Absorption, Pharmacokinetics, and Urinary Excretion of Pyridines After Consumption of Coffee and Cocoa-Based Products Containing Coffee in a Repeated Dose, Crossover Human Intervention Study.

SCOPE: The present study assesses the absorption, pharmacokinetics, and urinary excretion of coffee pyridines and their metabolites after daily regular exposure to specific dosages of coffee or cocoa-based products containing coffee (CBPCC), considering different patterns of consumption. METHODS AND RESULTS: In a three-arm, crossover, randomized trial, 21 volunteers are requested to randomly consume for 1 month: one cup of espresso coffee per day, three cups of espresso coffee per day, or one cup of espresso coffee plus two CBPCC twice per day. The last day of the one-month treatment, blood and urine samples are collected for 24 h. Trigonelline, N-methylpyridinium, N-methylnicotinamide, and N-methyl-4-pyridone-5-carboxamide are quantified. Trigonelline and N-methylpyridinium absorption curves and 24-h urinary excretion reflect the daily consumption of different servings of coffee or CBPCC, showing also significant differences in main pharmacokinetic parameters. Moreover, inter-subject variability due to sex and smoking is assessed, showing sex-related differences in the metabolism of trigonelline and smoking-related ones for N-methylpyridinium. CONCLUSION: The daily exposure to coffee pyridines after consumption of different coffee dosages in a real-life setting is established. This data will be useful for future studies aiming at evaluating the bioactivity of coffee-derived circulating metabolites in cell experiments, mimicking more realistic experimental conditions.

Oral Glucose Tolerance and Tryptophan Metabolism in Non-Obese and Non-Insulin-Dependent Diabetic Goto-Kakizaki Rats Fed High-Tryptophan Diets.

We investigated oral glucose tolerance and tryptophan (Trp) metabolism in non-obese and non-insulin-dependent diabetic Goto-Kakizaki (GK) rats fed high-Trp diets. Five-week-old male Wistar and GK rats were fed a 20% casein diet (control diet) or the same diet supplemented with 1%, 2%, 3%, or 5% Trp for 58 d. Oral glucose tolerance tests were performed on Days 14 and 28 of the experimental period. Urine as well as livers and blood were collected on the last day of the experiment. The glucose concentration and the amount of Trp metabolites were measured. On Day 14 of the experiment, the incremental blood glucose concentrations integrated over a period of 2 h (ΔAUC0-2h) of blood glucose in rats fed the 3% and 5% Trp diets had decreased by 13% and 18%, respectively, compared with that of the control-GK rats. However, no significant differences were found in the rats fed +1% or +2% Trp diets compared with control-GK rats. On Day 28, there were no significant differences found in the ΔAUC0-2h of blood glucose levels in any group including the control-GK group. On the last day, the concentrations of plasma glucose, total cholesterol, and triglyceride did not show differences in any group. There were no specific phenomena observed in the metabolism of Trp in GK rats even when fed an excess of Trp, compared with that of Wistar rats. Oral Trp administration and its continuous use may not improve blood glucose levels in type 2 diabetic rats.

Urinary Excretion of Niacin Metabolites in Humans After Coffee Consumption.

SCOPE: Coffee is a major natural source of niacin in the human diet, as it is formed during coffee roasting from the alkaloid trigonelline. The intention of our study was to monitor the urinary excretion of niacin metabolites after coffee consumption under controlled diet. METHODS AND RESULTS: We performed a 4-day human intervention study on the excretion of major niacin metabolites in the urine of volunteers after ingestion of 500 mL regular coffee containing 34.8 µmol nicotinic acid (NA) and 0.58 µmol nicotinamide (NAM). In addition to NA and NAM, the metabolites N1 -methylnicotinamide (NMNAM), N1 -methyl-2-pyridone-5-carboxamide (2-Py), and nicotinuric acid (NUA) were identified and quantified in the collected urine samples by stable isotope dilution analysis (SIVA) using HPLC-ESI-MS/MS. Rapid urinary excretion was observed for the main metabolites (NA, NAM, NMNAM, and 2-Py), with tmax values within the first hour after ingestion. NUA appeared in traces even more rapidly. In sum, 972 nmol h-1 of NA, NAM, NMNAM, and 2-Py were excreted within 12 h after coffee consumption, corresponding to 6% of the ingested NA and NAM. CONCLUSION: The results indicate regular coffee consumption to be a source of niacin in human diet.

