Maternal Nutrient Restriction Disrupts Gene Expression and Metabolites Associated with Urea Cycle, Steroid Synthesis, Glucose Homeostasis, and Glucuronidation in Fetal Calf Liver.

This study aimed to understand the mechanisms underlying the effects of maternal undernutrition (MUN) on liver growth and metabolism in Japanese Black fetal calves (8.5 months in utero) using an approach that integrates metabolomics and transcriptomics. Dams were fed 60% (low-nutrition; LN) or 120% (high-nutrition; HN) of their overall nutritional requirements during gestation. We found that MUN markedly decreased the body and liver weights of the fetuses; metabolomic analysis revealed that aspartate, glycerol, alanine, gluconate 6-phosphate, and ophthalmate levels were decreased, whereas UDP-glucose, UDP-glucuronate, octanoate, and 2-hydroxybutyrate levels were decreased in the LN fetal liver (p ≤ 0.05). According to metabolite set enrichment analysis, the highly different metabolites were associated with metabolisms including the arginine and proline metabolism, nucleotide and sugar metabolism, propanoate metabolism, glutamate metabolism, porphyrin metabolism, and urea cycle. Transcriptomic and qPCR analyses revealed that MUN upregulated QRFPR and downregulated genes associated with the glucose homeostasis (G6PC, PCK1, DPP4), ketogenesis (HMGCS2), glucuronidation (UGT1A1, UGT1A6, UGT2A1), lipid metabolism (ANGPTL4, APOA5, FADS2), cholesterol and steroid homeostasis (FDPS, HSD11B1, HSD17B6), and urea cycle (CPS1, ASS1, ASL, ARG2). These metabolic pathways were extracted as relevant terms in subsequent gene ontology/pathway analyses. Collectively, these results indicate that the citrate cycle was maintained at the expense of activities of the energy metabolism, glucuronidation, steroid hormone homeostasis, and urea cycle in the liver of MUN fetuses.

Maternal Undernutrition during Pregnancy Alters Amino Acid Metabolism and Gene Expression Associated with Energy Metabolism and Angiogenesis in Fetal Calf Muscle.

To elucidate the mechanisms underlying maternal undernutrition (MUN)-induced fetal skeletal muscle growth impairment in cattle, the longissimus thoracis muscle of Japanese Black fetal calves at 8.5 months in utero was analyzed by an integrative approach with metabolomics and transcriptomics. The pregnant cows were fed on 60% (low-nutrition, LN) or 120% (high-nutrition, HN) of their overall nutritional requirement during gestation. MUN markedly decreased the bodyweight and muscle weight of the fetus. The levels of amino acids (AAs) and arginine-related metabolites including glutamine, gamma-aminobutyric acid (GABA), and putrescine were higher in the LN group than those in the HN group. Metabolite set enrichment analysis revealed that the highly different metabolites were associated with the metabolic pathways of pyrimidine, glutathione, and AAs such as arginine and glutamate, suggesting that MUN resulted in AA accumulation rather than protein accumulation. The mRNA expression levels of energy metabolism-associated genes, such as PRKAA1, ANGPTL4, APLNR, CPT1B, NOS2, NOS3, UCP2, and glycolytic genes were lower in the LN group than in the HN group. The gene ontology/pathway analysis revealed that the downregulated genes in the LN group were associated with glucose metabolism, angiogenesis, HIF-1 signaling, PI3K-Akt signaling, pentose phosphate, and insulin signaling pathways. Thus, MUN altered the levels of AAs and expression of genes associated with energy expenditure, glucose homeostasis, and angiogenesis in the fetal muscle.

Effects of low and high levels of maternal nutrition consumed for the entirety of gestation on the development of muscle, adipose tissue, bone, and the organs of Wagyu cattle fetuses.

