Glutamate metabolism in a human intestinal epithelial cell layer model.

Plasma glutamate concentrations are constant despite dynamic changes in diets. Most likely, virtually all the dietary glutamate is metabolized in the gut. The present study investigated permeability and metabolism of dietary glutamate in a Caco-2 intestinal epithelial cell layer model by tracing the fate of [U-13C] or [15N]glutamate added to the apical medium. For comparison, several other labelled essential and non-essential amino acids were tested as well. Almost all the labelled glutamate in the apical medium (98% and 96% at 24 h of the culture, respectively) was incorporated in the cell layer, while it barely appeared at the basolateral side, indicating an almost complete utilization of glutamate. Indeed, the 13C was incorporated into alanine, proline, ornithine, and glutamine, and the 15N was incorporated into alanine, glutamine, ornithine, proline, branched chain amino acids and also found as ammonia indicative of oxidation. In contrast, substantial apical-to-basolateral transport of amino acids (8-85% of uptake) other than glutamate and aspartate was evident in studies using amino acid tracers labelled with 13C, 15N or D. These results suggest that the intestinal epithelial cell monolayer utilizes dietary glutamate which adds to maintaining glutamate homeostasis in the body.

Genotoxicity and repeated-dose toxicity evaluation of dried Wolffia globosa Mankai.

Wolffia is a genus of protein-rich aquatic plants. Mankai, a cultivated strain of Wolffia globosa, contains more than 40 % protein based on dry matter evaluation. Furthermore, Mankai is nutritionally excellent as a food material, and is expected to be applicable to various products as a substitute for animal protein. A battery of toxicological studies was conducted on the dried product of Mankai (Dry Mankai), with the expectation to utilize it as a raw material for food applications. Dry Mankai was not genotoxic in a bacterial reverse mutation test and in vitro micronucleus assay. In the subchronic toxicity study, rats were provided Dry Mankai in the diet at levels of 0 %, 5 %, 10 %, or 20 % (w/w), equivalent to 0, 3.18, 6.49, and 13.16 g/kg/day for males and 0, 3.58, 7.42, and 15.03 g/kg/day for females, respectively. No adverse effects that could be attributable to treatment were observed in clinical observations, body weight, food consumption, ophthalmology, hematology and blood chemistry, urinalysis, and macroscopic and microscopic findings. According to the repeated-dose study in rats, the no observed adverse effect level of Dry Mankai was 20 % (w/w) for both sexes (13.16 and 15.03 g/kg/day for males and females, respectively).

In vitro and in vivo genotoxicity studies on monosodium L-glutamate monohydrate.

In response to the lack of authenticated mutagenicity/genotoxicity studies on MSG monohydrate, a series of genotoxicity studies conducted under GLP and according to globally accepted test guidelines (e.g., OECD) was performed. A bacterial reverse mutation test using Salmonella typhimurium (TA100, TA1535, TA98 and TA1537) and Escherichia coli (WP2 uvrA) at concentrations up to 5000 µg/plate, an in vitro chromosomal aberration test in CHL/IU cells at concentrations up to 10 mmol/L (1.9 mg/mL), a mouse lymphoma tk assay at concentrations up to 10 mmol/L (1.9 mg/mL), an in vitro micronucleus test in human peripheral blood lymphocytes at concentrations up to 10 mmol/L (1871 µg/mL), and an in vivo micronucleus test in bone marrow of rats that were gavaged with up to 2000 mg/kg bw were investigated. MSG monohydrate did not cause mutagenicity in any bacterial strain, did not induce chromosomal aberrations in CHL/IU cells or gene mutation in mouse lymphoma cells, was not clastogenic or aneugenic to human lymphocytes, and did not induce micronuclei in erythrocytes of rats when compared with vehicle controls. These results show that MSG is not mutagenic or genotoxic under the study conditions.