Niacin (Vitamin B3) Supplementation in Patients with Serotonin-Producing Neuroendocrine Tumor.

BACKGROUND/AIMS: Tryptophan is the precursor of serotonin and niacin (vitamin B3). The latter is critical for normal cellular metabolism. Tryptophan and niacin can be deficient in patients with serotonin-producing neuroendocrine tumors (NETs). Niacin deficiency may lead to severe symptoms including pellagra. In patients with serotonin-producing NET, data on niacin status are scarce and niacin supplementation hardly receives attention. We aimed to assess the niacin status before and after supplementation in these patients. METHODS: We identified serotonin-producing NET patients who had received oral niacin supplementation (mean dose 144 mg daily) for tryptophan deficiency and/or pellagra-associated symptoms. Presupplementation plasma tryptophan levels and niacin status based on the urinary niacin metabolite N1-methylnicotinamide (N1-MN) before (n = 42) and after the start of the supplementation (in 34 paired samples) were assessed. Reference values for urinary N1-MN levels were established in 133 healthy individuals. RESULTS: The mean presupplementation plasma tryptophan level was 31.8 ± 9.7 µmol/l (reference value 40.0-70.0). Presupplementation urinary N1-MN levels were lower in patients (median 17.9 µmol/24 h, range 2.6-70.3) compared to healthy controls (median 43.7 µmol/24 h, range 9.5-169.3, p < 0.0001) and below normal in 45% of the patients. Niacin supplementation increased urinary N1-MN levels to high normal levels (median 55.5 µmol/24 h, range 7.4-489.0) in 86% of the niacin-deficient patients. CONCLUSION: In serotonin-producing NET patients, niacin deficiency is prevalent. Therefore, urinary N1-MN deserves to be included in their standard biochemical evaluation. Niacin supplementation normalizes the niacin status in most niacin-deficient serotonin-producing NET patients. A prospective study is warranted.

Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 induce different age-related metabolic profiles revealed by 1H-NMR spectroscopy in urine and feces of mice.

Age-related dysbioses of intestinal microbiota and decline in the overall metabolic homeostasis are frequently found in the elderly. Probiotic supplementation may represent a way to prevent or reduce the senescence-associated metabolic disorders. The present study evaluated the metabolic impact of Lactobacillus acidophilus La5 and Bifidobacterium lactis Bb12 supplementation in relation to age by analyzing urine and feces metabolic profiles using (1)H-nuclear magnetic resonance spectroscopy and multivariate analysis. Adult (3 mo old) and aged (16 mo old) mice received an oral supplementation of the 2 probiotics (1 × 10(9) colony-forming units/d each) or phosphate buffered saline (control) daily for 30 d. Urine and feces were collected for 48 h before the end of the study. Partial least squares-discriminant analysis showed that the urinary discriminant metabolites for the probiotic treatment included higher dimethylglycine in adult and aged mice, lower sarcosine and nicotinate in adult mice, higher N-methylnicotinamide in adult mice and lower N-methylnicotinamide in aged mice compared with their controls. These results indicate a probiotic-induced modulation of homocysteine and NAD metabolism pathways, which have important implications because these pathways are involved in essential cellular processes that can be altered in senescence. The probiotic supplementation also modified the fecal metabolic profiles, inducing in both adult and aged mice higher 4-hydroxyphenylacetate and lower xylose in treated mice compared with their control mice, whereas valerate was greater in treated adult mice and lower in treated aged mice compared with their controls. The ANOVA simultaneous component analysis on urinary and fecal metabolic profiling showed an age × treatment interaction (P < 0.05), confirming the age-related modulation of the metabolic response to probiotic supplementation. The results suggest that L. acidophilus and B. lactis may prevent or reduce age-related metabolic dysfunction.

