Quantitation Of Free Amino Acids Research Articles

Production of Taste Enhancers from Protein Hydrolysates of Porcine Hemoglobin and Meat Using Bacillus amyloliquefaciens γ-Glutamyltranspeptidase.

To improve the flavor of hydrolysates from porcine hemoglobin and meat, γ-glutamyltranspeptidase (GGT) from Bacillus amyloliquefaciens was added to catalyze the formation of kokumi γ-glutamyl peptides via a γ-glutamyl transfer reaction. Quantitation of free amino acids and γ-glutamyl dipeptides was carried out in combination with sensory analysis. Sensory perception, especially the thick, complex, continuous, and overall kokumi sensation of both hemoglobin and meat hydrolysates, was greatly enhanced by γ-glutamylation. Due to the higher amount of glutamine present in meat hydrolysates, γ-glutamylated hydrolysates from meat contained higher concentrations of γ-glutamyl dipeptides and showed stronger kokumi sensation than the hemoglobin counterpart without the addition of glutamine. For hydrolysates from both raw materials, extra addition of glutamine (10 and 20 mM) was beneficial for obtaining higher concentrations of γ-glutamyl dipeptides but contributed little to the kokumi sensation. This study revealed that the kokumi sensation of protein hydrolysates could be intensified by a γ-glutamyl transfer reaction, and the enhanced kokumi sensation could be related to the generation of γ-glutamyl peptides.

The quantification of free Amadori compounds and amino acids allows to model the bound Maillard reaction products formation in soybean products

The quantification of protein bound Maillard reaction products (MRPs) is still a challenge in food chemistry. Protein hydrolysis is the bottleneck step: it is time consuming and the protein degradation is not always complete. In this study, the quantitation of free amino acids and Amadori products (APs) was compared to the percentage of blocked lysine by using chemometric tools. Eighty thermally treated soybean samples were analyzed by mass spectrometry to measure the concentration of free amino acids, free APs and the protein-bound markers of the Maillard reaction (furosine, Nε-(carboxymethyl)-l-lysine, Nε-(carboxyethyl)-l-lysine, total lysine). Results demonstrated that Discriminant Analysis (DA) and Correlated Component Regression (CCR) correctly estimated the percent of blocked lysine in a validation and prediction set. These findings indicate that the measure of free markers reflects the extent of protein damage in soybean samples and it suggests the possibility to obtain rapid information on the quality of the industrial processes.

Simultaneous Quantitation of Free Amino Acids, Nucleosides and Nucleobases in Sipunculus nudus by Ultra-High Performance Liquid Chromatography with Triple Quadrupole Mass Spectrometry.

To evaluate the nutritional and functional value of Sipunculus nudus, a rapid, simple and sensitive analytical method was developed using ultra-high performance liquid chromatography coupled with a triple quadrupole mass detection in multiple-reaction monitoring mode for the simultaneous quantitative determination of 25 free amino acids and 16 nucleosides and nucleobases in S. nudus within 20 min, which was confirmed to be reproducible and accurate. The limits of detection (LODs) and quantification (LOQs) were between 0.003–0.229 μg/mL and 0.008–0.763 μg/mL for the 41 analytes, respectively. The established method was applied to analyze 19 batches of S. nudus samples from four habitats with two different processing methods. The results showed that S. nudus contained a variety of free amino acids, nucleosides and nucleobases in sufficient quantity and reasonable proportion. They also demonstrated that the contents of these compounds in different parts of S. nudus were significantly discriminating, which were in the order: (highest) coelomic fluid > body wall > intestine (lowest). The method is simple and accurate, and could serve as a technical support for establishing quality control of S. nudus and other functional seafoods. Moreover, the research results also laid foundation for further exploitation and development of S. nudus.

A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.

Branched-chain amino acids are required for the survival and virulence of Actinobacillus pleuropneumoniae in swine.

In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 micromol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM-BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

Analysis of 25 underivatized amino acids in human plasma using ion‐pairing reversed‐phase liquid chromatography/time‐of‐flight mass spectrometry

Amino acids in biological fluids have previously been shown to be detectable using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application of a recently described liquid chromatographic separation method was coupled with TOFMS to employ accurate mass for qualitative identification; resulting in additional qualitative data not available with standard single quadrupole data. In the current study, we evaluated 25 physiological amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids; in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves were linear over a range from 1.56 to 400 microM. The dynamic range was found to be within physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino acids was between 80-120% spike recovery and <10% relative standard deviation (RSD). The LC/MS technique described in this study relies on mass accuracy and is suitable for the quantitation of free amino acids in plasma.

Chromatographic Determination of Amino Acids in Foods

Amino acids in foods exist in a free form or bound in peptides, proteins, or nonpeptide bonded polymers. Naturally occurring L-amino acids are required for protein synthesis and are precursors for essential molecules, such as co-enzymes and nucleic acids. Nonprotein amino acids may also occur in animal tissues as metabolic intermediates or have other important functions. The development of bacterially derived food proteins, genetically modified foods, and new methods of food processing; the production of amino acids for food fortification; and the introduction of new plant food sources have meant that protein amino acids and amino acid enantiomers in foods can have both nutritional and safety implications for humans. There is, therefore, a need for the rapid and accurate determination of amino acids in foods. Determination of the total amino acid content of foods requires protein hydrolysis by various means that must take into account variations in stability of individual amino acids and resistance of different peptide bonds to the hydrolysis procedures. Modern methods for separation and quantitation of free amino acids either before or after protein hydrolysis include ion exchange chromatography, high performance liquid chromatography (LC), gas chromatography, and capillary electrophoresis. Chemical derivatization of amino acids may be required to change them into forms amenable to separation by the various chromatographic methods or to create derivatives with properties, such as fluorescence, that improve their detection. Official methods for hydrolysis and analysis of amino acids in foods for nutritional purposes have been established. LC is currently the most widely used analytical technique, although there is a need for collaborative testing of methods available. Newer developments in chromatographic methodology and detector technology have reduced sample and reagent requirements and improved identification, resolution, and sensitivity of amino acid analyses of food samples.