The Nitrogen Moieties of Dietary Nonessential Amino Acids Are Distinctively Metabolized in the Gut and Distributed to the Circulation in Rats.

Background: Although previous growth studies in rodents have indicated the importance of dietary nonessential amino acids (NEAAs) as nitrogen sources, individual NEAAs have different growth-promoting activities. This phenomenon might be attributable to differences in the nitrogen metabolism of individual NEAAs. Objective: The aim of this study was to compare nitrogen metabolism across dietary NEAAs with the use of their 15N isotopologues.Methods: Male Fischer rats (8 wk old) were given 1.0 g amino acid-defined diets containing either 15N-labeled glutamate, glutamine (amino or amide), aspartate, alanine, proline, glycine, or serine hourly for 5-6 h. Then, steady-state amino acid concentrations and their 15N enrichments in the gut and in portal and arterial plasma were measured by an amino acid analyzer and LC tandem mass spectrometry, respectively.Results: The intestinal 15N distribution and portal-arterial balance of 15N metabolites indicated that most dietary glutamate nitrogen (>90% of dietary input) was incorporated into various amino acids, including alanine, proline, and citrulline, in the gut. Dietary aspartate nitrogen, alanine nitrogen, and amino nitrogen of glutamine were distributed similarly to other amino acids both in the gut and in the circulation. In contrast, incorporation of the nitrogen moieties of dietary proline, serine, and glycine into other amino acids was less than that of other NEAAs, although interconversion between serine and glycine was very active. Cluster analysis of 15N enrichment data also indicated that dietary glutamate nitrogen, aspartate nitrogen, alanine nitrogen, and the amino nitrogen of glutamine were distributed similarly to intestinal and circulating amino acids. Further, the analysis revealed close relations between intestinal and arterial 15N enrichment for each amino acid. The steady-state 15N enrichment of arterial amino acids indicated that substantial amounts of circulating amino acid nitrogen are derived from dietary NEAAs.Conclusions: The present results revealed similarities and differences among NEAAs in terms of their intestinal nitrogen metabolism in rats and indicated substantial entry of dietary NEAA nitrogen into circulating amino acid nitrogen, presumably primarily through metabolism in the gut.

A 4-week toxicity study of methionine in male rats.

To examine 4-week toxicity of l-methionine (methionine), 5-week-old Fisher strain male rats were fed on diets containing 0, 0.1, 0.3, 0.9, 2.7 (w/w) of added methionine. Although no deaths were recorded, the highest dose of methionine (2.7% [w/w] of diet) reduced food intake and significantly suppressed growth rate. Growth suppression was characterized by an increase in hemolysis, splenic, and hepatic accumulation of hemosiderin, hemolytic anemia, and promotion of hematopoiesis. Other changes observed in the highest methionine intake group were a decrease in white blood cell count, thymus atrophy, and histological abnormalities in the adrenal gland and testis. Small, but significant, growth suppression, accompanied by some minor changes in plasma biochemical parameters, was also seen in rats fed on a test diet containing 0.9% (w/w) of additional methionine. Thus, no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect level (LOAEL) of diet-added methionine were determined at 0.3% and 0.9% (w/w), corresponding to 236 and 705 mg/kg/d body weight, respectively. Since the basal diet contained protein-bound methionine at 0.5% (w/w), NOAEL and LOAEL of total dietary methionine were estimated at 0.8% and 1.4% (w/w) of diet.

Measurement of (15)N enrichment of glutamine and urea cycle amino acids derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate using liquid chromatography-tandem quadrupole mass spectrometry.

