Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxylysine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different collagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromatography (HILIC) coupled on-line to electrospray ionization mass spectrometry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qualitative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u).     

Analysis of amino acids in individual wheat embryonic protoplast

Summary. Amino acids analysis in single wheat embryonic protoplast was performed using capillary electrophoresis equipped with laser-induced fluorescence (CE-LIF), combination with tissue culture technique. Reagent fluorescein isothiocyanate (FITC) was introduced into living protoplasts by electroporation for intracellular derivatization. A special osmotic buffer (0.6 mol/L mannitol, 5 mmol/L CaCl₂) was used to keep the osmotic balance of embryonic protoplasts during the protoplasts derivatization. After completion of the derivatization reaction in the protoplasts, a single protoplast was drawn into the capillary tip by electroosmotic flow. Then a 0.1 M NaOH lysing solution was injected by diffusion. The derivatized amino acids were separated by capillary electrophoresis and detected by laser-induced fluorescence detection after the protoplast was lysed Nine amino acids were quantitatively and qualitatively determined and compared in lysate and single protoplast of wheat embryonic cells respectively, with mean concentrations of amino acids ranging from 2.68×10⁻⁵ mol/L to 18.18×10⁻⁵ mol/L in single protoplast.     

Strategies for comprehensive analysis of amino acid biomarkers of oxidative stress

Summary. Despite the wide interest in using modified amino acids as putative biomarkers of oxidative stress, many issues remain as to their overall reliability for early detection and diagnosis of diseases. In contrast to conventional single biomarker studies, comprehensive analysis of biomarkers offers an unbiased strategy for global assessment of modified amino acid metabolism due to reactive oxygen and nitrogen species. This review examines recent analytical techniques amenable for analysis of modified amino acids in biological samples reported during 2003–2007. Particular attention is devoted to the need for validated methods applicable to high-throughput analysis of multiple amino acid biomarkers, as well as consideration of sample pretreatment protocols on artifact formation for improved clinical relevance.     

Relationships between the sedative and hypnotic effects of intracerebroventricular administration of L-serine and its metabolites, pyruvate and the derivative amino acids contents in the neonatal chicks under acute stressful conditions

Summary. Intracerebroventricular (i.c.v.) injection of L-serine was shown to have sedative and hypnotic effects on neonatal chicks under acute stressful conditions. To clarify the central mechanism of these effects of L-serine, two experiments were done. First, we focused on the glycogenic pathway in which L-serine is converted into pyruvate and finally glucose. I.c.v. administration of pyruvate (0.84 μmol) did not induce any behavioral and endocrinological changes, while L-serine and glucose triggered sedative and hypnotic effects. Secondly, the relationship between the sedation by L-serine and the metabolism into other amino acids which have sedative effects was investigated in the telencephalon and diencephalon. In both brain areas, a dose-dependent increase was seen in L-serine, although other amino acids were not changed. In the present study, it was concluded that the sedative action of L-serine was not due to the action of its metabolite pyruvate, or to the action of other amino acids. Authors’ address: M. Furuse, PhD, Laboratory of Advanced Animal and Marine Bioresources, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan     

Metabolite analysis in positron emission tomography studies: examples from food sciences

Summary. Substances of various chemical structures can be labelled with appropriate positron emitting isotopes and applied as tracer compounds in PET examinations. Using dynamic data acquisition protocols, time-activity curves of radioactivity uptake in organs can be derived and the measurements of tissue tracer concentrations can be translated into quantitative values of tissue function. However, analysis of metabolites of these tracers regarding their nature and distribution in the living organism is an essential need for the quantitative analysis of PET measurements. In addition, metabolite analysis contributes to the interpretation of the images obtained as well as to the identification of pathological changes in metabolic pathways. This paper reports on representative examples of radiolabelled compounds which might be of importance in food science (e.g., amino acids, polyphenols, and model compounds for advanced glycation end products (AGEs)). Typical procedures of analysis (radio-HPLC, radio-TLC) including pre-analytical sample preparation are described. Specific challenges of the method, e.g., trace amounts of radiolabelled compounds and the influence of the often very short half-lives of positron-emitting nuclides used are highlighted. Representative results of analyses of plasma, urine, and tissue samples are presented and discussed in terms of the metabolic fate of the tracers.     

A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), for the enantioresolution of amino acids on a teicoplanin phase under the elution of the methanol-based solvent mixture

Summary. A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), is chosen to functionalize amino acids in alkaline medium before their HPLC resolution. FCC reacts with both primary and secondary amino acids to produce stable and highly fluorescent derivatives suitable for sensitive and efficient chromatographic determination and resolution on a teicoplanin chiral stationary phase (CSP) using the methanol-based solvent mixture as the mobile phase. The detection limit is in the picomole range and approximately 0.01% of the d-enantiomer in an excess of the l-enantiomer is detectable. However, the resolution is not reproducible under the elution of either the water- or the acetonitrile-based mobile phase. The increase in solubility of analyte in the mobile phase seems to be responsible. Upon comparison under the optimal chromatographic conditions, the resolution is better than that for the 9-fluorenylmethyl chloroformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives reported previously.     