Chromatographic quantitation of free amino acids: S‐methylmethionine, methionine and lysine in corn

Abstract The Pickering lithium gradient system was used with a modified Hewlett‐Packard HPLC and post‐column ninhydrin derivatization to separate and visualize the free amino acids and other ninhydrin‐positive substances in corn (Zea mays). About 50 major and minor components were discernible, with generally very good separation. Analytical focus was on S‐methylmethionine, principal precursor of the volatile flavor component dimethyl sulfide, in several sweet corn genotypes. The standard Pickering gradient program gave excellent separation of S‐methylmethionine from the other basic amino acids, permitting detection of as little as 20 pmoles. Quantitation was highly reproducible when canavanine was used as internal standard. Free methionine and lysine were also measured.

Chromatographic quantitation of free amino acids: S‐methylmethionine, methionine and lysine in corn

Abstract The Pickering lithium gradient system was used with a modified Hewlett‐Packard HPLC and post‐column ninhydrin derivatization to separate and visualize the free amino acids and other ninhydrin‐positive substances in corn (Zea mays). About 50 major and minor components were discernible, with generally very good separation. Analytical focus was on S‐methylmethionine, principal precursor of the volatile flavor component dimethyl sulfide, in several sweet corn genotypes. The standard Pickering gradient program gave excellent separation of S‐methylmethionine from the other basic amino acids, permitting detection of as little as 20 pmoles. Quantitation was highly reproducible when canavanine was used as internal standard. Free methionine and lysine were also measured.

Quantitation of free amino acids in plasma and muscle samples in healthy subjects and uremic patients by high-performance liquid chromatography and fluorescence detection

An automatic on-line high-performance liquid chromatographic (HPLC) method is developed to quantitate free amino acids in the biological samples. The method is based on pre-column derivatization of amino acids with orthophthalaldehyde (OPA) in presence of 2-mercaptoethanol (2-ME). The derivatized amino acids were separated on a 5 μm C 18 column using a multi-step gradient with two solvents and the detection was made at E ex = 340 nm and E em = 450 nm. The results were highly reproducible with a relative standard deviation (RSD) between 0.5–2% for all amino acids. Each chromatographic run was completed within 40 min to separate 24 amino acids. The optimized method was applied to evaluate the levels of free amino acids in plasma and muscle samples of eight healthy subjects and 13 uremic patients under fasting conditions.

Quantitation of free amino acids in biological samples by high-performance liquid chromatography : Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis

An automatic on-line high-performance liquid chromatographic method based on a precolumn derivatization with o-phthalaldehyde has been developed to quantitate levels of free amino acids in cerebrospinal fluid (CSF) and plasma samples from 12 patients with multiple sclerosis (MS) and 12 controls. The analytical method gave reproducible results with relative standard deviations of 0.5–3% for all amino acids. The separation of 24 amino acids was performed on a reversed-phase C 18 column, using two solvents and a multiple-step gradient. Each chromatographic experiment was completed within 40 min. The results showed higher levels of Glu, Gln, Gly and Ala and lower levels of Met, Val, Phe and Lys in plasma of MS patients. In CSF, increased levels of Gln, Arg, Ser and Tyr and decreased levels of Asp, Glu, Met, γ-aminobutyric acid and Phe were found in MS patients, whereas the levels of other amino acids remained more or the less same in both groups.

Ligand-exchange chromatography on copper(II)-modified silica gel : Improvements and use for screening of protein hydrolyzate and quantitation of dipeptides and amino acid fractions

Improvements in the copper(II) modification of silica gel, the stability of the analytical column by use of a guard column, the convenient UV detection at 254 nm of amino acids and peptides as their copper(II) complexes and the use of gradients make ligand-exchange chromatography a simple method for qualitative screening of protein hydrolyzates and quantitation of free amino acids and dipeptides in a complex polypeptide mixture. Examples are given of the chromatography of polyenzymatic ovalbumin hydrolyzates.

Free Amino Acids in Citrullus vulgaris (Watermelon)

To the Editor.— Currently accepted dietary management of citrullinemia and other urea cycle disorders includes protein restriction, sodium benzoate, and dietary supplements of keto acids or essential amino acids with postblock intermediates such as arginine in citrullinemia and arginino-succinic aciduria. When a child survives the neonatal period on such a regimen and solid foods are introduced into the diet, there is at least one fruit that should be avoided, Citrullus Vulgaris, commonly known as watermelon. Quantitation of free amino acids extracted from 1 g wet weight of watermelon fruit yielded the following (in mmoles per gram wet weight): Phenylalanine, 1.25; histidine, 0.24; tryptophan, 0.35; lysine, 0.82; ornithine, 0.32; arginine, 11.36; aspartic acid, 0.97; threonine, 0.74; serine, 1.05; glutamine, 3.86; glutamic acid, 1.38; citrulline, 23.68; alanine, 1.15; valine, 0.17; isoleucine, 1.24; leucine, 0.24.