Amino Acids In Physiological Fluids Research Articles

Genetic regulatory element based whole-cell biosensors for the detection of metabolic disorders

Clinicians require simple, and cost-effective diagnostic tools for the quantitative determination of amino acids in physiological fluids for the detection of metabolic disorder diseases. Besides, amino acids also act as biological markers for different types of cancers and cardiovascular diseases. Herein, we applied an in-silico based approach to identify potential amino acid-responsive genetic regulatory elements for the detection of metabolic disorders in humans. Identified sequences were further transcriptionally fused with GFP, thus generating an optical readout in response to their cognate targets. Screening of genetic regulatory elements led us to discover two promoter elements (pmetE::GFP and ptrpL::GFP) that showed a significant change in the fluorescence response to homocysteine and tryptophan, respectively. The developed biosensors respond specifically and sensitively with a limit of detection of 3.8 μM and 3 μM for homocysteine and tryptophan, respectively. Furthermore, the clinical utility of this assay was demonstrated by employing it to identify homocystinuria and tryptophanuria diseases through the quantification of homocysteine and tryptophan in plasma and urine samples within 5 h. The precision and accuracy of the biosensors for disease diagnosis were well within an acceptable range. The general strategy used in this system can be expanded to screen different genetic regulatory elements present in other gram-negative and gram-positive bacteria for the detection of metabolic disorders.

Capillary Electrophoresis of Free Amino Acids in Physiological Fluids Without Derivatization Employing Direct or Indirect Absorbance Detection.

Whole blood and/or plasma amino acids are useful for monitoring whole-body protein and amino acid metabolism in an organism under various physiological and pathophysiological conditions. Various methodological procedures are in use for their measurement in biological fluids. From the time when capillary electrophoresis was introduced as a technology offering rapid separation of various ionic and/or ionizable compounds with low sample and solvent consumption, there were many attempts to use it for the measurement of amino acids present in physiological fluids. As a rule, these methods require derivatization procedures for detection purposes.Here, we present two protocols for the analysis of free amino acids employing free zone capillary electrophoresis. Main advantage of both methods is an absence of any derivatization procedures that permits the analysis of free amino acid in physiological fluids. The method using direct detection and carrier electrolyte consisting of disodium monophosphate (10mM at pH2.90) permits determination of compounds that absorb in UV region (aromatic and sulfur containing amino acids, as well as some peptides such as carnosine, reduced, and oxidized glutathione). The other method use indirect absorbance detection, employing 8mM p-amino salicylic acid buffered with sodium carbonate at pH10.2 as running electrolyte. It permits quantification of 30 underivatized physiological amino acids and peptides. In our experience factorial design represents a useful tool for final optimization of the electrophoretic conditions if it is necessary.

Design and characterization of auxotrophy-based amino acid biosensors.

Efficient and inexpensive methods are required for the high-throughput quantification of amino acids in physiological fluids or microbial cell cultures. Here we develop an array of Escherichia coli biosensors to sensitively quantify eleven different amino acids. By using online databases, genes involved in amino acid biosynthesis were identified that – upon deletion – should render the corresponding mutant auxotrophic for one particular amino acid. This rational design strategy suggested genes involved in the biosynthesis of arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, and tyrosine as potential genetic targets. A detailed phenotypic characterization of the corresponding single-gene deletion mutants indeed confirmed that these strains could neither grow on a minimal medium lacking amino acids nor transform any other proteinogenic amino acid into the focal one. Site-specific integration of the egfp gene into the chromosome of each biosensor decreased the detection limit of the GFP-labeled cells by 30% relative to turbidometric measurements. Finally, using the biosensors to determine the amino acid concentration in the supernatants of two amino acid overproducing E. coli strains (i.e. ΔhisL and ΔtdcC) both turbidometrically and via GFP fluorescence emission and comparing the results to conventional HPLC measurements confirmed the utility of the developed biosensor system. Taken together, our study provides not only a genotypically and phenotypically well-characterized set of publicly available amino acid biosensors, but also demonstrates the feasibility of the rational design strategy used.

