To assess the use of plasma free amino acids (PFAAs) as biomarkers for metabolic disorders, it is essential to identify genetic factors that influence PFAA concentrations. PFAA concentrations were ...absolutely quantified by liquid chromatography-mass spectrometry using plasma samples from 1338 Japanese individuals, and genome-wide quantitative trait locus (QTL) analysis was performed for the concentrations of 21 PFAAs. We next conducted a conditional QTL analysis using the concentration of each PFAA adjusted by the other 20 PFAAs as covariates to elucidate genetic determinants that influence PFAA concentrations. We identified eight genes that showed a significant association with PFAA concentrations, of which two, SLC7A2 and PKD1L2, were identified. SLC7A2 was associated with the plasma levels of arginine and ornithine, and PKD1L2 with the level of glycine. The significant associations of these two genes were revealed in the conditional QTL analysis, but a significant association between serine and the CPS1 gene disappeared when glycine was used as a covariate. We demonstrated that conditional QTL analysis is useful for determining the metabolic pathways predominantly used for PFAA metabolism. Our findings will help elucidate the physiological roles of genetic components that control the metabolism of amino acids.
•We developed an automated precolumn derivatization amino acid analytical method.•This method achieved the separation of physiological amino acids within 8min.•We validated this method for 21 major ...types of free amino acids in human plasma.•Our developed method should be suitable for use in the clinical field.
The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.
Recently, the association of plasma free amino acid (PFAA) profile and lifestyle-related diseases has been reported. However, few studies have been reported in large Asian populations, about the ...usefulness of PFAAs for evaluating disease risks. We examined the ability of PFAA profiles to evaluate lifestyle-related diseases in so far the largest Asian population.
We examined plasma concentrations of 19 amino acids in 8589 Japanese subjects, and determined the association with variables associated with obesity, blood glucose, lipid, and blood pressure. We also evaluated the PFAA indexes that reflect visceral fat obesity and insulin resistance. The contribution of single PFAA level and relevant PFAA indexes was also examined in the risk assessment of lifestyle-related diseases.
Of the 19 amino acids, branched-chain amino acids and aromatic amino acids showed association with obesity and lipid variables. The PFAA index related to visceral fat obesity showed relatively higher correlation with variables than that of any PFAA. In the evaluation of lifestyle-related disease risks, the odds ratios of the PFAA index related to visceral fat obesity or insulin resistance with the diseases were higher than most of those of individual amino acid levels even after adjusting for potential confounding factors. The association pattern of the indexes and PFAA with each lifestyle-related disease was distinct.
We confirmed the usefulness of PFAA profiles and indexes as markers for evaluating the risks of lifestyle-related diseases, including diabetes mellitus, metabolic syndrome, dyslipidemia, and hypertension in a large Asian population.
Previous studies demonstrated independent contributions of plasma free amino acids (PFAAs) and high uric acid (UA) concentrations to increased risks of lifestyle-related diseases (LSRDs), but the ...important associations between these factors and LSRDs remain unknown. We quantified PFAAs and UA amongst Japanese subjects without LSRDs (no-LSRD, n = 2805), and with diabetes mellitus (DM, n = 415), dyslipidemia (n = 3207), hypertension (n = 2736) and metabolic syndrome (MetS, n = 717). The concentrations of most amino acids differed significantly between the subjects with and without hyperuricemia (HU) and also between the no-LSRD and LSRD groups (p < 0.05 to 0.001). After adjustment, the logistic regression analyses revealed that lysine in DM, alanine, proline and tyrosine in dyslipidemia, histidine, lysine and ornithine in hypertension, and lysine and tyrosine in MetS demonstrated significant positive associations with HU among the patients with LSRDs only (p < 0.05 to 0.005). By contrast, arginine, asparagine and threonine showed significant inverse associations with HU in the no-LSRD group only (p < 0.05 to 0.01). For the first time, we provide evidence for distinct patterns of association between PFAAs and HU in LSRDs, and postulate the possibility of interplay between PFAAs and UA in their pathophysiology.
Studies on the association of plasma-free amino acids with gout are very limited and produced conflicting results. Therefore, we sought to explore and characterize the plasma-free amino acid (PFAA) ...profile in patients with gout and evaluate its association with the latter.