Supplementing healthy women with up to 5.0 g/d of L-tryptophan has no adverse effects.

Because of the frequent use of L-tryptophan (L-Trp) in dietary supplements, determination of the no-observed-adverse-effect-level is desirable for public health purposes. We therefore assessed the no-observed-adverse-effect-level for L-Trp and attempted to identify a surrogate biomarker for excess L-Trp in healthy humans. A randomized, double-blind, placebo-controlled, crossover intervention study was performed in 17 apparently healthy Japanese women aged 18-26 y with a BMI of ≈ 20 kg/m(2). The participants were randomly assigned to receive placebo (0 g/d) or 1.0, 2.0, 3.0, 4.0, or 5.0 g/d of L-Trp for 21 d each with a 5-wk washout period between trials. Food intake, body weight, general biomarkers in blood and urine, and amino acid composition in blood and urine were not affected by any dose of L-Trp. Administration of up to 5.0 g/d L-Trp had no effect on a profile of mood states category measurement. The urinary excretion of nicotinamide and its catabolites increased in proportion to the ingested amounts of L-Trp, indicating that participants could normally metabolize this amino acid. The urinary excretion of L-tryptophan metabolites, including kynurenine (Kyn), anthranilic acid, kynurenic acid, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and quinolinic acid (QA), all of which are intermediates of the L-TRP→Kyn→QA pathway, was in proportion to L-Trp loading. The response of 3-HK was the most characteristic of these L-Trp metabolites. This finding suggests that the urinary excretion of 3-HK is a good surrogate biomarker for excess L-Trp ingestion.

Dietary quinic acid supplied as the nutritional supplement AIO + AC-11® leads to induction of micromolar levels of nicotinamide and tryptophan in the urine.

Hippuric acid is synthesized and produced primarily by the gastrointestinal (GI) microflora. However, there is no known health benefit for hippuric acid except its catabolic conjugation of benzene-type compounds via glycine and subsequent excretion in the urine. For years the GI tract microflora were known to metabolize quinic acid to hippuric acid. Recently it was also proposed that DNA repair was strongly enhanced by quinic acid. In order to explain these quinic acid effects, Pero and colleagues have examined whether tryptophan and nicotinamide were also enhanced by quinic acid levels in urine. They were indeed, and so another study was designed using a natural supplement source of quinic acid called AIO + AC-11®, and then the effects of intervention were measured after only 21 days. It was possible to show profound increases in quinic acid that were again paralleled by increases in tryptophan and nicotinamide urinary levels. Because the high pressure liquid chromatography (HPLC) methods differed greatly between the two studies, differences in chemical analyses probably did not contribute to the data base.

Antioxidant metabolism induced by quinic acid. Increased urinary excretion of tryptophan and nicotinamide.

For over 50 years, hippuric/quinic acids were believed to have no biological efficacy. Here data are presented to support the hypothesis that quinic acid is not responsible for any efficacy, but rather that quinic acid nutritionally supports the synthesis of tryptophan and nicotinamide in the gastrointestinal (GI) tract, and that this in turn leads to DNA repair enhancement and NF-kB inhibition via increased nicotinamide and tryptophan production.Moreover, it is shown that quinic acid is a normal constituent of our diet, capable of conversion to tryptophan and nicotinamide via the GI tract microflora, thus providing an in situ physiological source of these essential metabolic ingredients to humans. The concentrations of quinic and hippuric acids in the diet were dependent on each other when analysed in urine, as was evidenced by a significant linear regression analysis that included unsupplemented control subjects (n = 45, p < 0.001). Thus, these ingredients were identified as major dietary components, and not simply originating from environmental pollution as previously had been thought.

Identification of N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-5-carboxamide as components in urine extracts of individuals consuming coffee.