Quantitative analysis of amino acids in physiological fluids by UPLC-MS/MS with stable isotope internal standards

Amino acid analysis is an essential service for the diagnosis and management of inborn errors of metabolism (IEM). For over 50 years the gold standard technique has been lithium ion exchange chromatography (IEC) with post-column ninhydrin derivatisation and photometric detection. This technique is laborious and expensive with typical run times over 120 minutes and requires dedicated instrumentation. Reverse phase chromatography with pre-column derivatisation and either fluorimetric or photometric detection reduces runs times but can suffer from interference. Many biochemical genetics laboratories already have tandem mass spectrometry (MS/MS) equipment which can be linked to ultra performance liquid chromatography (UPLC). The combination of these two techniques gives the opportunity to produce highly specific quantitative assays with high throughput and we report on our experience with a UPLC-MS/MS system for amino acid analysis in physiological fluids. Twenty-three physiological amino acids and 13 stable isotope internal standards, using 34 mass pairs are analysed within a 20 minute run utilising a simple acetonitrile/water gradient with tridecafluoroheptanoic acid ion pairing reagent. Sample preparation is limited to ultrafiltration to remove protein and dilution with internal standard mixture in mobile phase. The method correlates well with IEC results and gives comparable precision and external QAP performance.

Advances in amino acid analysis

Amino acids are important targets for metabolic profiling. For decades, amino acid analysis has been accomplished by either cation-exchange or reversed-phase liquid chromatography coupled to UV absorbance or fluorescence detection of pre-column or post-column-derivatized amino acids. Recent years have seen great progress in the development of direct-infusion or hyphenated mass spectrometry in the analysis of free amino acids in physiological fluids, because mass spectrometry not only matches optical detection in sensitivity, but also offers superior selectivity. The advent of cryo-probes has also brought NMR spectroscopy within the detection limits required for the analysis of free amino acids. But there is still room for further improvement, including expansion of the analyte spectrum, reduction of sample preparation and analysis time, automation, and synthesis of affordable isotope standards.

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2±0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm×75 μm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 μmol/l (median 6.71 μmol/l). The method was linear within the 50–200 μmol/l concentration range ( r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.

Ion-exchange chromatography for physiological fluid amino acid analysis

In the past l0 years, reversed-phase high-performance liquid chromatography (RP-HPLC) has been widely accepted as a preferred method for the amino acid analysis of protein hydrolysates. L ~ In contrast, physiological samples are often so varied and complex that application of RP-HPLC to their analysis has always resulted in poor peak resolution. 4-7 Therefore, the reliability of RP-HPLC in the quantitative determination of physiological amino acids remains controversial. 2~-H Analysis of physiological amino acids is traditionally carried out by ion-exchange chromatography (IEC) followed by post-column ninhydrin ~47 or o-phthalaldehyde ~'~ derivatization, instrumentation required for IEC has had the disadvantage of being more expensive and less versatile than RP-HPLC systems. Recently, with the advances in instrumental design a new generation of IE analyzers (System Gold TM analyzer, Beckman Instruments, San Ramon, USA) has emerged. This system offers the advantage of ease of operation and is highly adaptable for analyses of substances other than amino acids. Although the manufacturer of the System Gol& M' analyzer has made strong claims for its wide applicability in amino acid analysis, there have been no reports fully describing the use of the System Gold '~ analyzer for the analysis of free amino acids in physiological fluids. An additional complication to the development of a satisfactory system arises from the manufacturer's unwillingness to disclose the composition of the buffers recommended for use in the system. Thus the user has to purchase commercial buffers at high cost for routine operation. In this study, we describe the preparation of lithium citrate buffers and their application in physiological amino acid analysis on the System Gold :'~ analyzer. Quantitative analysis of results obtained for physiological amino acids was examined in terms of accuracy and precision.

Applications of automated amino acid analysis using 9-fluorenylmethyl chloroformate

A rapid and sensitive fully automated method for the determination of primary and secondary amino acids in different matrices is described. Amino acids are derivatized with 9-fluorenylmethyl chloroformate using an automated precolumn derivatization technique. Data are presented to show that the technique is both reproducible and highly sensitive. Applications of the technique are presented, including the analysis of peptide and protein hydrolysates and the profiling of free amino acids in physiological fluids.

