High-performance liquid chromatographic method for profiling 2-oxo acids in urine and its application in evaluating vitamin status in rats.

B-group vitamins are involved in the catabolism of 2-oxo acids. To identify the functional biomarkers of B-group vitamins, we developed a high-performance liquid chromatographic method for profiling 2-oxo acids in urine and applied this method to urine samples from rats deficient in vitamins B1 and B6 and pantothenic acid. 2-Oxo acids were reacted with 1,2-diamino-4,5-methylenebenzene to produce fluorescent derivatives, which were then separated using a TSKgel ODS-80Ts column with 30 mmol/L of KH2PO4 (pH 3.0):acetonitrile (7:3) at a flow rate of 1.0 mL/min. Vitamin B1 deficiency increased urinary levels of all 2-oxo acids, while vitamin B6 deficiency only increased levels of sum of 2-oxaloacetic acid and pyruvic acid, and pantothenic acid deficiency only increased levels of 2-oxoisovaleric acid. Profiles of 2-oxo acids in urine samples might be a non-invasive way of clarifying the functional biomarker of B-group vitamins.

Moderate food restriction suppresses the conversion of L-tryptophan to nicotinamide in weaning rats.

Calorie restriction leads to a change in the metabolism of nutrients. Nicotinamide is biosynthesized from L-tryptophan. We attempted to determine the effects of food restriction on the biosynthesis of nicotinamide from L-tryptophan. Weaning male rats were fed a conventional chemically defined diet without preformed niacin for 63 d. However, the food intake was restricted to 80 and 65% of the intake of the ad libitum-fed control group of rats. The 24-h urine samples were periodically collected, and the urinary excretion of nicotinamide and its catabolites was measured. The conversion percentages were lower in both restricted groups than in the ad libitum-fed control group during the experimental period (control group, 1.37 ± 0.24%; 80%-restricted group, 0.20 ± 0.04%; 65%-restricted group, 0.15 ± 0.02%; control vs. restricted groups, p < 0.01). Food restriction, even at mild level, suppressed the conversion of L-tryptophan to nicotinamide when compared to the ad libitum-fed control group.

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.

Pantothenic acid deficiency may increase the urinary excretion of 2-oxo acids and nicotinamide catabolites in rats.

Pantothenic acid (PaA) is involved in the metabolism of amino acids as well as fatty acid. We investigated the systemic metabolism of amino acids in PaA-deficient rats. For this purpose, urine samples were collected and 2-oxo acids and L-tryptophan (L-Trp) and its metabolites including nicotinamide were measured. Group 1 was freely fed a conventional chemically-defined complete diet and used as an ad lib-fed control, which group was used for showing reference values. Group 2 was freely fed the complete diet without PaA (PaA-free diet) and used as a PaA-deficient group. Group 3 was fed the complete diet, but the daily food amount was equal to the amount of the PaA-deficient group and used as a pair-fed control group. All rats were orally administered 100 mg of L-Trp/kg body weight at 09:00 on day 34 of the experiment and the following 24-h urine samples were collected. The urinary excretion of the sum of pyruvic acid and oxaloacetic acid was higher in rats fed the PaA-free diets than in the rats fed pair-fed the complete diet. PaA deficiency elicited the increased urinary excretion of anthranilic acid and kynurenic acid, while the urinary excretion of xanthurenic acid decreased. The urinary excretion of L-Trp itself, 3-hydroxyanthranilic acid, and quinolinic acid revealed no differences between the rats fed the PaA-free and pair-fed the complete diets. PaA deficiency elicited the increased excretion of N(1)-methylnicotinamide, N(1)-methyl-2-pyridone-5-carboxamide, and N(1)-methyl-4-pyridone-3-carboxamide. These findings suggest that PaA deficiency disturbs the amino acid catabolism.

The effects of glycine, L-threonine, and L-cystine supplementation to a 9% casein diet on the conversions of L-tryptophan to nicotinamide and to serotonin in rats.

Nicotinamide and serotonin are synthesized from L-tryptophan in mammals. It is important to know the nutritional factors affecting the synthesis of nicotinamide and serotonin. We investigated the effects of amino acid composition. Young adult rats were fed ad libitum for 21 d a low-protein (9% casein) diet([1] control), or one of the low protein diets supplemented with following amino acids: [2] glycine, L-threonine, and L-cystine, [3] L-threonine and L-cystine, [4] glycine and L-cystine, and [5] glycine and L-threonine. The amounts of glycine, L-threonine and L-cystine supplementations were 2%, 0.078%, and 0.2%, respectively, and the amino acid contents of all diet were adjusted with supplementation of L-glutamic acid. The body weight gain, food efficiency ratio, and the amino acid nutrition biomarker, which is the urinary excretion ratio of (N(1)-methyl-2-pyridone-5-carboxamide+N(1)-methyl-4-pyridone-3-carboxamide)/N(1)-methylnicotinamide, improved by adding the amino acids glycine, L-threonine and L-cystine to a 9% casein diet. The conversion percentage of L-tryptophan to nicotinamide decreased with the addition of the amino acids glycine, L-threonine and L-cystine to a 9% casein diet, while the concentrations of serotonin in the brain, stomach and small intestine were not affected at all. The effects of each amino acid on body weight gain and the conversion ratios were also investigated. Glycine did not affect these variables. L-Cystine improved the body weight gain, the food efficiency ratio and the urine ratio, and decreased the conversion percentage. L-Threonine did not affect body weight gain or food efficiency ratio; however, it improved the urine ratio and decreased the conversion percentage.