Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN⁻). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.     

Quantitative determination of free intracellular amino acids in single human polymorphonuclear leucocytes: Recent developments in sample preparation and high-performance liquid chromatography

The described procedure allows quantitative, highly precise and reproducible analysis of free amino acid concentrations in single polymorphonuclear leucocytes (PMLs). This method is superior to previously described procedures with regard to sample size, PML separation, sample preparation and stability, as well as the chosen fluorescence high-performance liquid chromatography procedure, and can satisfy the high demands for ultra-sensitive and comprehensive amino acid analysis, especially for the continuous surveillance of severe diseases and organ dysfunction.     

Isolation and quantitation of isotopically labeled amino acids from biological samples

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phtaaldialdehyde (OPA), which is known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Becuase the OPA probe cannot be removed after isolation of the derivative, we used 9-fluorenylmethylchloroformate (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken byb gas-phase acid hydrolysis (103% recovery after 5 h at 150°C: S.D = 3.5%, n = 14). Run time (injection to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 μl could be injcted, representing approximately 200 μl of deproteinized plasma. The methods were linear up to injection of 0.5 μmol of all amino acids (OPA: r²=0.995−0.999; FMOC: r²=0.992−0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50–500 nmol), was less than 3% above 100 mmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.     

Comparative analysis of amino acid metabolism and transport in CHO variants with different levels of productivity

Chinese hamster ovary (CHO) cells are widely used for the production of biopharmaceuticals; however, our understanding of several physiological elements that contribute to productivity is limited. One of these is amino acid transport and how its limitation and/or regulation might affect productivity. To further our understanding, we have examined the expression of 40 mammalian amino acid transporter genes during batch cultures of three CHO cell lines: a non-producer and two antibody-producing cell lines with different levels of productivity. In parallel, extracellular and intracellular levels of amino acids were quantified. The aim was to identify differences in gene regulation between cell lines and within culture. Our results show that three transporters associated with transport of taurine and β-alanine, acidic amino acids and branched chain amino acids, are highly upregulated in both antibody-producing cell lines but not in the non-producer. Additionally, genes associated with the transport of amino acids related to the glutathione pathway (alanine, cysteine, cystine, glycine, glutamate) were found to be highly upregulated during the stationary phase of cell culture, correlating well with literature data on the importance of the pathway. Our analysis highlights potential markers for cell line selection and targets for process optimization.     

A new approach for sample preparation of protein hydrolyzates for amino acid analysis

Sample preparations of protein hydrolyzates for amino acid analysis by ion-exchange chromatography has been accomplished without the removal of hydrochloric acid which was used for the hydrolysis. The technique involves partial neutralization of the available hydrochloric acid after hydrolysis with a solution which neutralizes and dilutes the sample hydrolyzate at the same time. The resulting sample solution which is employed for amino acid analysis produces an amino acid chromatogram having the same elution times and resolution as compared to a mixture of amino acids prepared in pH 2.2 sodium citrate buffer. Experimental data is also presented which shows that the amount of available hydrochloric acid in the final sample solution employed for amino acid analysis can affect both the resolution and elution time of many of the amino acids found in a protein hydrolyzate.     

Balancing the amino acid needs of the dairy cow

In order to maximize milk protein production, one must present sufficient amounts of the essential amino acids to the intestinal tract in forms that can be absorbed. We do not know the specific tissue-level amino acid requirements of lactating cows, but they are likely to be similar to the amino acid content of milk protein with requirements for other metabolic functions similar to those in nonruminants. Formulating diets to meet these amino acid requirements is complicated because much of the dietary crude protein is converted to rumen microbial protein. Knowing the amount of dietary crude protein converted to ruminal microbial protein and the amino acid content of the rumen microbes; and the proportion of ruminally undegradable protein, its postruminal digestibility and amino acid content will allow one to make a reasonable estimate of the quality of protein presented for gastrointestinal digestion and absorption. Hypothetical calculations indicate potential dietary differences in quality of protein presented for absorption. Many of these differences correspond very well with production responses observed in research trials. Failure of this system to explain production results in other studies points to areas where additional information is still needed.     

Regulation of amino acid transport in the renal epithelial cell line NBL-1

Summary The activities of the transport systems A, B° and X_AG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and X_AG- in a protein-synthesis dependent process. In the case of System X_AG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.     

