Xterra RP18柱高效液相色谱法快速分离测定氨基酸

建立了一种用XterraRP1 8色谱柱快速分离测定水解氨基酸的方法。所采用的色谱条件是 :WatersAlliance系统 ,柱温 5 6℃ ,流速 1 .8ml/min ,检测波长 2 4 8nm ,梯度分离 ,运行周期 2 5min,柱反压低于 2 0 0 0Psi。在 1 7.5min内分离了包括AMQ、NH3 和牛磺酸在内的 2 1种氨基化合物 ,适应于复合氨基酸注射液、含牛磺酸的氨基酸口服液及水解氨基酸样品的分析测定     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Measurement of amino acid release from cultured cerebellar granule cells by an improved high performance liquid chromatography procedure

Endogenous amino acid release was examined in rat cerebellar primary cultures comprising more than 95% of glutamatergic granule cells. Eighteen amino acids were determined in the cell extracts and in the release fractions by high performance liquid chromatography, using precolumn derivatization witho-phthaldialdehyde and separation on a reverse-phase column using a multi-step gradient system of two solvents (0.1 M Na⁺acetate, pH 7.2/methanol: tetrahydrofuran, 97:3). The fluorimetric response was linear, at least in the range of 2–162 pmol, for all the amino acids analysed, with a detection limit of 1 pmole. We observed a good reproducibility in within-assay and between-assay coefficients of variation of the retention times and fluorescence yield. When cultured granule cells were exposed to the excitatory amino acid receptor agonist quisqualic acid (50 M), we observed a net increase in the release of glutamate (3 fold over the baseline) and a smaller increase in that of aspartate (2 fold) and taurine (1.6 fold). Other amino acids were not significantly affected. GABA levels were below detection limits, due to the minimal number of GABAergic neurons present in the cultures.     

Rapid and Precise Measurement of Serum Branched-Chain and Aromatic Amino Acids by Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry

Background

Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming.

Methods

An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C₁₈ column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode.

Results

Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%.

Conclusion

A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.     

Rapid microbial metabolism of non-protein amino acids in the sea

The non-protein amino acids, -alanine and -aminobutyric acid, frequently dominate the amino acid composition of deep-sea sediments. This accumulation is most likely due to the slower decomposition of non-protein amino acids by microorganisms or to the preferential adsorption of non-protein amino acids by clay minerals. We investigated relative rates of heterotrophic uptake of alanine, -ala, and -aba in sea water to see if there were different rates of microbial assimilation and respiration between these protein and non-protein amino acids. Heterotrophic uptake was rapid for all three amino acids with turnover times of hours in productive coastal waters and days in more oligotrophic open-ocean waters. Uptake of the non-protein amino acids was significantly slower than uptake of alanine, particularly in anoxic waters. However, the difference in uptake rates is probably not great enough to cause significant preferential accumulation of non-protein amino acids.     

The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns

Amino acids were reacted with o-phthalaldehyde and 2-mercaptoethanol and were separated using a simple linear gradient from 10 to 65% methanol over 15 min on an octyl silica (C8) column by reversed-phase chromatography. The separation obtained was found to be sensitive to the pH, ionic strength, and tetrahydrofuran concentration of aqueous solvent A [THF: sodium acetate (45 mM), pH 5.7, (4:96)]. These effects were characterized and used to design a rapid (17 min) separation of the amino acids commonly found in acid hydrolysates of proteins. A more involved procedure was used to separate the more complex mixture of amino acids that are found in enzymatic hydrolysates of proteins or in physiological fluids. The simplicity of the methods allows their use on different chromatographic systems with little or no alteration.     

Amino acid uptake by Lemna gibba by a mechanism with affinity to neutral L-and D-amino acids

Earlier work suggested that amino acid uptake by Lemna gibba cells is a H⁺-cotransport mechanism driven by a proton-electrochemical gradient at the plasmalemma. The present investigations of the transient membrane depolarizations elicited by amino acids and tracer-uptake experiments show that all neutral -L-amino acids, D-alanine and analogues, like -alanine and p-fluorophenylalanine, are transported by the same system. It remains to be seen if there are separate mechanisms for the uptake of acidic and basic amino acids.     