Data from a total of 819 subjects (including 34 patients with gout) undergoing an annual health examination program in Shimane, Japan were considered for this study. Venous blood samples were collected from the subjects and concentrations of 19 plasma amino acids were determined by high-performance liquid chromatography-electrospray ionization-mass spectrometry. Student's t-test was applied for comparison of variables between patient and control groups. The relationships between the presence or absence of gout and individual amino acids were investigated by logistic regression analysis controlling for the effects of potential demographic confounders.
Among 19 amino acids, the levels of 10 amino acids (alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, tryptophan, valine) differed significantly (P < .001 to .05) between the patient and control groups. Univariate logistic regression analysis revealed that plasma levels of alanine, isoleucine, leucine, phenylalanine, tryptophan and valine had significant positive associations (P < .005 to .05) whereas glycine and serine had significant inverse association (P < .05) with gout.
The observed significant changes in PFAA profiles may have important implications for improving our understanding of pathophysiology, diagnosis and prevention of gout. The findings of this study need further confirmation in future large-scale studies involving a larger number of patients with gout.
Metabolic flux analysis using
13C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to ...typical industrial conditions, such as batch and fed-batch cultivations, a novel method of
13C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of
Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.
Dipeptides are expected to improve the physical properties of amino acid, to have a new physical description and a physiological function. However, there was almost no practical method for a ...comprehensive analysis of dipeptides, and there were only few reports that examined those existences, behavior or functions. In the present study, we developed a comprehensive analytical method for dipeptides, using pre-column derivatization coupled to reversed-phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (LC/MS/MS). Dipeptides samples were derivatized with phenyl isocyanate (PIC), which is able to react under the mild condition to the free amino group. The resulting typical derivatives were analyzed by tandem mass spectrometry and specific cleavages in the collision induced dissociation (CID) were observed. These fragmentation patterns have been enabled for practical analysis combined with the predicted selected reaction monitoring (pSRM), precursor ion scan or constant neutral loss scan. With these developed methods, we demonstrated to be able to show dipeptide profiles of food samples, such as cheese and wine.
► The kokumi peptide γ-Glu-Val-Gly in scallop foods was determined using LC/MS/MS. ► Scallop foods contained γ-Glu-Val-Gly at 0.08–0.77μg/g. ► This is the first report to confirm the existence of ...γ-Glu-Val-Gly in foodstuff.
The determination of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in raw scallop and processed scallop products was carried out using high pressure liquid chromatography–tandem mass spectrometry (LC/MS/MS). The detection of γ-Glu-Val-Gly was achieved using a multiple reaction monitoring (MRM) method. The optimised condition enabled the precise determination of γ-Glu-Val-Gly. Raw scallop contained 0.08μg/g γ-Glu-Val-Gly, and the γ-Glu-Val-Gly levels in processed scallop products, such as dried-scallop and scallop extract, were measured to be 0.64 and 0.77μg/g, respectively. This is the first report to confirm the existence of γ-Glu-Val-Gly in foodstuff.
Our aim is to construct a practical dynamic-simulation system that can model the metabolic and regulatory processes involved in the production of primary metabolites, such as amino acids. We have ...simulated the production of glutamate by transient batch-cultivation using a model of
Escherichia coli central metabolism. Kinetic data were used to produce both the metabolic parts of the model, including the phosphotransferase system, glycolysis, the pentose-phosphate pathway, the tricarboxylic acid cycle, the glyoxylate shunt, and the anaplerotic pathways, and the regulatory parts of the model, including regulation by transcription factors, cyclic AMP receptor protein (CRP), making large colonies protein (Mlc), catabolite repressor/activator (Cra), pyruvate dehydrogenase complex repressor (PdhR), and acetate operon repressor (IclR). RNA polymerase and ribosome concentrations were expressed as a function of the specific growth rate,
μ, corresponding to the changes in the growth rate during batch cultivation. Parameter fitting was performed using both extracellular concentration measurements and
in vivo enzyme activities determined by
13C flux analysis. By manual adjustment of the parameters, we simulated the batch fermentation of glucose or fructose by a wild-type strain (MG1655) and a glutamate-producing strain (MG1655 Δ
sucA). The differences caused by the carbon source, and by wild-type and glutamate-producing strains, were clearly shown by the simulation. A sensitivity analysis revealed the factors that could be altered to improve the production process. Furthermore, an
in silico deletion experiments could suggested the existence of uncharacterized regulation. We concluded that our simulation model could function as a new tool for the rational improvement and design of metabolic and regulatory networks.
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