Branched arabino-oligosaccharides isolated from sugar beet arabinan

Sugar beet arabinan consists of an α-(1,5)-linked backbone of l-arabinosyl residues, which can be either single or double substituted with α-(1,2)- and/or α-(1,3)-linked l-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an α-(1,5)-linked backbone of l-arabinosyl residues. One series carries single substituted α-(1,3)-linked l-arabinosyl residues at the backbone, whereas the other series consists of a double substituted α-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.     

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.     

Oligosaccharide analysis by capillary-scale high-pH anion-exchange chromatography with on-line ion-trap mass spectrometry

A capillary-scale high-pH anion-exchange chromatography (HPAEC) system for the analysis of carbohydrates was developed, in combination with two parallel on-line detection methods of sub-picomolar sensitivity: (1) pulsed amperometric detection (PAD); (2) capillary-scale desalting followed by electrospray ion-trap (IT) mass spectrometry (MS). The capillary chromatographic system combined the superb selectivity of HPAEC that allows routine separation of isomeric oligosaccharides with the information on monosaccharide sequence and linkage positions obtained by MS/MS fragmentation using the IT-MS. The applicability of the system in biomedical research was demonstrated by its use for the analysis of a urine sample of a GM1-gangliosidosis patient. Isomeric glycans in the sample could be resolved by HPAEC and assigned on the basis of the monosaccharide linkage information revealed by on-line IT-MS/MS.     

STUDIES OF POLYSACCHARIDES FROM THREE EDIBLE SPECIES OF NOSTOC (CYANOBACTERIA) WITH DIFFERENT COLONY MORPHOLOGIES: STRUCTURAL CHARACTERIZATION AND EFFECT ON THE COMPLEMENT SYSTEM OF POLYSACCHARIDES FROM NOSTOC COMMUNE

The cyanobacterium Nostoc commune Vaucher produces quite complex extracellular polysaccharides. The cyanobacterium is nitrogen fixing, and on growing the cyanobacterium in media with and without nitrogen, different types of extracellular polysaccharides were obtained. These were also different from the polysaccharides present in N. commune collected in the field. High pH anion exchange chromatography (HPAEC) of weak acid hydrolysates of the culture-grown material demonstrated that, in this case, HPAEC was useful for comparison of the different polymers. The main differences between the polymers from the field group and the culture-grown samples were the presence of substantial amounts of arabinose, 2- O -methylglucose, and glucuronic acid in the latter. Methylation studies also revealed a difference in the branching points on the glucose units between the field and cultured samples, being 1,4,6 for the first and 1,3,6 for the latter. The field acidic fraction gave, on weak acid hydrolysis and separation on BioGel P2 and HPAEC, 12 oligosaccharide fractions that were isolated and studied by different mass spectroscopy techniques. The structures of the oligosaccharides were determined, and two different series that can originate from two repeating pentamers were identified: GlcA1-4/6GlcM1-4Gal1-4Glc1-4Xyl and GlcA1-4/6Glc1-4Gal1-4Glc1-4Xyl. The difference between these oligosaccharides lies in the methyl substituent on carbon 2 of the glucose unit next to the nonreducing glucuronic acid unit. The polysaccharides from field material were shown to have a strong effect on the complement system.     

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.     

Simultaneous determination of five flavonoids from Scutellaria Barbata extract in rat plasma by LC-MS/MS and its application to pharmacokinetic study

A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the determination of five flavonoids including scutellarin, naringenin, apigenin, luteoline and wogonin in rat plasma using sulfamethalazole as internal standard (IS). Plasma samples were pretreated with liquid-liquid extraction procedure and acid hydrolysis method was used for converting conjugated flavonoids to their respective free forms. The chromatographic separation was performed on a C(18) column with a linear gradient elution using a mobile phase consisted of 0.01% acetic acid and methanol. The detection was accomplished by multiple-reaction monitoring (MRM) scanning with electrospray ionization (ESI) source operating in the negative ionization mode. The optimized mass transition ion-pairs (m/z) monitored for scutellarin, naringenin, apigenin, luteoline, wogonin and IS were 461.1/285.1, 271.0/119.0, 269.0/117.0, 285.0/132.9, 283.0/268.0 and 252.0/155.9, respectively. The method was linear for all analytes over investigated ranges with all correlation coefficients greater than 0.9915. The lower limit of quantitation (LLOQ) of scutellarin was 9.15 ng/mL and other compounds were all less than 2.0 ng/mL. The proposed method showed appropriate accuracy and repeatability and was suitable for pharmacokinetic studies of the five flavonoids after oral administration of Scutellaria Barbata extract.     