6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is an amino acid-specific derivatizing reagent that has been used for sensitive amino acid quantification by liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS). In this study, we aimed to evaluate the ability of this method to measure the isotopic enrichment of amino acids and to determine the positional (15)N enrichment of urea cycle amino acids (i.e., arginine, ornithine, and citrulline) and glutamine. The distribution of the M and M+1 isotopomers of each natural AQC-amino acid was nearly identical to the theoretical distribution. The standard deviation of the (M+1)/M ratio for each amino acid in repeated measurements was approximately 0.1%, and the ratios were stable regardless of the injected amounts. Linearity in the measurements of (15)N enrichment was confirmed by measuring a series of (15)N-labeled arginine standards. The positional (15)N enrichment of urea cycle amino acids and glutamine was estimated from the isotopic distribution of unique fragment ions generated at different collision energies. This method was able to identify their positional (15)N enrichment in the plasma of rats fed (15)N-labeled glutamine. These results suggest the utility of LC-MS/MS detection of AQC-amino acids for the measurement of isotopic enrichment in (15)N-labeled amino acids and indicate that this method is useful for the study of nitrogen metabolism in living organisms.

Long-term ingestion of monosodium L-glutamate did not induce obesity, dyslipidemia or insulin resistance: a two-generation study in mice.

The use of monosodium glutamate (MSG) as a flavor enhancer spans more than 100 y and there are many studies indicating the safety of general use of MSG. Recently, however, Collison et al. (2010) reported a two-generation study with a low dose of MSG that caused abdominal obesity, insulin resistance and dyslipidemia in mice. Due to public health concerns over metabolic syndrome, their report merits careful analysis. The present study attempted to repeat the Collison et al. findings. Groups of male or female C57BL/6J mice were fed a control diet or one supplemented with high-fructose corn syrup (HFCS) at a level of 20%. Drinking water control was provided or treatment groups were given 0.064% MSG solution (w/v). Diets and MSG administration continued throughout mating and during gestation and lactation periods. To further investigate the effects of ingestion of MSG, the offspring were continued on the same dosing conditions until they reached 32 wk of age. MSG administration in mice fed a normal or a HFCS diet throughout gestation and for 32 wk after birth, did not affect growth, girth size, abdominal fat weight or body composition. This study reports that MSG did not trigger insulin resistance, dyslipidemia or hepatic steatosis, regardless of the diet, not reproducing the results of the above-mentioned study (Collison et al., 2010).

Leucine and protein metabolism in obese Zucker rats.

Branched-chain amino acids (BCAAs) are circulating nutrient signals for protein accretion, however, they increase in obesity and elevations appear to be prognostic of diabetes. To understand the mechanisms whereby obesity affects BCAAs and protein metabolism, we employed metabolomics and measured rates of [1-(14)C]-leucine metabolism, tissue-specific protein synthesis and branched-chain keto-acid (BCKA) dehydrogenase complex (BCKDC) activities. Male obese Zucker rats (11-weeks old) had increased body weight (BW, 53%), liver (107%) and fat (∼300%), but lower plantaris and gastrocnemius masses (-21-24%). Plasma BCAAs and BCKAs were elevated 45-69% and ∼100%, respectively, in obese rats. Processes facilitating these rises appeared to include increased dietary intake (23%), leucine (Leu) turnover and proteolysis [35% per g fat free mass (FFM), urinary markers of proteolysis: 3-methylhistidine (183%) and 4-hydroxyproline (766%)] and decreased BCKDC per g kidney, heart, gastrocnemius and liver (-47-66%). A process disposing of circulating BCAAs, protein synthesis, was increased 23-29% by obesity in whole-body (FFM corrected), gastrocnemius and liver. Despite the observed decreases in BCKDC activities per gm tissue, rates of whole-body Leu oxidation in obese rats were 22% and 59% higher normalized to BW and FFM, respectively. Consistently, urinary concentrations of eight BCAA catabolism-derived acylcarnitines were also elevated. The unexpected increase in BCAA oxidation may be due to a substrate effect in liver. Supporting this idea, BCKAs were elevated more in liver (193-418%) than plasma or muscle, and per g losses of hepatic BCKDC activities were completely offset by increased liver mass, in contrast to other tissues. In summary, our results indicate that plasma BCKAs may represent a more sensitive metabolic signature for obesity than BCAAs. Processes supporting elevated BCAA]BCKAs in the obese Zucker rat include increased dietary intake, Leu and protein turnover along with impaired BCKDC activity. Elevated BCAAs/BCKAs may contribute to observed elevations in protein synthesis and BCAA oxidation.