Indispensable amino acid concentrations decrease in tissues of stomachless fish,common carp in response to free amino acid- or peptide-based diets

Summary. The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.     

The role of growth hormone and amino acids on brain protein synthesis in aged rats given proteins of different quantity and quality

Summary. The purpose of the present study was to determine whether the regulation of brain protein synthesis was mediated through changes in the plasma concentrations of insulin and growth hormone (GH), and whether the concentrations of amino acids in the brain and plasma regulate the brain protein synthesis when the quantity and quality of dietary protein is manipulated. Two experiments were done on three groups of aged rats given diets containing 20% casein, 5% casein or 0% casein (Experiment 1), and 20% casein, 20% gluten, or 20% gelatin (Experiment 2) for 1 d (only one 5-h period) after all rats were fed the 20% casein diet for 10 d (only 5-h feeding per day). The aggregation of brain ribosomes, the concentration in plasma GH, and the branched chain amino acids in the plasma and cerebral cortex declined with a decrease of quantity and quality of dietary protein. The concentration of plasma insulin did not differ among groups. The results suggest that the ingestion of a higher quantity and quality of dietary protein increases the concentrations of GH and several amino acids in aged rats, and that the concentrations of GH and amino acids are at least partly related to the mechanism by which the dietary protein affects brain protein synthesis in aged rats.     

Identification of the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney

Summary. Glucocorticoid hormones enhance the reabsorptive capacity of filtered amino acids in rat kidney, as it was shown in previous in vivo clearance experiments. In the present study, the site of glucocorticoid action on neutral amino acid transport in superficial nephrons of rat kidney was investigated using in vivo micropuncture technique. Adult female Wistar rats were treated with dexamethasone (DEX), and fractional excretion of L-glutamine (L-Gln) and L-leucine (L-Leu) were determined and related to inulin after microinfusion into different nephron segments. DEX reduced fractional excretion of both neutral amino acids as a sign of enhanced reabsorptive capacity. The site of main DEX action on L-Leu reabsorption has been localized in the proximal straight tubule. However, in the case of L-Gln, the inhibition of γ-glutamyltranspeptidase (γ-GT) by administration of acivicin indicated the importance of this brush border enzyme in reduced L-Gln excretion. DEX enhanced γ-GT activity by tubular acidification. It can be presumed a DEX-inducible transport system for neutral amino acids mainly localized in proximal straight tubules of rat kidney. Received July 8, 1999     

Determination of L- and D-amino acids in foodstuffs by coupling of high-performance liquid chromatography with enzyme reactors

Summary. A technique is described for the enantiomeric determination of L- and D-amino acids. It works on the principle that the separation efficiency of high-performance liquid chromatography is coupled with the specificity of enzymes and the sensitivity of electrochemical detection. After separation on a lithium cation-exchange column the amino acids are converted into keto acids and hydrogen peroxide under catalyzation of L- or D-amino acid oxidase. Hydrogen peroxide is detected amperometrically. The method has been tested by the analysis of beer, port, sherry, wine and fruit juice. A main emphasis was put onto the determination of D-alanine which can serve as an indicator for bacterial contamination. It is shown that a coupling of HPLC with enzyme reactors is a suitable technique for the rapid detection of this marker. Received April 14, 1999, Accepted September 15, 1999     

Molecular cloning of a cDNA encoding mouse D-aspartate oxidase and functional characterization of its recombinant proteins by site-directed mutagenesis

Summary. The cDNA encoding D-aspartate oxidase (DASPO) was cloned from mouse kidney RNA by RT–PCR. Sequence analysis showed that it contained a 1023-bp open reading frame encoding a protein of 341 amino acid residues. The protein was expressed in Escherichia coli with or without an N-terminal His-tag and had functional DASPO activity that was highly specific for D-aspartate and N-methyl-D-aspartate. To investigate the roles of the Arg-216 and Arg-237 residues of the mouse DASPO (mDASPO), we generated clones with several single amino acid substitutions of these residues in an N-terminally His-tagged mDASPO. These substitutions significantly reduced the activity of the recombinant enzyme against acidic D-amino acids and did not confer any additional specificity to other amino acids. These results suggest that the Arg-216 and Arg-237 residues of mDASPO are catalytically important for full enzyme activity.     

Multi-layered network structure of amino acid (AA) metabolism characterized by each essential AA-deficient condition

Summary. The concentrations of free amino acids in plasma change coordinately and their profiles show distinctive features in various physiological conditions; however, their behavior can not always be explained by the conventional flow-based metabolic pathway network. In this study, we have revealed the interrelatedness of the plasma amino acids and inferred their network structure with threshold-test analysis and multilevel-digraph analysis methods using the plasma samples of rats which are fed diet deficient in single essential amino acid. In the inferred network, we could draw some interesting interrelations between plasma amino acids as follows: 1) Lysine is located at the top control level and has effects on almost all of the other plasma amino acids. 2) Threonine plays a role in a hub in the network, which has direct links to the most number of other amino acids. 3) Threonine and methionine are interrelated to each other and form a loop structure.     