Caffeine metabolites were extracted from urine samples collected 4 h after consumption of a cup of coffee and were separated by high-performance liquid chromatography (HPLC) on a C18 (5 microm) reverse-phase column using an acetonitrile (5%), acetic acid (0.05%) solution as the mobile phase. The elution profiles indicated the constant presence of a major and a minor components eluting between the caffeine metabolites 5-acetamido-6-formyl-3-methyluracil (AFMU) and 7-methylxanthine (7X) in an approximate nine. A procedure was developed for the isolation of the major component in an apparent pure form, and the yield was 10-20 mg from 400 ml of urine. The minor component was isolated in an apparent pure form by this procedure as well, and the yield was 0.5 mg from 200 ml of urine. The average ratio of the two components in urine, UV absorption and 1H-NMR spectra of the two components, and 13C-NMR spectrum, mass spectrum and elemental analysis of the major component identified the major and minor components as N(1)-methyl-2-pyridone-5-carboxamide and N(1)-methyl-4-pyridone-5-carboxamide, respectively, two major metabolites of the vitamin niacin present in a significant amount in coffee beans. The two metabolites were present in the same average amount in urine extracts of individuals irregardless of coffee consumption. The findings are briefly discussed in relation to the nutritional sources of niacin and to current procedures for measuring amounts of the two metabolites in urine samples.

Lipid peroxidation in nicotinamide-deficient and nicotinamide-supplemented rats.

Supplementation or deficiency of nicotinamide in rats may interfere with the oxidative balance, with excess leading to greater lipid peroxidation, measured by TBARS, and deficiency causing a greater consumption of antioxidants such as vitamin E and glutathione. Urinary N-methylnicotinamide excretion was much more marked in the supplemented group, whereas the difference between deficient and control animals was nonsignificant.

Simultaneous determination of nicotinic acid and its metabolites in rat urine by micellar electrokinetic chromatography with photodiode array detection.

Nicotinic acid, nicotinamide and their possible metabolites were successfully separated within 17 min by micellar electrokinetic chromatography using 50 mM borate buffer (pH 9.0) containing 150 mM sodium dodecyl sulfate as the running buffer. Calibration curves for all compounds showed good linearity in a range of 5 microg/ml and 250 microg/ml with good correlation. The present method did not require any clean-up procedures and made it possible to determine all metabolites without interference on a photodiode array detector. Urine samples collected from Wistar male rats were analyzed after high-dose oral or intravenous administration of nicotinic acid or nicotinamide. Metabolic pathways of nicotinic acid in male Wistar rats are also discussed.

[Body niacin status in experimental diabetes mellitus; effect of protein level in the ration]. / Niatsinovyi status organizma pri éksperimental'nom sakharnom diabete; vliianie urovnia belka v ratsione.

Administration of a high-protein diet providing 7-7.8 g of tryptophan per kg of the ration to rats with streptozotocin and alloxan diabetes mellitus resulted in development of a trend to increased liver content of nicotinamide coenzymes and in increased 1-methylnicotinamide excretion with the urine in both groups of animals, this reflecting increased niacin synthesis from tryptophan. Sugar-reducing effect of high-dose nicotinamide was not potentiated by increase of protein share in the ration. These results permitted the authors to suggest that intensification of endogenous niacin synthesis from tryptophan contained in the ration may be one of the mechanisms of a protective effect of high-protein diets in diabetes.

[Jejunal electrogenic transport of glucose in rats with niacin deficiency]. / Transporte jejunal eletrogênico da glicose em ratos com deficiência de niacina.

The active electrogenic absorption of glucose was studied in 12 niacin deficient rats using a method for measuring changes in transmural potential difference across jejunal mucosa. The glucose was infused in 6 different concentrations (2.5; 5.0; 10.0; 20.0; 50.0 and 100.0 mM/L) at a constant rate of 1.7 ml per minute. The apparent kinetic parameters (Km and Pdmax) of active electrogenic transport were obtained graphically from curves of glucose transfer potentials. The results were compared with that obtained in a control group. The curve of glucose transfer potential in niacin deficient group was significantly lower than that of the control group. The apparent Km of niacin deficient group was greater than in the control group (16.1 x 12.7 mM/L). Furthermore, the Pdmax of the deficient group was lower than that of the control group (12.5 x 19.4 mV). The results showed that in niacin deficiency occurs a decreasing of the active electrogenic glucose absorption. One of the possible interpretation of the differences in the kinetic characteristics of electrogenic glucose transport would be a depleted energy supplement for the active transport in the enterocyte of the niacin deficient rats.

