Mitsue Sano

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.

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.

Fluorometric Determination of 2-Oxoadipic Acid, a Common Metabolite of Tryptophan and Lysine, by High-Performance Liquid Chromatography with Pre-Chemical Derivatization

2-Oxoadipic acid, a key metabolite of tryptophan and lysine, reacted with 1,2-diamino-4,5-methylenebenzene in an acidic solution to produce a fluorescent derivative. The reaction product was separated using a Tosoh ODS-80Ts column with 20 mmol/L of KH2PO4–K2HPO4 buffer (pH 7.0) containing 26% methanol at a flow rate 0.8 mL/min. The excitation wavelength of detection was 367 nm, and the emission wavelength was 446 nm. The limit of quantification was 1 pmol per injection, sufficiently sensitive for the determination of 2-oxoadipic acid in human and experimental animal urine.

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

Abstractl-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 15N2-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.

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.

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.

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.

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.

Effects of ethanol consumption on the B-group vitamin contents of liver, blood and urine in rats.

Several studies have shown that blood vitamin levels are lower in alcoholic patients than in control subjects. Acute ethanol exposure enhances the release of vitamins from liver cells in vitro. The aim of the present study is to confirm the effects of ethanol consumption on vitamin contents in vivo. We compared the contents of B-group vitamins in the liver, blood and urine between ethanol-fed and control rats fed a diet containing a sufficient- and low-vitamin mixture. The experimental rats were fed a 15 % ethanol solution freely for 28 d, and then 24 h urine samples were collected, after which the animals were killed. The B-group vitamin contents in the liver, blood and urine were measured. No differences in liver, blood and urine contents were observed between the control and ethanol-fed rats fed a diet containing a sufficient-vitamin mixture. On the contrary, in rats fed a diet containing a low-vitamin mixture, consumption of ethanol caused a decrease in the contents of vitamins B₁, B₂ and pantothenic acid in the liver; however, the contents of the other vitamins did not decrease. In the blood, the contents of vitamins B₁, B₂, B₆ and pantothenic acid were lower in the ethanol-fed rats than in the controls. Urinary excretion of the B-group vitamins, except for niacin, was lower in the ethanol-fed rats. These results show that ethanol consumption affects the absorption, distribution and excretion of each of the vitamins in rats fed a diet containing a low-vitamin mixture.