An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.
Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca2+-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.
BackgroundRecently, 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.MethodsWe 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.ResultsOf 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.ConclusionsWe 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.
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