[The effect of alimentary factors on the status of nicotinamide nucleotides in acute phosphorus poisoning]. / Vliianie alimentarnykh faktorov na sostoianie nikotinamidnykh nukleotidov pri ostroi fosfornoi intoksikatsii.

It has been shown that the status condition of pyridine nucleotides is significantly deteriorated in the hepatic tissues of animals with experimental acute phosphorus intoxication. The content of reduced forms of these nucleotides in hepatocytes is increased, while the concentration of oxidized potentials is lowered. It has been established that balanced and vitamin-enriched diets produce a modifying effect on the recovery of the functions of the above coenzymes, that should be taken into consideration when medico-prophylactic diets are composed for workers engaged in phosphorus production.

Interaction of niacin and zinc metabolism in patients with alcoholic pellagra.

The effect of zinc supplementation on the metabolism of tryptophan conversion to niacin was studied in 14 alcoholic patients with pellagra and in 7 male control subjects aged 21-45 y. The pellagrins received chemically defined diets based on crystalline amino acids through an enteral tube for 7 d. Patients were divided into two groups (A and B), both receiving a diet from which tryptophan, Zn, and niacin were excluded. Patients in group B, however, received 220 mg Zn sulfate orally. Upon admission the pellagra patients had low plasma Zn levels and low urinary excretion values of N'methylnicotinamide (N'MN) and N'methyl-2-pyridone-5-carboxamide (2-PYR) in relation to the control subjects (p less than 0.01). During the experimental period there was an increase in plasma Zn levels (p less than 0.005) and in urinary N'MN (p less than 0.05) and 2-PYR (p less than 0.05) excretion in the patients receiving Zn supplementation (group B). These results suggest that Zn interacts with niacin metabolism in alcoholic patients with pellagra through a probable mediation by vitamin B-6.

Effect of supplementing low protein diets with the limiting amino acids on the excretion of N1-methylnicotinamide and its pyridones in rats.

We have hypothesized that the ratio of the excreted by-products of niacin metabolism, N1-methyl-2-pyridone-5-carboxamide (2-pyr) + N1-methyl-4-pyridone-3-carboxamide (4-pyr)/N1-methylnicotinamide (MNA), might be useful as an index to assess the adequacy of amino acid intake in rats. The experiment reported herein was performed to test this hypothesis. When a 10, 20 or 40% casein diet supplemented with 0.1, 0.2 or 0.4% L-methionine, respectively, was fed to rats, the urinary excretion of MNA decreased, and that of 4-pyr increased, as the level of dietary casein and methionine increased. Therefore, the ratio of (2-pyr + 4-pyr)/MNA increased with increasing dietary casein and methionine levels. When the limiting amino acids of casein or soy protein isolate were added to a low casein or low soy protein isolate diet, the urinary ratio of (2-pyr + 4-pyr)/MNA also increased. These results indicate that the increased urinary ratio of (2-pyr + 4-pyr)/MNA can serve as a biological marker for adequate amino acid intake.

Biochemical markers for assessment of niacin status in young men: urinary and blood levels of niacin metabolites.

Biochemical markers of niacin status were studied in healthy young men fed 6.1 to 32 niacin equivalents (NE) per day over an 11-wk period while residing in a metabolic unit. Methylated metabolites of niacin, N1-methylnicotinamide (NMN) and N1-methyl-2-pyridone-5-carboxamide (2-pyr), in urine and plasma were determined during periods of low (6.1 or 10.1 NE per day), adequate (19 NE per day = 1 RDA) and high (25 or 32 NE per day) niacin intakes and after small test doses of nicotinamide. Urine excretion of less than 1.2 mg/d of either NMN or 2-pyr was a reliable indicator of subjects receiving the lowest intake of 6.1 NE/d, but the NMN metabolite was a better marker of subjects ingesting 10.1 NE/d. The ratio of 2-pyr/NMN in urine was not as good a measure of the 6.1 NE/d intake as the individual metabolite excretions and was not responsive to the 10.1 NE/d intake. Plasma niacin metabolites were generally not as reliable as urinary metabolites for identifying subjects receiving low niacin intakes, however, values for plasma 2-pyr dropped quickly and were eventually nondetectable. After a 1 RDA oral dose of nicotinamide, increases in urine and plasma 2-pyr levels above pre-dose baseline values were significantly decreased in subjects receiving low, as compared to adequate, niacin intake. A leucine supplement had no effect on the rate of repletion of niacin-deficient subjects nor on the level of methylated niacin metabolites in urine or plasma.

