.     

Hepatic pyridine nucleotides content in rat--a better indicator for determining available niacin values of food.

After 10 days depletion period with niacin free diet, weanling rats were repleted with either reference diets containing niacin at four levels (2, 4, 8 and 12 mg/kg diet) or test diets containing test material at two levels as source of niacin. Gains in body weight and hepatic pyridine nucleotides content increased with increase in niacin intake. The correlation coefficient for hepatic pyridine nucleotides content and niacin intake was 0.98, whereas for weight gain and niacin intake was 0.89. Dried skim milk and pearl millet were taken as test materials. Dried skim milk with most of its niacin in free form showed 98% niacin equivalent to be bioavailable whereas in pearl millet niacin was in bound form and bioavailability equivalent was only 48 per cent.     

The impact of PLA2G4A and PTGS2 gene polymorphisms,and red blood cell PUFAs deficit on niacin skin flush response in schizophrenia patients

We investigated the etiology of the attenuated niacin skin flush response in schizophrenia patients. Skin response to topical niacin of 0.1 M, 0.01 M, 0.001 M, and 0.0001 M concentrations was rated using method of volumetric niacin response (VNR) and correlated to two functional A/G polymorphisms in genes: phospholipase A2 group IVA (BanI of the PLA2G4A), and rs689466 of the prostaglandin synthase-2 (PTGS2). We further tested the possible correlation between niacin response and fatty acid (FA) content of red blood cells (RBCs). We detected statistically significant but weak impact of both polymorphisms on niacin flush response in schizophrenia patients. The dosage of the G alleles of both polymorphisms was associated with higher VNR values, although each polymorphic variant accounted for only 1% of the overall flush response variability. Regarding FA content, both n?3 and n?6 polyunsaturated FAs (PUFAs) were significantly reduced in the patient group, but an association with niacin sensitivity was not detected.     

Metabolism of 4-n-nonylphenol by non-modified and CYP1A1- and CYP1A2-transgenic cell cultures of tobacco

The metabolism of 14C-4-n-nonylphenol (l4C-4-n-NP), as a model for the xenoestrogen nonylphenol, was investigated in three types of tobacco cell suspension cultures: one genetically non-modified culture (NT) and two cultures constitutively expressing human cytochrome P450 CYP1A1 or CYP1A2. With 1 mg l(-1) of 14C-4-n-NP and 24 h of incubation, the xenobiotic was transformed almost completely to glycosides. After glycosidic cleavage, 14C-4-n-NP and several primary metabolites of 4C-4-n-NP were liberated. Portions of the primary metabolites were 29.3% (NT culture), 34.3% (CYP1A1 culture), and 50.7% of applied 14C (CYP1A2 culture). Thus, the endogenous capacity of the tobacco cells to form primary metabolites of 4-n-NP was noticeably higher than that of CYP1A1 or CYP1A2. The results however clearly suggest that 4-n-NP is - even though a poor - substrate of CYP1A1 and CYP1A2. In order to examine metabolic profiles of 4-n-NP in the NT, CYP1A1 and CYP1A2 cultures, the suspensions were exposed to 10 mg 1(-1) of 14C-4-n-NP using a two-liquid-phase system with carrier n-hexadecane and 192 h of incubation. Results obtained resembled those of the low concentration study. The oxidative metabolic profiles determined after hydrolytic cleavage using GC-EIMS were similar in the NT, CYP1A1 and CYP1A2 cultures. Main metabolites were side-chain mono-hydroxylated derivatives of 4-n-NP with 6'-, 7'- and 8'-OH-4-n-NP as prominent metabolites. In addition, olefinic side-chain hydroxy, ring methoxylated, keto and ring hydroxylated derivatives were observed. The lack of differences in metabolic profiles among the CYP1A1, CYP1A2 and NT cultures was referred to the low enzymatic activity of CYP1A1 and CYP1A2 as compared to the higher endogenous oxidative capacity of tobacco, as well as to similar metabolic profiles of 4-n-NP produced by CYP1A1 and CYP1A2 and tobacco itself.