This study aimed to investigate the effects of high and low levels of energy intake during the entire gestation period on the skeletal muscle development, organ development, and adipose tissue accumulation in fetuses of Wagyu (Japanese Black) cows, a breed with highly marbled beef. Cows were allocated to a high-nutrition (n = 6) group (fed 120% of the nutritional requirement) or low-nutrition (n = 6) group (fed 60% of the nutritional requirement). The cows were artificially inseminated with semen from the same sire, and the fetuses were removed by cesarean section at 260 ± 8.3 days of fetal age and slaughtered. The whole-body, total muscle, adipose, and bone masses of the fetal half-carcasses were significantly higher in the high-nutrition group than the low-nutrition group (p = 0.0018, 0.009, 0.0004, and 0.0362, respectively). Fifteen of 20 individual muscles, five of six fat depots, nine of 17 organs, and seven of 12 bones that were investigated had significantly higher masses in the high-nutrition group than the low-nutrition group. The crude components and amino acid composition of the longissimus muscle significantly differed between the low- and high-nutrition groups. These data indicate that maternal nutrition during gestation has a marked effect on the muscle, bone, and adipose tissue development of Wagyu cattle fetuses.

Amino Acid Composition of Amniotic Fluid during the Perinatal Period Reflects Mother's Fat and Carbohydrate Intake.

Dietary content during pregnancy is important because it is necessary for the growth of the fetus. With the assumption that the nutritional status of the fetus can be monitored by measuring amino acid concentrations in the amniotic fluid, we investigated whether the habitual dietary intake of pregnant women affected the composition of the amniotic fluid and the significance of performing amniotic fluid analysis. The subjects were 34 mothers who delivered full-term babies by cesarean section. Three biological samples were collected from the mothers: blood, cord blood, and amniotic fluid. At the same time, the mothers' prenatal nutritional intake information was also recorded. When the amino acid contents of the samples were compared with the mothers' nutrient intake, many amino acids in the amniotic fluid were positively correlated with lipid intake, but not with protein intake. There was a negative correlation between lipid intake and carbohydrate intake, and the amino acid contents of the amniotic fluid were also negatively correlated with carbohydrate intake. The results of this study were consistent with those found in animal models, suggesting that the analysis of amniotic fluid may be a useful method to investigate the effects of habitual diet during human pregnancy on the fetus.

Maternal Nutrition During Gestation Alters Histochemical Properties, and mRNA and microRNA Expression in Adipose Tissue of Wagyu Fetuses.

We hypothesized that maternal low or high nutrition would give unique effects to morphological and molecular dynamics in adipose tissue of fetus of fatty breed Wagyu (Japanese Black) cattle which produce highly marbled beef. This study aimed to determine the effects of maternal energy intake in Wagyu cows, during gestation on fetal adipose tissue development, histochemical properties, and gene and microRNA (miRNA) expression. Cows were allocated to one of two nutritional energy groups: 120% (HIGH) or 60% nutritional requirements of (LOW). Fetuses (n = 6 per treatment) were removed from pregnant cows by cesarean section at fetal age 260 ± 8 days and euthanized. Subcutaneous adipose tissue (SAT), thoracic cavity visceral adipose tissue (TVAT), and perirenal adipose tissue (PAT) were collected for analysis. In histochemical analysis, in SAT and PAT, HIGH fetuses had greater diameter of adipocytes than LOW fetuses (P<0.05). Only in SAT, LOW fetuses had more Leptin (LEP) mRNA and tended to have more Peroxisome Proliferator-Activated Receptor gamma (PPARG) CCAAT-enhancer-binding proteins alpha (CEBPA) and Glucose transporter (GLUT) 4 mRNA(P<0.10). In all SAT, TVAT, and PAT, LOW fetuses had higher levels of the brown adipose tissue (BAT) biomarkers Uncoupling Protein (UCP) 1 and PPARG coactivator (PGC) 1α mRNA than HIGH fetuses (P<0.08). Meanwhile, in the other adipose tissue, LOW fetuses had lower PPARG, CEBPA, and Zinc Finger Protein (ZFP) 423 (in TVAT and PAT), FASN (in TVAT), LEP and GLUT4 mRNA (in PAT; P<0.10). In particular, in TVAT and PAT, LOW fetuses exhibited lower expression of WAT biomarkers (PPARG and ZFP423). Differential expression of various miRNAs related to adipogenesis between the LOW and HIGH fetuses was detected in an adipose tissue-specific manner (P<0.10). Based on adipose tissue-specific effects of maternal nutrition, these findings suggested that poor maternal nutrition in Wagyu cattle increased BAT development in SAT, TVAT and PAT, while elevated maternal nutrition stimulated fetal SAT development compared with that of TVAT and PAT.