Ethanol and leucine as possible stress factors in rats marginally deficient in niacin and vitamin B-6.

Young rats were fed a purified diet containing no niacin (and 0.85 g/kg tryptophan) and only 0.5 mg/kg vitamin B-6. Three supplements were tested in a 2 x 2 x 2 multifactorial experiment: 1) 25 mg/kg niacin plus 7.3 mg/kg vitamin B-6; 2) 15 g/kg L-leucine; and 3) 12.15% (wt/wt) ethanol in the drinking water. The ethanol, for those receiving it, contributed approximately 15% of the rats' total energy intake, and their diets had some of the carbohydrate omitted to maintain approximately the same proportion of energy to other nutrients. The vitamin supplements stimulated 4-wk weight gain (and gain-feed ratio), but ethanol depressed it, and to a significantly greater extent in the absence of the vitamin supplement. Excretion of N1-methylnicotinamide was increased only by the vitamin supplement. Leucine had no significant effects either by itself or by interaction with the other supplements.

The role of column switching in analysing complex samples.

Our objective in using column switching is primarily to achieve the desired separation in the minimum analysis time. Complimentary to this aim is the need for sample and column cleanup followed by column re-equilibration. Finally, all operations should be capable of automation. Fundamental to column switching methodology is the concept of Zone cutting, where part of the chromatogram is transferred to another column. This forms the basis of sample cleanup and is a very versatile and powerful methods. Multiple zone cutting is also possible to further increase to scope of cleanup or to minimise analysis time. Zone cutting is also complimentary to the techniques of trace enrichment and recycling. Examples will be given involving the use of these techniques in the analysis of complex matrices such as urine, plant extracts, wine and serum. The latter will be used to propose a novel approach to the quantitative analysis of anti-convulsants in serum using hexobarbital as internal standard.

Effect of tryptophan intake on oxidation of [7a-14C]tryptophan and urinary excretion on N1-methylnicotinamide in the rat.

Oxidation of tryptophan and urinary excretion of niacin metabolites were studied in weanling rats fed ad libitum for 12 to 16 days niacin-free and nicotinamide-supplemented (20 mg/kg of diet) diets containing 15% of crystalline amino acids and from 0.04 to 1.0% of L-tryptophan. N1-methylnicotinamide was measured in urine collected during the last 3 days of the feeding period. After the feeding period, rats were placed in metabolic cages, and each rat was given 4 g (dry weight) of diet containing a tracer dose of DL-[7a-14C]tryptophan. Expired CO2 was collected hourly for 10 hours. In both the niacin-free and nicotinamide-supplemented groups, weight gains increased with increasing increments of tryptophan up to about 0.16% L-tryptophan. With more than 0.16% of L-tryptophan, the amount of N1-methylnicotinamide excreted per day by both groups of rats increased linearly with increasing tryptophan intake, but proportionately less N1-methylnicotinamide was excreted when the dietary tryptophan level was 1.0%. The amount of tryptophan oxidized per 10 hours increased linearly with increasing dietary tryptophan levels between 0.16 and 1.0%. Therefore, the decline in excretion of N1-methylnicotinamide with the 1.0% tryptophan level could not be accounted for by increased oxidation. These results suggest that after the tryptophan requirement for growth is met, the amount of tryptophan oxidized and converted to nicotinamide is directly proportional to the level of tryptophan intake up to about 3 times the requirement for growth. The tryptophan requirement of the growing rat fed niacin-free and nicotinamide-supplemented diets estimated from the inflection points of N1-methylnicotinamide excretion curves was in good agreement with the requirement of about 100 mumoles/day determined from the growth curve.