Scaling up a microbore separation for semipreparative analysis: differential recoveries of radiolabeled amino acids

Application of a high-sensitivity microbore system designed to separate and quantify nonderivatized amino acids by anion exchange chromatography and amperometric detection for determination of amino acid-specific activities in biological samples requires high capacity to recover sufficient labeled material for adequate count statistics. Scale up from a low (25-1000 pmol) to a high (500-15,000 pmol) working range was achieved by use of a thick working electrode gasket to reduce sensitivity and eliminate peak splitting and tailing and by modification of the wash procedure to eliminate carryover. Analysis of recoveries of labeled amino acids revealed that specific amino acids are either selectively retained on the column or partially degraded during analysis and that assessment of purities of labeled compounds and metabolic labeling patterns requires careful analysis of recoveries of labeled compounds in the appropriate eluate fraction.     

Molecular regulation of amino acid biosynthesis in plants

Summary Our understanding of amino acid biosynthesis in plants has grown by leaps and bounds in the last decade. It appears that most of the amino acid biosynthesis takes place in the chloroplast. Recent demonstration of glutamine synthetase and DAHP synthase in the vascular tisuue has added a new dimension in the complexity of the nitrogen cycle in plants. Isolation of various genes and transformation of plants with the modified forms of the genes are providing tools for understanding the regulation of various pathways. Plant transformation approaches are also going to provide the food of the future with an improved amino acid composition.     

Effects of carbohydrate feedings on plasma free tryptophan and branched-chain amino acids during prolonged cycling

Brain serotonin (5-hydroxytryptamine, 5-HT) has been suggested to be involved in central fatigue during prolonged exercise. Changes in the ratio of plasma free tryptophan (free Trp) to branched-chain amino acids (BCAA) are associated with altered brain 5-HT synthesis. The purposes of this study were to describe systematically the effects of prolonged exercise on changes in plasma free Trp and BCAA and to examine the effects of carbohydrate (CHO) feedings on these same variables. Eight well-trained men [VO2max = 57.8 (SE 4.1) ml kg-1 min-1] cycled for up to 255 min at a power output corresponding to VO2 at lactate threshold (approximately 68% VO2max) on three occasions separated by at least 1 week. Subjects drank 5 ml kg-1 body wt-1 of either a water placebo, or a liquid beverage containing a moderate (6% CHO) or high (12% CHO) concentration of carbohydrate beginning at min 14 of exercise and every 30 min thereafter. Exercise time to fatigue was shorter in subjects receiving placebo [190 (SE 4) min] as compared to 6% CHO [235 (SE 10) min] and 12% CHO [234 (SE 9) min] (P < 0.05). Glucose and insulin decreased in the placebo group, and free Trp, free-Trp/BCAA, and free fatty acids increased approximately five- to sevenfold (P < 0.05). These changes were attenuated in a dose-related manner by the carbohydrate drinks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sample preparation for amino acid determination by integrated pulsed amperometric detection in foods

This report describes a new sample preparation method for food which allows a complete separation of carbohydrates and amino acids prior to their analysis by anion-exchange chromatography and integrated pulsed amperometric detection. Food samples with high carbohydrate concentrations are applied to solid-phase extraction columns containing a strong cation-exchange resin. Carbohydrates are recovered initially; retained amino acids are eluted with 0.2 M CaC l(2) subsequently. The carbohydrate and the amino acid fractions are analyzed. The recovery calculated for 21 amino acids was in the range from 84 to 126%. The sample preparation was tested for amino acid concentrations between 4.2 and 84.0 nmol of each amino acid (between 2.1 and 42.0 nmol of cystine) and correlation coefficients between 0.84 and 0.99 were obtained. The capacity of the solid-phase extraction columns employed was up to 3.7 micro mol. Sample preparation was evaluated with four different food samples: sourdough, skim milk, lemon juice, and potato.     

Single-step enantioselective amino acid flux analysis by capillary electrophoresis using on-line sample preconcentration with chemical derivatization

Capillary electrophoresis (CE) represents a versatile platform for integrating sample pretreatment with chemical analysis because of its ability to tune analyte electromigration and band dispersion properties in discontinuous electrolyte systems. In this article, a single-step method that combines on-line sample preconcentration with in-capillary chemical derivatization is developed for rapid, sensitive, and enantioselective analysis of micromolar levels of amino acids that lack intrinsic chromophores by CE with UV detection. Time-resolved electrophoretic studies revealed two distinct stages of amino acid band narrowing within the original long sample injection plug occurring both prior to and after in-capillary labeling via zone passing by ortho-phthalaldehyde/N-acetyl l-cysteine (OPA/NAC). This technique enabled direct analysis of d-amino acids in a 95% enantiomeric excess mixture with sub-micromolar detection limits and minimal sample handling, where the capillary functions as a preconcentrator, microreactor, and chiral selector. On-line sample preconcentration with chemical derivatization CE (SPCD-CE) was applied to study the enantioselective amino acid flux in Escherichia coli bacteria cultures, which demonstrated a unique l-Ala efflux into the extracellular medium. New strategies for high-throughput analyses of low-abundance metabolites are important for understanding fundamental physiological processes in bacteria required for screening the efficacy of new classes of antibiotics as well as altered metabolism in genetically modified mutant strains.     