Stimulation of renal amino acid reabsorption after treatment with triiodothyronine or dexamethasone in amino acid loaded rats

Summary In adult female anaestetized rats, the influence of triiodothyronine or dexamethasone on renal amino acid handling was investigated in leucine (20mg/100g b.wt.) or glutamine (45mg/100g b.wt.) loaded animals. Bolus injections of both amino acids were followed by temporary increase in fractional excretion of the administered amino acids as well of the endogenous amino acids which were not administered.Under load conditions (leucine and glutamine), dexamethasone treatment (60 g/100 g b.wt. for 3 days, i.p. once daily) was followed by a stimulation of renal amino acid reabsorption. The increase in fractional amino acid excretion after amino acid load was significantly lower than in untreated rats. The effect of triiodothyronine (20,g/1008 b.wt. for 3 days, i.p. once daily) was different in leucine and glutamine loaded animals: after leucine bolus injection a comparable stimulatory effect as shown for dexamethasone could be demonstrated, but after glutamine administration the stimulatory action of T3 was masked. T3 even increases fractional amino acid excretion in glutamine loaded rats as a sign of enhanced house-keeping in the renal tubular cells. These results confirm previous findings and indicate different effects of both hormones on the renal handling of amino acids.     

The assimilation and allocation of nutrients by symbiotic and aposymbiotic pea aphids, Acyrthosiphon pisum

The failure of a nutritionally balanced diet to ameliorate the impact of symbiont disruption in the pea aphid Acyrthosiphon pisum (Harris) was investigated using two approaches. The assimilation of dietary nutrients by aphids was investigated using chemically-defined diets containing ³ H-labelled inulin and ¹⁴C-labelled sucrose or amino acids. Symbiotic aphids (i.e., aphids containing their bacteria) had a high sucrose demand and assimilated 72% of sucrose ingested in the diet, whereas the assimilation of sucrose by aposymbiotic aphids (in which the bacteria had been disrupted), was significantly reduced to 47%. The assimilation of individual dietary amino acids by symbiotic aphids varied between 61 and 92%, and there was no impact on the feeding or assimilation rate when the aphids were fed a phloem sap-like diet containing a reduced amount of essential amino acids. Consequently, the absolute amount of each essential amino acid assimilated by symbiotic aphids feeding on a phloem sap-like diet was reduced by 36–59%. Aposymbiotic aphids consistently assimilated a lower proportion of ingested amino acids, and lysine in particular was poorly assimilated from the diet. In a second experiment, the allocation of free amino acids in the haemocoel to aphid embryos was investigated following microinjection of ¹⁴C-labelled amino acids. After 2 h, radiolabel could be detected at varying levels from the embryo complement of both symbiotic and aposymbiotic aphids, indicating rapid but selective uptake by the embryos. The essential amino acids phenylalanine and lysine were incorporated into the protein fraction of embryo tissues, but the rate of incorporation per unit biomass was approximately 4-fold higher in the embryos of aposymbiotic aphids, possibly reflecting increased demand due to the lack of amino acid provisioning from the symbiotic bacteria.     

Changes of amino acid gradients in brain tissues induced by microwave irradiation and other means

Focused microwave irradiation to the head (FMI) has been used extensively by neurochemists for rapid inactivation of enzymatic activity in brain tissues and the preservation, for in vitro analysis, of in vivo substrate concentrations. Periodically the suitability of this technique for regional studies has been questioned. Evidence has now been obtained, on the basis of altered concentration gradients for GABA and taurine from the Substantia Nigra (SN) to an Adjacent Dorsal Area (ADJ), that FMI not only inactivates enzymes, but also facilitates rapid diffusion of small molecules from areas of high concentrations to adjacent areas of lower concentration. To a lasser extent, the implantation of plastic injection cannulas also decreased these concentration gradients. These results offer clear evidence that FMI is ill suited and unreliable for studies designed to map and compare the in vivo regional concentrations of diffusible organic molecules (such as amino acids) in brain tissues. Any invasive technique that compromises membrane barriers is likely to produce smaller similar effects.     