Maternal and fetal tryptophan metabolism in gestating rats: effects of intrauterine growth restriction.

L-Tryptophan (L-Trp) is a precursor for serotonin (5-HT) and nicotinamide adenine dinucleotide (NAD) synthesis. Both 5-HT and NAD may impact energy metabolism during gestation given that recent studies have demonstrated that increased 5-HT production is crucial for increasing maternal insulin secretion, and that sirtuin, an NAD(+)-dependent protein deacetylase, regulates endocrine signaling. Infants born with intrauterine growth restriction (IUGR) are at a higher risk of metabolic disease once they reach adulthood. IUGR is associated with altered maternal-fetal amino acid transfer. Whether IUGR affects L-Trp metabolism in mother and fetus has not been fully elucidated. Recently, we developed an analytical method using stable isotope-labeled L-Trp to explore the metabolism of L-Trp and its main metabolites, L-kynurenine (L-Kyn), 5-HT and quinolinic acid (QA). In this study, dams submitted to dietary protein restriction throughout gestation received intravenous infusions of stable isotope-labeled (15)N2-L-Trp to determine whether L-Trp metabolism is affected by IUGR. Samples were obtained from maternal, fetal and umbilical vein plasma, as well as the amniotic fluid (AF), placenta and liver of the mother and the fetus after isotope infusion. We observed evidence for active L-Trp transfer from mother to fetus, as well as de novo synthesis of 5-HT in the fetus. Plasma 5-HT was decreased in undernourished mothers. In IUGR fetuses, maternal-fetal L-Trp transfer remained unaffected, but conversion to QA was impaired, implying that NAD production also decreased. Whether such alterations in tryptophan metabolism during gestation have adverse consequences and contribute to the increased risk of metabolic disease in IUGR remains to be explored.

Amino acids inhibit kynurenic acid formation via suppression of kynurenine uptake or kynurenic acid synthesis in rat brain in vitro.

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor at endogenous brain concentrations. Recent studies have suggested that increase of brain KYNA levels is involved in psychiatric disorders such as schizophrenia and depression. KYNA-producing enzymes have broad substrate specificity for amino acids, and brain uptake of kynurenine (KYN), the immediate precursor of KYNA, is via large neutral amino acid transporters (LAT). In the present study, to find out amino acids with the potential to suppress KYNA production, we comprehensively investigated the effects of proteinogenic amino acids on KYNA formation and KYN uptake in rat brain in vitro. Cortical slices of rat brain were incubated for 2 h in Krebs-Ringer buffer containing a physiological concentration of KYN with individual amino acids. Ten out of 19 amino acids (specifically, leucine, isoleucine, phenylalanine, methionine, tyrosine, alanine, cysteine, glutamine, glutamate, and aspartate) significantly reduced KYNA formation at 1 mmol/L. These amino acids showed inhibitory effects in a dose-dependent manner, and partially inhibited KYNA production at physiological concentrations. Leucine, isoleucine, methionine, phenylalanine, and tyrosine, all LAT substrates, also reduced tissue KYN concentrations in a dose-dependent manner, with their inhibitory rates for KYN uptake significantly correlated with KYNA formation. These results suggest that five LAT substrates inhibit KYNA formation via blockade of KYN transport, while the other amino acids act via blockade of the KYNA synthesis reaction in brain. Amino acids can be a good tool to modulate brain function by manipulation of KYNA formation in the brain. This approach may be useful in the treatment and prevention of neurological and psychiatric diseases associated with increased KYNA levels.