Analysis of glycosphingolipid-derived oligosaccharides by high pH anion exchange chromatography

As an adjunct to existing thin layer and column chromatographic methods for the identification of glycolipids a method that utilizes the high pH anion chromatographic (HPAEC) analysis of the oligosaccharides released from the glycolipids by endoglycoceramidase has been developed. Using a Dionex Carbo Pak PA1 column and elution with a linear gradient of sodium acetate in 0.2M NaOH, the elution times of eight neutral and fourteen acidic oligosaccharides derived from glycolipids were determined. Under these conditions the neutral oligosaccharides were well separated from each other but some of the acidic oligosaccharides had overlapping elution times. The ganglioside-derived oligosaccharides could be further identified by treating them with sialidase or by mild acid hydrolysis and reanalysing the products by HPAEC. The method was applied to the analysis of mixed bovine brain gangliosides. The procedure provides an additional approach for the initial identification of glycolipids by analysing the component oligosaccharides rather than the intact glycolipids.     

Liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for simultaneous determination of venlafaxine and its active metabolite O-desmethyl venlafaxine in human plasma

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for simultaneous quantification of venlafaxine (VEN) and O-desmethyl venlafaxine (ODV) in human plasma. The analytes were extracted from human plasma by using solid-phase extraction (SPE) technique. Escitalopram (ESC) was used as the internal standard. A Betasil C18 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The mass transition ion-pair has been followed as m/z 278.27-->121.11 for VEN, m/z 264.28-->107.10 for ODV and m/z 325.00-->262.00 for ESC. The method involves a solid phase extraction from plasma, simple isocratic chromatography conditions and mass spectrometric detection that enables detection at nanogram levels. The proposed method has been validated with linear range of 3-300 ng/ml for VEN and 6-600 ng/ml for ODV. The intrarun and interrun precision and accuracy values are within 10%. The overall recoveries for VEN and ODV were 95.9 and 81.7%, respectively. Total elution time as low as 3 min only.     

Effects of solvent on chiral and enantiomeric separation of Koga bases using derivatized amylose chiral stationary phase

The separation of R,R‐, S,S‐, and meso‐Koga bases on derivatized amylose chiral stationary phases (CSP) has been studied using different alcohol and alcohol‐hexane mixtures as eluant. Straight‐chain and branched alcohols with carbon numbers from one to four were investigated. The carbon number and geometry of the alcohol impacts the separation of Koga bases. The optimal separations were obtained using a mixture of methanol with linear or branched alcohol. Also, the elution order of meso‐ and R,R‐Koga base was switched as content of branched alcohol increases in cosolvent. The study of acidic and basic additive effects demonstrated that maintaining analytes in the free base state is crucial in order to achieve retention and separation. TEA alone or TEA and TFA mixture were used in the studies. Chirality 2010. © 2009 Wiley‐Liss, Inc.     

Interpreting size-exclusion data for highly branched biopolymers by reverse monte carlo simulations

Size-exclusion chromatography with multiple detection provides data on the distributions of various properties in a branched polymer sample, for example, distributions of the number, average mass, mean-squared mass, and branching fraction against hydrodynamic volume. A method is developed that provides a basis to use such data for obtaining structural and biosynthetic information on highly branched polymers, such as amylopectin. We generate by simulation a reference distribution of randomly branched polymers from the experimental distribution of debranched chains of the target polymer. We then select from these simulated chains a set with the same number (or other) distribution as the actual polymer sample, using reverse Monte Carlo simulations. Properties of these model polymers are used to interpret the differences with experiment as due to correlations in branching structure. The same methodology can be applied to data from other separation techniques such as field-flow fractionation and high-performance anionic exchange chromatography.     