Rapid Analysis of Nutritionally Important Free Amino Acids in Serum and Organs (Liver, Brain, and Heart) by Liquid Chromatography of Precolumn Phenylisothiocyanate Derivatives

An amino acid analysis method for protein hydrolysates, using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography, was modified for its application in rapid analysis of commonly occurring free amino acids in serum and other physiological samples. The modifications included changes in column temperature (47.5 degrees C compared to 25-35 degrees C used in analyzing protein hydrolysates), method of preparing standard and test samples, and gradient conditions. By using a Waters Pico-Tag amino acid analysis 15 cm long column (which is also used for analyzing protein hydrolysates), separation of 27 PTC-amino acids in human serum and rat liver, brain, or heart was completed in 20 min by the modified method. The total time for analysis and equilibration was 30 min. The modified method was much faster than the traditional ion-exchange methods (2-3 h) or the existing liquid chromatographic methods using PITC derivatization (66-80 min) for determining nutritionally important free amino acids in physiological fluids and tissues. Variability of the method (expressed as coefficients of variation) for the determination (including deproteinization, derivatization, and liquid chromatography) of all amino acids was less than 5%, which compared favorably with the reproducibility of ion-exchange methods.

A review of quantitative ion exchange, high performance liquid and gas chromatographic analysis of amino acids in physiological fluids

AbstractMethodologies for quantitative analysis of amino acids in physiological fluids based on classical ion exchange, high performance liquid chromatography, and gas chromatography are analyzed. As judged by the relative number of reports on these techniques, it appears that classical ion exchange continues to be the main method of choice for amino acid determinations and only limited advantage has been taken of the benefits (e.g. lower capital outlay and running costs and shorter analysis times) offered by other techniques. More importantly, however, there appears to be insufficient quantitative evaluation of the methodologies used. As a result, the validity of many reports based on data from amino acid analysis of physiological samples may be questioned.

Inexpensive, Computer-Automated HPLC for Ion Exchange Separation and Quantification of Amino Acids in Physiological Fluids

Abstract An inexpensive, computer-automated HPLC for separation and quantification of amino acids in physiological fluids is described. The system offers fully automated equipment control, data collection, processing and storage capabilities. The component nature of the system and the software flexibility permit extensive system modification, accomodating a wide variety of different separatory procedures which are not possible with many dedicated amino acid analysers. The system uses a lithium-based ion exchange column with post-column o-phthalaldehyde derivatization. A time of 128 minutes, including column regeneration, is required for separation of all amino acids through to arginine. The advantages of post-column derivatization over pre-column derivatization methods for post-separatory amino acid techniques are discussed. Accurate quantification of radiolabel in amino acids is demonstrated.

Problems in the analysis of low levels of amino acids in physiological fluids and tissues using o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography with electrochemical detection

Problems in the analysis of low levels of amino acids in physiological fluids and tissues using o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography with electrochemical detection

Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

Abstract An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.

31 Evaluation of waters HPLC analyzer for quantitation of amino acids in physiological fluids

31 Evaluation of waters HPLC analyzer for quantitation of amino acids in physiological fluids

Application of high-performance liquid chromatography to the determination of free amino acids in physiological fluids

Multiple-step gradient systems were used for the analysis of free amino acids in physiological fluids by high-performance liquid chromatography with fluorescence detection on two reversed-phase C 18 columns. Standard amino acids, plasma or urine samples were subjected to derivatization with ophthalaldehyde in the presence of mercaptoethanol before the separation was performed. More than 22 amino acids were separated in less than 1 h on either 5-μm Ultrasphere-ODS columns or 5-μm Resolve C 18 columns by using a two-solvent system. The correlation of the integrated peak areas with the concentration of all amino acids showed a linear relationship between 10 and 150 pmol per 20-μl injection. The method has a lower detection limit of less than 1 pmol per 20-μl injection for all amino acids. Quantitative analysis of micro amounts of amino acids in plasma and urine by the internal standard method gave highly reproducible results with a mean coefficient of variation of less than 3% and r 2 = 0.999. Because of the simplicity of the method its application provides a better means for closely monitoring the patients undergoing dialysis and treatment for renal disorders. These results are compared with those from classical ion-exchange chromatography.