Time-dependent effects of L-tryptophan administration on urinary excretion of L-tryptophan metabolites.

We have previously reported that dietary supplementation with up to 5.0 g/d of L-tryptophan (L-Trp) for 21 d has no adverse effects, judging from the levels of general blood variables, in healthy women. We performed a randomized, double-blind, placebo-controlled, crossover intervention study in 17 apparently healthy Japanese women. The subjects were randomly assigned to receive a 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. We examined the 24-h urine profiles on days -1 (1 d before starting L-Trp), 7, 14, and 21 to determine whether administration of L-Trp at doses of up to 5.0 g/d affects time-dependent urinary excretion of L-Trp or its metabolites in healthy women. The urinary excretion of L-Trp, kynurenic acid, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, N(1)-methylnicotinamide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide increased in an L-Trp dose-dependent manner on day 7. The amount of urinary excretion of these compounds was unchanged on days 14 and 21. The urinary excretion of serotonin, 5-hydroxyindole-3-acetic acid, 2-oxoadipic acid, and nicotinamide was unaffected by L-Trp at any of the doses tested. L-Trp doses had weak effects on the urinary excretion of kynurenine and anthranilic acid. Based on these findings, we conclude that there are no time-dependent effects of L-Trp administration in urinary excretion of L-Trp metabolites. Additionally, L-Trp and its metabolites do not accumulate in the body.

The urinary ratio of 3-hydroxykynurenine/3-hydroxyanthranilic acid is an index to predicting the adverse effects of D-tryptophan in rats.

The adverse effects of D-tryptophan and the possibility of it being a surrogate index for predicting adverse effects in rats were investigated. Male rats were fed one of several test diets (20% casein diets with 0% (control), 0.1%, 0.2%, 0.3%, 0.4%, and 0.5% D-tryptophan) for 21 d, and 24-h urine samples on the final day of the experiment were collected. Analyses of food intake and body-weight changes revealed adverse effects to be observed in the group fed the 0.3% D-tryptophan diet. We propose urinary levels of 3-hydroxykynurenine/3-hydroxyanthranilic acid to be surrogate indicators for predicting the adverse effects of D-tryptophan from the break point of body-weight gains and urinary levels of D-tryptophan metabolites. The reaction 3-hydroxykynurenine→3-hydroxyanthranilic acid is catalyzed by the pyridoxal phosphate-dependent enzyme kynureninase. Increasing urinary 3-hydrokykynurenine indicates kynureninase deficiency. Intake of D-tryptophan in rats fed the 0.3% D-tryptophan diet was 0.21 g/kg body weight and feeding of the 0.3% D-tryptophan diet did not elicit adverse effects. Thus, the safe level of D-tryptophan was less than 0.2% in the diet, 0.15 g/kg body weight, in rats.

Simultaneous measurement of nicotinamide and its catabolites, nicotinamide N-oxide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-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, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-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.

Contributions of tryptophan 2,3-dioxygenase and indoleamine 2,3-dioxygenase to the conversion of D-tryptophan to nicotinamide analyzed by using tryptophan 2,3-dioxygenase-knockout mice.

We investigated the contribution percentage of tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) to the conversion of D-tryptophan to nicotinamide in TDO-knockout mice. The calculated percentage conversions indicated that TDO and IDO oxidized 70 and 30%, respectively, of the dietary L-tryptophan. These results indicate that both TDO and IDO biosynthesize nicotinamide from D-tryptophan and L-tryptophan in mice.

Simultaneous detection of stable isotope-labeled and unlabeled L-tryptophan and of its main metabolites, L-kynurenine, serotonin and quinolinic acid, by gas chromatography/negative ion chemical ionization mass spectrometry.