The L-Arabinan utilization system of Geobacillus stearothermophilus

Geobacillus stearothermophilus T-6 is a thermophilic soil bacterium that has a 38-kb gene cluster for the utilization of arabinan, a branched polysaccharide that is part of the plant cell wall. The bacterium encodes a unique three-component regulatory system (araPST) that includes a sugar-binding lipoprotein (AraP), a histidine sensor kinase (AraS), and a response regulator (AraT) and lies adjacent to an ATP-binding cassette (ABC) arabinose transport system (araEGH). The lipoprotein (AraP) specifically bound arabinose, and gel mobility shift experiments showed that the response regulator, AraT, binds to a 139-bp fragment corresponding to the araE promoter region. Taken together, the results showed that the araPST system appeared to sense extracellular arabinose and to activate a specific ABC transporter for arabinose (AraEGH). The promoter regions of the arabinan utilization genes contain a 14-bp inverted repeat motif resembling an operator site for the arabinose repressor, AraR. AraR was found to bind specifically to these sequences, and binding was efficiently prevented in the presence of arabinose, suggesting that arabinose is the molecular inducer of the arabinan utilization system. The expression of the arabinan utilization genes was reduced in the presence of glucose, indicating that regulation is also mediated via a catabolic repression mechanism. The cluster also encodes a second putative ABC sugar transporter (AbnEFJ) whose sugar-binding lipoprotein (AbnE) was shown to interact specifically with linear and branched arabino-oligosaccharides. The final degradation of the arabino-oligosaccharides is likely carried out by intracellular enzymes, including two α-l-arabinofuranosidases (AbfA and AbfB), a β-l-arabinopyranosidase (Abp), and an arabinanase (AbnB), all of which are encoded in the 38-kb cluster.     

Enantioselective Separation and Simultaneous Determination of Tolperisone and Eperisone in Rat Plasma by LC‐MS/MS

Tolperisone and eperisone used as muscle relaxants possess one chiral center each and exist as two optical isomers for each drug. Therefore, enantioselective assays to measure each enantiomer in biological matrices are of great importance. In the present study a simple and complete reverse‐phase liquid chromatography tandem mass spectrometric method for separation and enantioselective determination of tolperisone and eperisone in rat plasma was developed. The analytes were extracted from rat plasma by a simple protein precipitation method with acetonitrile as the extraction solvent. The enantioselective separation of analytes was achieved on a Cellulose Tris (4‐chloro‐3‐methylphenylcarbamate) chiral column with a mobile phase of acetonitrile: 10 mM ammonium acetate in an isocratic mode of elution and mass spectrometric detection. The calibration curve for each enantiomer was found to be linear over 0.2 to 20 ng/mL for each enantiomer. The proposed method exhibited good intra‐ and interday precision (% CV) ranged between 0.95–6.05% and 1.11–8.21%, respectively. The intra‐ and interday accuracy for the proposed assay method ranged between 94.0–100.5% and 92.7–102.1%, respectively. The proposed method was validated as per regulatory guidelines. Chirality 25:622–627, 2013. © 2013 Wiley Periodicals, Inc.     

Electrophoretic mobility of human red blood cells coated with poly(ethylene glycol)

Poly(ethylene glycol), abbreviated as PEG, was covalently attached to the surface of human red blood cells (RBC) and the effects of such coating on the regions near the cell's glycocalyx were explored by means of cell electrophoresis. RBC electrophoretic mobilities were measured, in polymer-free buffers of various ionic strengths, as functions of PEG molecular mass (3.35, 18.5, 35.0, 35.9 kDa), geometry, (linear or 8-arm branched) and polymer/RBC ratio during attachment. The results indicate marked decreases of the mobility (up to 85%) which were affected by polymer molecular mass and geometry. Since PEG is neutral and its covalent attachment only removes positively-charged amino groups on the cell membrane, such decreases of mobility likely reflect structural changes near and within the RBC glycocalyx. Experimental results were analyzed using an extended "hairy sphere" model to consider friction and thickness of the polymer layer. Calculated polymer layer thickness increased with molecular mass for linear PEGs and was less extended for a branched PEG of similar molecular mass. Friction within the polymer layer increased with polymer/RBC ratio and for the linear PEGs was inversely related to molecular mass; friction was greatest for the branched PEG. Our results are consistent with the effects of attached PEGs on RBC aggregation and surface antigenic site masking, and suggest the usefulness of electrophoretic mobility techniques for studies of bound neutral polymers.     