Separation of peptides by reverse-phase high-performance liquid chromatography using propyl- and cyanopropylsilyl supports

The separation of peptides and proteins by reverse-phase high-performance liquid chromatography with cyanopropylsilyl and large-pore propylsilyl supports, together with aqueous trifluoroacetic acid/acetonitrile gradients, was studied. Operating parameters (trifluoroacetic acid concentration, flow rate, and gradient slope) were evaluated using different enzymatic digests of horse cytochrome c and bovine serum albumin. Peptides ranging in size from five amino acids to 68 kDa could be separated on the propylsilyl column in a single chromatographic run. The cyanopropylsilyl column is suitable as a supplement to the use of the large-pore column for medium size (5-20 amino acids) peptides. The chromatographic supports and conditions presented here offer a simple, sensitive, and rapid separation system for a wide size range of peptides and proteins. They extend the versatility of separation methodology for these molecules.     

乙醇系统HPLC分析PTC－氨基酸的条件优化

首次报道用乙醇系统分析ＰＴＣ－氨基酸的新方法中各衍生物获得最佳分离的建立过程。ＰＴＣ－氨基酸衍生后溶于Ａ溶液,然后进样于４μｍＮｏｖａＰａｋＣ１８柱（３．９ｍｍ×１５０ｍｍ）。系统的优化步序包括全面调控流动相的ｐＨ值与ＴＥＡ浓度、乙醇梯度程序、柱温等诸多影响ＨＰＬＣ色谱行为的因素。最适条件为：Ａ溶液含０．１４ＭＧ酸钠、０．７５ｍｌ／ＬＴＥＡ、ＰＨ６．３５;Ｂ溶液为１００％乙醇;柱温３０℃。通过优化的乙醇梯度最终在约４４ｍｉｎ内将１５种ＰＴＣ－氨基酸很好地分离。此法可用于替代代表新科技水平的ＰＴＣ－氨基酸乙腈分析系统。     

Effect of administration of 5-hydroxytryptophan and an inhibitor ofl-aromatic amino acid decarboxylase on glucose metabolism in rat brain

The effect of 5-hydroxytryptophan (5-HTP)—the precursor of serotonin (5-hydroxytryptamine, 5-HT)—and of an inhibitor,N-(dl-seryl)-N-(2,3,4-trihydroxybenzyl)hydrazine (Ro4-4602), ofl-aromatic amino acid decarboxylase on the metabolism of glucose to amino acids in brain tissue was investigated. Labeled glucose (20 Ci, 0.24 mg in 0.2 ml 0.9% saline) was injected intravenously into fed rats pretreated with Ro4-4602 (50 mg/kg intraperitoneally) either alone or in combination with 5-HTP (30 mg/kg intravenously) or with the appropriate vehicle. After the injection of Ro4-4602 plus 5-HTP, the concentrations of 5-HT and 5-HTP in brain were increased, but the increase of 5-HTP that Ro4-4602 slightly inhibits the reaction of decarboxylation in the brain, although at the dose used the drug is usually considered to act only peripherally. After administration of Ro4-4602 alone or combined with 5-HTP, the concentration of glucose in plasma was not significantly increased. However, the concentration of glucose in brain was markedly increased with such treatments. The administration of Ro4-4602 alone or combined with 5-HTP reduced the flux of¹⁴C from labeled glucose to amino acids in brain. The concentrations of amino acids in brain were little changed by these treatments.     

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.     