A method for the detection of unlabeled and (15)N2 -labeled L-tryptophan (L-Trp), L-kynurenine (L-Kyn), serotonin (5-HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. L-[(13)C11, (15)N2]-Trp, methyl-serotonin and 3,5-pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter-assay repeatability were found to be approximately 5.2% for L-Trp and (15)N2-Trp, 17.1% for L-Kyn, 16.9% for 5-HT and 5.8% for QA (n = 2). We used this method to determine isotope enrichments in plasma L-Trp over the course of a continuous, intravenous infusion of L-[(15) N2 ]Trp in pregnant rat in the fasting state. Plasma (15)N2-Trp enrichment reached a plateau at 120 min. The free Trp appearance rate (Ra) into plasma was 49.5 ± 3.35 µmol/kg/h. The GC/MS method was applied to determine the enrichment of (15)N-labeled L-Trp, L-Kyn, 5-HT and QA concurrently with the concentration of non-labeled L-Trp, L-Kyn, 5-HT and QA in plasma. This method may help improve our understanding on L-Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of L-Trp metabolism.

Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na(+)-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na(+)-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

L-tryptophan metabolism in pregnant mice fed a high L-tryptophan diet and the effect on maternal, placental, and fetal growth.

Excess L-tryptophan (L-Trp) in the diet decreases fetal body weight. However, the relationship between L-Trp concentration and its effects on maternal, placental, and fetal growth are not well-understood. We investigated the effects of excess L-Trp intake on maternal, placental, and fetal growth. Female mice were fed a 20% casein diet (control diet) or control diet plus 2% or 5% L-Trp during gestation. Pup weights did not differ between the control (L-Trp intake: 0.04 g/kg body weight (BW)/day) and 2% L-Trp groups (L-Trp intake: 3.3 g/kg BW/day), but were significantly lower in the 5% L-Trp group (L-Trp intake: 7.0 g/kg BW/day) than in the control and 2% L-Trp groups. These results show that less than 3.3 g/kg BW/day L-Trp intake in pregnant mice during gestation does not affect fetal growth or L-Trp homeostasis in the placenta or fetus.

Urinary excretion levels of water-soluble vitamins in pregnant and lactating women in Japan.

Recent studies have shown that the urinary excretion levels of water-soluble vitamins can be used as biomarkers for the nutritional status of these vitamins. To determine changes in the urinary excretion levels of water-soluble vitamins during pregnant and lactating stages, we surveyed and compared levels of nine water-soluble vitamins in control (non-pregnant and non-lactating women), pregnant and lactating women. Control women (n=37), women in the 2nd (16-27 wk, n=24) and 3rd trimester of pregnancy (over 28 wk, n=32), and early- (0-5 mo, n=54) and late-stage lactating (6-11 mo, n=49) women took part in the survey. The mean age of subjects was ~30 y, and mean height was ~160 cm. A single 24-h urine sample was collected 1 d after the completion of a validated, self-administered comprehensive diet history questionnaire to measure water-soluble vitamins or metabolites. The average intake of each water-soluble vitamin was ≍ the estimated average requirement value and adequate intake for the Japanese Dietary Reference Intakes in all life stages, except for vitamin B6 and folate intakes during pregnancy. No change was observed in the urinary excretion levels of vitamin B2, vitamin B6, vitamin B12, biotin or vitamin C among stages. Urine nicotinamide and folate levels were higher in pregnant women than in control women. Urine excretion level of vitamin B1 decreased during lactation and that of pantothenic acid decreased during pregnancy and lactation. These results provide valuable information for setting the Dietary Reference Intakes of water-soluble vitamins for pregnant and lactating women.

Changes in B-group vitamin status in adenine-induced chronic renal failure rats.

The purpose of this study was to determine the effects of chronic renal failure (CRF) on B-group vitamin status using model rats in which adenine-induced CRF. We measured B-groups vitamins in the urine, blood, liver, and kidney. These results showed that renal failure affected the distribution, metabolism, and renal clearance of water-soluble vitamins, and that the effects were different with each vitamin.

The niacin required for optimum growth can be synthesized from L-tryptophan in growing mice lacking tryptophan-2,3-dioxygenase.