Effect of natural amino alcohols on the yield of essential amino acids and the amino acid pattern in stressed barley

Summary By application with 2-aminoethanol (2-AE) and choline chloride (CC) in pot experiments the effect of drought stress on barley plants was diminished. In treated plants an increase of the grain yield by 14% (2-AE) and 40% (CC) and a decrease of the stress metabolites glycine betaine and trigonelline was observed. Additionally, treated barley plants showed higher yields of essential amino acids as well. The contents of proline (stress indicator) and arginine (precursor of the stress metabolite putrescine) of treated plants were by 12% and 22% respectively, lower than in untreated plants.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

Differential diagnosis of (inherited) amino acid metabolism or transport disorders

Summary Disorders of amino acid metabolism or transport are most clearly expressed in urine. Nevertheless the interpretation of abnormalities in urinary amino acid excretion remains difficult. An increase or decrease of almost every amino acid in urine can be due to various etiology. To differentiate between primary and secondary aminoacido-pathies systematic laboratory investigation is necessary. Early diagnosis of disorders of amino acid metabolism or transport is very important, because most of them can be treated, leading to the prevention of (further) clinical abnormalities. In those disorders, which cannot be treated, early diagnosis in an index-patient may prevent the birth of other siblings by means of genetic counseling and prenatal diagnosis.Primary aminoacidopathies can be due to genetically determined transport disorders and enzyme deficiencies in amino acid metabolism or degradation. Secondary aminoacidopathies are the result of abnormal or deficient nutrition, intestinal dysfunction, organ pathology or other metabolic diseases like organic acidurias.A survey of amino acid metabolism and transport abnormalities will be given, illustrated with metabolic pathways and characteristic abnormal amino acid chromatograms.     

The amino acid composition of mammalian and bacterial cells

Summary High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, andEscherichia coli andStaphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions ofEscherichia coli andStaphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.     

The regularities of the changes of amino acid physico-chemical properties within the genetic code

Summary All the codons of the genetic code can be arranged into the closed one-step mutation ring, containing three periods of the same sequence of mutations (2,3,3,3,1,3,3,3,1,3,3,3,1,3,3,3,2,3,3,3). The codons of Gly play a role of the connecting element between the end of the third, and the beginning of the first period of the genetic code. The reactivity of amino acids, expressed by the reaction rates of aminolysis reaction of N-hydroxysuccinimide esters of protected amino acids with p-anisidine, changes periodically with the respect to the mutation periods of the genetic code. Chou-Fasman P as well as P conformational parameters of amino acids, and also the compositional frequencies of amino acids in proteins, demonstrate the pseudosymmetry pattern with respect to the center of one-step mutation ring, which is situated between Thr ACY and ACR codons.     

Renal handling of amino acids in 5/6-nephrectomized rats: Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone under amino acid load

Summary In anaesthetized adult female rats, the renal amino acid handling was measured six days after 5/6 nephrectomy (5/6NX). The distinct rise in blood urea nitrogen as well as the significant reduction in urine flow and GFR indicate an impairment of kidney function. In principle, in 5/6NX rats amino acid plasma concentrations were comparable to those of control animals with two intact kidneys, whereas the fractional excretions (FE_AA) of most endogenous amino acids measured were significantly enhanced. After bolus injection of leucine or taurine (each 20 mg/100 g b.wt.) or glutamine (90 mg/ 100 g b.wt.), dissolved in 2m1 normal saline per 100 g b.wt., the FE_AA of both the amino acids administered and the endogenous amino acids increased as a sign of overloaded amino acid reabsorption capacity. This effect was more pronounced in 5/6NX rats than in controls. As early as one hour after amino acid load, plasma concentrations and FE_AA returned to baseline values of 5/6NX rats. A pretreatment with triiodothyronine (20,µg/100 g b.wt.) or dexamethasone (60 µg/100 g b.wt.), both given intraperitoneally once daily for 3 days, stimulated the renal amino acid transport capacity in 5/6NX rats: the increase in FE_AA after amino acid load was significantly lower compared to non-pretreatred animals. This stimulation could be shown for the bolus amino acids and the endogenous amino acids and was more distinct in 5/6NX rats than in controls with two intact kidneys.