Branched-chain α-keto acid analysis in biological fluids: Preparative clean-up by anion-exchange and analysis by capillary gas chromatography

A method is given for the quantitative analysis of the α-keto derivatives of the branched- chain amino acids in physiological fluids. A sample containing α-ketovalerate and α-ketocaproate as internal standards is passed through a week anion-exchange resin at neutral pH. After washing the resin with distilled water, the α-keto acids are eluted with 4 M hydrochloric acid-ethanol (50:50). Quinoxalinol derivatives are prepared directly in the eluent, extracted with methylene chloride, and trimethylsilylated. Separation of the derivatives is by capillary gas chromatography on a 30 m fused-silica SE-30 column. Chromatographic separation is superior to that reported for packed column methods, thereby permitting the use of α-ketovalerate and α-ketocaproate as internal standards.

High-performance liquid chromatographic analysis of amino acids in physiological fluids: On-line precolumn derivatization with o-phthaldialdehyde

The reaction between primary amines and o-phthaldialdehyde in the presence of a thiol was exploited to measure the concentrations of 21 amino acids in serum by high-performance liquid chromatography with fluorescent detection. The method described here includes an automatic on-line precolumn procedure for derivatizing the amino acids, permitting full automation and avoiding problems due to time-dependent decay of fluorescence of the derivatives. The total analysis time is less than 60 min and limits of sensitivity are about 100 fmol. Proline, hydroxyproline, and cysteine are not detected. Comparison with data generated by a standard amino acid analyzer shows this technique to be reliable.

Use of d-glucosaminic acid as an internal standard in single-column accelerated amino acid analysis of physiological fluids

d-Glucosaminic acid (DGA) was investigated as an early-eluting internal standard under single-column cation-exchange chromatography conditions because it elutes early in the analysis between urea and aspartic acid and obeys Beer's law. The precision and accuracy of data obtained with this internal standard were indistinguishable from those obtained with the conventionally used, late-eluting internal standard S-β(4-pyridylethyl)- l-cysteine (4-pec). DGA was stable when stored over 6 months at −20 or −70°C but was not stable to acid hydrolysis. The use of d-glucosaminic acid as an internal standard provides considerable flexibility in the development of analytic methodologies for rapidly analyzing selected amino acids in physiological fluids.

Contribution to clean-up procedures for serum amino acids

Most studies dealing with gas chromatographic determinations of amino acids in biological samples involve proteinaceous starting material. PhysioIogical fluids requiring analysis for free amino acid are usually pretreated with a denaturating agent such as picric acid’*‘, suKosalicyclic acid=, ethanol’** or chloroformg. Following removal of the precipitated protein by centrifugation the amino acids are isolated by cation-exchange ciean-up. However, variable recoveries of the free plasma amino acids, caused mainly by co-precipitation of the basic amino acids omittine, lysine and arginine, found by other workers “‘-“, led to a search for an alternative procedure. A simple handy technique, involving dih~tion of the sample with acetic acid and, thus, elimination of the protein precipitation, was su_ggested by PelIizari et al.“, and in combination with cationexchange clean-up it was used for determination of free amino acids in physiological fluids ‘z-‘* Even when the results obtained with the _ acetic acid method proved to be more consistent with those obtained by the amino acid analyzer technique than those obtained by the picrate denaturation12, the yields of some plasma amino acids were higher with the picrate method, making the acetic acid procedure less attractive”. This fact ied us to investigate the acetic acid procedure more comprehensively, i.e. with respect to the ion-exchange material and the inherent isolation process. It was found that the rate of cross-linking of the ion-exchange polystyrene matrix with the diviuylbenzeue (DVB) iutlueuced yields of some amino acids markedly, and that by reversing the mechanism of uptake toward the elution a further improvement occurred. Moreover, pieces of polyethylene tubing were found to be a convenient substitute for the ordinary glass columns.