Blood amino acids concentration during insulin induced hypoglycemia in rats: the role of alanine and glutamine in glucose recovery

Summary. Our purpose was to determine the blood amino acid concentration during insulin induced hypoglycemia (IIH) and examine if the administration of alanine or glutamine could help glycemia recovery in fasted rats. IIH was obtained by an intraperitoneal injection of regular insulin (1.0 U/kg). The blood levels of the majority of amino acids, including alanine and glutamine were decreased (P < 0.05) during IIH and this change correlates well with the duration than the intensity of hypoglycemia. On the other hand, the oral and intraperitoneal administration of alanine (100 mg/kg) or glutamine (100 mg/kg) accelerates glucose recovery. This effect was partly at least consequence of the increased capacity of the livers from IIH group to produce glucose from alanine and glutamine. It was concluded that the blood amino acids availability during IIH, particularly alanine and glutamine, play a pivotal role in recovery from hypoglycemia.     

Detecting oxidative post-translational modifications in proteins

Summary. Oxidative stress induces various post-translational modifications (PTM); some are reversible in vivo via enzymatic catalysis. The present paper reviews specific procedures for the detection of oxidative PTM in proteins, most of them including electrophoresis. Main topics are carbonylated and glutathionylated proteins as well as modification of selected amino acids (Cys, Tyr, Met, Trp, Lys).     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Laccase-induced derivatization of unprotected amino acid L-tryptophan by coupling with p-hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide

Summary. We have studied the enzymatic derivatization of amino acids by use of the polyphenol oxidase laccase. Derivatization of L-tryptophan was achieved by enzymatic crosslinking with the laccase substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. The main product (yield up to 70%) was identified as the quinoid compound 2-[2-(2-hydroxy-ethylcarbamoyl)-3,6-dioxo-cyclohexa-1,4-dienylamino]-3-(1H-indol-3-yl)- propionic acid and demonstrates that laccase-catalyzed C–N-coupling occurred on the amino group of the aliphatic side chain. These enzyme based reactions provide a simple and fast method for the derivatization of unprotected amino acids.     

When quinones meet amino acids: chemical, physical and biological consequences

Summary. Quinones and amino acids are usually compartmentally separated in living systems, however there are several junctions in which they meet, react and influence. It occurs mainly in wounded, cut or crushed plant material during harvest, ensiling or disintegrating cells. Diffusing polyphenols are oxidized by polyphenol oxidases (PPOs) to quinonic compounds, which associate reversibly or irreversibly with amino acids and proteins. The reaction takes place with the free nucleophilic functional groups such as sulfhydryl, amine, amide, indole and imidazole substituents. It results in imine formation, in 1,4-Michael addition via nitrogen or sulphur and in Strecker degradation forming aldehydes. The formation and activity of quinone–amino acids conjugates influences the colour, taste, and aroma of foods. Physical and physiological phenomena such as browning of foods, discoloration of plants during processing, alteration of solubility and digestibility, formation of humic substances, germicidal activity, cytotoxicity and more occur when quinones from disintegrating cells meet amino acids. The mechanisms of toxicity and the pathways by which PCBs may be activated and act as a cancer initiator include oxidation to the corresponding quinones and reaction with amino acids or peptides. Sclerotization of insect cuticle is a biochemical process involving also the reaction between quinones and amino acid derivatives.     

Bio-available amino acids and mineral nitrogen forms in soil of moderately mown and abandoned mountain meadows

Summary. The abandonment of traditional mowing methods of mountain meadows in the Czech Republic at the end of the last century has resulted in secondary re-colonization of these areas. Altered accumulation of plant biomass resulted in a deceleration of N turnover. A mountain meadow may be regarded as a N-limited ecosystem in which plant nutrition is dependent on direct uptake of soil amino acids. The composition and distribution of ammonium ions, nitrate ions and the 16 bio-available proteinaceuous amino acids were investigated in the top 7 cm of the Ah horizon of a Gleyic Luvisol in a long-term moderately mown meadow and an eleven year old, abandoned or uncut meadow. Ammonium N has a dominant role in both ecosystems. The moderately mown meadow showed accelerated N-turnover and higher net ammonization. The plant community showed a dependence on this form. Plant utilization of nitrates and amino acids appeared to be negligible. The uncut or abandoned meadow showed net ammonization from May (start of the experiment) through August, after which plant N-uptake consisted only of amino acids due to microbial immobilization. The release of bio-available nitrogen from spring until the beginning of summer in the Ah horizon was too low to explain total plant N-uptake. Glutamic acid, arginine and aspartic acids had the highest concentrations of any of the amino acids analyzed. Authors’ address: Pavel Formánek, Department of Geology and Pedology, Mendel University of Agriculture and Forestry, Zemedelska 3, 613 00 Brno, Czech Republic