In mammals, nicotinamide (Nam) is biosynthesized from l-tryptophan (l-Trp). The enzymes involved in the initial step of the l-Trp→Nam pathway are l-Trp-2,3-dioxygenase (TDO) and indoleamine-2,3-dioxygenase (IDO). We aimed to determine whether tdo-knockout (tdo(-/-)) mice fed a diet without preformed niacin can synthesize enough Nam to sustain optimum growth. Wild-type (WT) and tdo(-/-) mice were fed a chemically defined 20% casein diet with or without preformed niacin (30 mg nicotinic acid/kg) for 28 d. Body weight, food intake, and liver NAD concentrations did not differ among the groups. In the groups of mice fed the niacin-free diet, urinary concentrations of the upstream metabolites kynurenine (320% increase, P < 0.0001), kynurenic acid (270% increase, P < 0.0001), xanthurenic acid (770% increase, P < 0.0001), and 3-hydroxyanthranilic acid (3-HA; 450% increase, P < 0.0001) were higher in the tdo(-/-) mice than in the WT mice, while urinary concentrations of the downstream metabolite quinolinic acid (QA; 50% less, P = 0.0010) and the sum of Nam and its catabolites (10% less, P < 0.0001) were lower in the tdo(-/-) mice than in the WT mice. These findings show that the kynurenine formed in extrahepatic tissues by IDO and subsequent enzymes can be metabolized up to 3-HA, but not into QA. However, the tdo(-/-) mice sustained optimum growth even when fed the niacin-free diet for 1 mo, suggesting they can synthesize the minimum necessary amount of Nam from l-Trp, because the liver can import blood kynurenine formed in extrahepatic tissues and metabolize it into Nam via NAD and the resulting Nam is then distributed back into extrahepatic tissues.

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.

Increased conversion of tryptophan to nicotinamide in rats by dietary valproate.

Valproic acid (VPA) is a short-chained, branched fatty acid that is widely used in humans as an anticonvulsant and mood stabilizer, and has been reported to increase the liver NAD concentration. We investigated the effects of VPA on the conversion of tryptophan to nicotinamide. Rats were fed diets containing various amounts of VPA (0, 0.5, and 1.0% in the diets) for 14 d, 24-h urine samples were collected, and tryptophan and its catabolites were measured. We found that the conversion of tryptophan to nicotinamide was increased by feeding a diet containing VPA (p<0.01; 0% vs. 1.0% VPA). Of the intermediates formed during the conversion of tryptophan to nicotinamide, the tryptophan to 3-hydroxyanthranilic acid step was not affected by the administration of VPA, while such metabolites beyond quinolinic acid as nicotinamide and its catabolites were significantly increased (p<0.01; 0% vs. 1.0% VPA). This increase was dependent on the intake of VPA.

Urinary excretion of B-group vitamins reflects the nutritional status of B-group vitamins in rats.

We have reported previously that the urinary excretion of B-group vitamins reflects recent dietary intakes of these vitamins. We also proposed reference values for the urinary levels of B-group vitamins for human subjects, and used these for evaluating human nutritional status. However, the question arises as to whether the urinary excretion of B-group vitamins in animals or human subjects decreases immediately before they become B-group vitamin insufficient or when fed a diet low in vitamins. In the present study, rats were fed a vitamin-free diet for 5 d, and changes in the levels of B-group vitamins in urine and blood were monitored. Urinary excretion of vitamin B1, vitamin B2, 4-pyridoxic acid (a catabolite of vitamin B6), pantothenic acid, folate and biotin steeply decreased, and all of the values reached zero within 1-2 d. With respect to blood, the concentrations of only three of the eight B-group vitamins (vitamin B1, pyridoxal phosphate and biotin) decreased to 15 % (P < 0·0001), 7 % (P < 0·0001) and 2 % (P < 0·0001) on day 5, respectively, compared with the values at the beginning of the experiment. The decrease was more rapid and the changes were greater in the urine samples than in the blood samples. The present data complement our previous proposal that the urinary excretion of B-group vitamins reflects the nutritional status of these vitamins.