Electrically induced release of amino acids formed from (U14C) glucose in rat brain cortex slices,studied by a simplified dansylation procedure

The nonessential amino acids glutamate, aspartate, glutamine, -minobutyrate (GABA), alanine, glycine, and proline present in rat thin brain cortex slices were labeled by in vitro incubation of these with [U-¹⁴C]glucose, and the efflux of such endogenous radioactive amino acids and of lactate was studied in a superfused system, under control conditions or when the slices were depolarized by various procedures. When electrical stimuli known to induce selective neurotransmitter release (1 or 1.5 volt, sine wave 60 Hz) were applied for 10 sec to the slices, no significant increase in amino acid efflux was found. When more intense stimuli (4 volt, 60 Hz) were applied for 60 sec, or extracellular potassium was raised to 56 mM, both conditions being known to induce nonselective substance release, the efflux of essentially all amino acids and of lactate was markedly increased. Increases in efflux were proportionately larger for glutamate, aspartate, and -aminobutyrate, and this could be accounted for by their greater intracellular chemical (or electrochemical) potentials, but not because of a selective release mechanism for them. Amino acids were analyzed as their 1-dimethylaminonaphthalene-5-sulfonyl (dansyl) derivatives, by a modification of existing procedures in which the dansyl (DNS) derivatives were efficiently extracted from acidified incubation fluid into an organic phase. This rapidly desalted the derivatives and allowed their concentration and chromatographic separation on thin-layer silica gel sheets with little loss.     

The role of amino acids in phagostimulation in the shallow-water omnivorous Antarctic sea star Odontaster validus

Experiments were carried out to study the behavioural responses of the omnivorous Antarctic sea star Odontaster validus to natural food odour, several single amino acids and their mixtures. Starved sea stars responded to natural food stimulus by rapid rise of metabolic rate, locomotory activity directed towards signal source, cessation of movement after reaching it and initiation of feeding. All single amino acids that were tested were detected by experimental animals, although there were marked differences in the sea stars reaction to them. Amino acids with narrow and broad scope of influence were distinguished, with glutamic acid being the most potent sea star stimulant. It was also found that the same sea star reaction (e.g. metabolic rate increase or locomotory activity) can be caused by several different amino acids. The effects of amino acid mixtures were significantly stronger than that of single amino acids, with >80% of animals reaching signal source. O. validus seems well adapted to using both single and complicated food signals in its foraging behaviour.     

Permeation of membranes by the neutral form of amino acids and peptides: Relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10⁵ slower than facilitated inward transport across biological membranes. This suggests that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10¹⁰ times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g., for certain signal sequences, toxins and thylakoid proteins) in vivo.Abbreviations LUV large unilamellar vesicle - pH transmembrane pH gradient - PAH polyaromatic hydrocarbon Correspondence to: A.C. Chakrabarti     

Age dependence of cowpea protoplasts for uptake of spermidine and infectibility by alfalfa mosaic virus

Cowpea protoplasts were prepared from plants of different ages and examined for their ability to take up polyamines and for their infectibility by alfalfa mosaic virus. A lag period of 20 h was necessary before the onset of rapid polyamine uptake; the occurrence of this rapid uptake depended on the age of the leaves used for protoplast preparation. The percentage of infection of cowpea protoplasts by alfalfa mosaic virus, and the amount of virus produced also depended on the age of the plants used for protoplast preparation. In contrast, the uptake of amino acids was rapid in all cowpea protoplasts tested.     

Simultaneous determination of primary and secondary d- and l-amino acids by reversed-phase high-performance liquid chromatography using pre-column derivatization with two-step labelling method

This work describes a method for the simultaneous determination of primary d- and l-amino acids and secondary amino acids such as d- and l-proline. In order to remove interferences in the simultaneous determination of primary and secondary amines, the primary amines were derivatized with o-phthalaldehyde/N-acetyl-l-cysteine (OPA/NAC) and subsequently with 1-(9-fluorenyl)ethyl chloroformate (FLEC) for secondary amines, in a pre-column separation derivatization technique. These fluorescent diastereomers of the amino acids were obtained within 3 min at room temperature and determined simultaneously by changing wavelengths during analysis in a single eluting run in the high-performance liquid chromatography column. This method, referred to as the “two-step labelling method,” is effective for the simultaneous determination of d- and l-amino acids.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.