Nitrogen in dietary glutamate is utilized exclusively for the synthesis of amino acids in the rat intestine.

Although previous studies have shown that virtually the entire carbon skeleton of dietary glutamate (glutamate-C) is metabolized in the gut for energy production and amino acid synthesis, little is known regarding the fate of dietary glutamate nitrogen (glutamate-N). In this study, we hypothesized that dietary glutamate-N is an effective nitrogen source for amino acid synthesis and investigated the fate of dietary glutamate-N using [(15)N]glutamate. Fischer male rats were given hourly meals containing [U-(13)C]- or [(15)N]glutamate. The concentration and isotopic enrichment of several amino acids were measured after 0-9 h of feeding, and the net release of each amino acid into the portal vein was calculated. Most of the dietary glutamate-C was metabolized into CO(2), lactate, or alanine (56, 13, and 12% of the dietary input, respectively) in the portal drained viscera (PDV). Most of the glutamate-N was utilized for the synthesis of other amino acids such as alanine and citrulline (75 and 3% of dietary input, respectively) in the PDV, and only minor amounts were released into the portal vein in the form of ammonia and glutamate (2 and 3% of the dietary input, respectively). Substantial incorporation of (15)N into systemic amino acids such as alanine, glutamine, and proline, amino acids of the urea cycle, and branched-chain amino acids was also evident. These results provide quantitative evidence that dietary glutamate-N distributes extensively to amino acids synthesized in the PDV and, consequently, to circulating amino acids.

A 90-day Feeding Toxicity Study of l-Serine in Male and Female Fischer 344 Rats.

A subchronic feeding study of l-serine (l-Ser) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0, 0.06, 0.5, 1.5 or 5.0% concentrations of l-Ser for 90 days. There were no toxicologically significant, treatment-related changes with regards to body weight, food intake, water intake or urinalysis data. In several of the hematology, serum biochemistry and organ weight parameters, significant changes were observed between some of the treated groups and the controls. All these changes, however, were subtle and lacked any corresponding pathological findings. In addition, the increased or decreased values remained within the range of the historical control values. In fact, histopathological assessment revealed only sporadic and/or spontaneous lesions. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Ser was, therefore, determined to be at least a dietary dose of 5.0% (2765.0 mg/kg body weight/day for males and 2905.1 mg/kg body weight/day for females) under the present experimental conditions.

Screening of toxicity biomarkers for methionine excess in rats.

Although many animal studies have reported that dietary excess of methionine causes toxic changes including growth suppression and hemolytic anemia, the biochemical mechanism and biomarkers for methionine toxicity have not been well elucidated. The present study aimed to identify toxicity biomarkers from plasma metabolites in rats fed excessive methionine. Young growing rats were fed graded doses of additional methionine for 2 wk. Cluster analysis of multivariate correlations was performed on the physiological and toxicity variables with plasma metabolites detected by GC/MS, amino acid analyzer, and thiol-specific analysis. Indicative variables for hemolysis such as splenic nonheme iron content and plasma bilirubin were grouped in the same cluster as many methionine metabolites. Homocysteine and some undefined metabolites in this cluster were found to be strong discriminators between nontoxic and toxic levels of methionine intake. Product-to-precursor ratios of each methionine metabolite demonstrated that excessive methionine intake caused a marked decrease only in the ratio of cystathionine to homocysteine, suggesting that metabolism from homocysteine to cystathionine would be rate limiting in the disposal of excessive methionine. Collectively from these results, homocysteine appeared to be the most plausible biomarker to assess methionine excess as a surrogate marker both for toxicity and for setting a metabolic upper limit.