Polyglycosylceramides recognized by Helicobacter pylori: analysis by matrix-assisted laser desorption/ionization mass spectrometry after degradation with endo-beta-galactosidase and by fast atom bombardment mass spectrometry of permethylated undegraded material

Human erythrocyte polyglycosylceramides (PGCs) are recognized by the gastric pathogen Helicobacter pylori and are based on a successively extended and highly branched N-acetyllactosamine core linked to ceramide and substituted by fucose and sialic acid. As a step in the identification of the binding epitope we earlier characterized intact PGCs by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF MS (Karlsson,H., Johansson,L., Miller-Podraza,H., and Karlsson,K-A. [1999] Glycobiology, 9, 765-778). In the present work, PGCs from human blood group O erythrocytes were digested with endo-ss-galactosidase (Bacterioides fragilis), an enzyme which cleaves the bond 3Galss1-4GlcNAc in linear but not branched poly-N-acetyllactosamine chains. The enzymatic digestion resulted in a mixture of neutral and sialic acid-containing glycolipids together with terminal and internal sequences of mainly neutral oligosaccharides. The products were analyzed by MALDI-TOF MS in both positive and negative ion mode which gave spectra where the ions could be assigned to structures of the neutral and acidic components, respectively. Among glycolipids found were [structure in text] where R could be H, Fuc or NeuAc. Also observed were structures as [structure in text] which indicated linear extension along both branches. Observed at higher masses were fully branched structures obtained by stepwise extension with [structure in text] where R could be H, Fuc or NeuAc. Most probably further branching may occur along both the (1-->3)- and the (1-->6)-linked branches to give a partly dendritic structure. Structures with more than one sialic acid substituted could not be observed in the MALDI spectrum. Complementary information of the terminal sequences was obtained by FAB-MS analysis of permethylated undegraded PGCs. High-temperature gas chromatography/mass spectrometry of reduced and permethylated products from enzyme hydrolysis documented that Fuc was present in a blood group O sequence, Fuc-Hex-HexN-. Fucose may be placed on short (monolactosamine) or longer branches, while sialic acid seems to be restricted to monolactosamine branches. The conclusion is that human erythrocyte PGCs display microheterogeneity within terminal and internal parts of the poly-N-acetyllactosamine chains. The first branch from the ceramide end may be located at the second or third Gal and possibly also on the first Gal. Other branches may occur on every N-acetyllactosamine unit in fully branched domains, or there may be linear extensions between branches resulting in incompletely branched structures. The extended linear sequences may be present in both 3- and 6-linked antennae. Terminal structures are based on one, two or maybe higher number of N-acetyllactosamine units.     

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS-HCl+0.03 M NaHCO(3) was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS-HCl+0.03 M NaHCO(3)) to 100% buffer B (A+1M NH(4)Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90+/-3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL(-1). In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.     

De facto molecular weight distributions of glucans by size-exclusion chromatography combined with mass/molar-detection of fluorescence labeled terminal hemiacetals

A major capability of polysaccharides in aqueous media is their tendency for aggregation and dynamic formation of supermolecular structures. Even extended dissolution processes will not eliminate these structures which dominate many analytical approaches, in particular absolute molecular weight determinations referring to light scattering data. An alternative approach for determination of de facto molecular weight for glucans with free terminal hemiacetal functionality (reducing end group) has been adjusted from carbohydrates for midrange and high-dp glucans: quantitative and stabilized labeling as aminopyridyl-derivatives (AP-glucans) and subsequent analysis of SEC-separated elution profiles based on simultaneously monitored mass and molar fractions by refractive index and fluorescence detection. SEC-DRI/FL of AP-glucans proved as an appropriate approach for determination of de facto molecular weight of constituting glucan molecules even in the presence of supermolecular structures for non-branched (pullulan), branched (dextran), narrow distributed and broad distributed and for mixes of compact and loose packed polymer coils (starch glucan hydrolizate).     

Purification and properties of two type-B alpha-L-arabinofuranosidases produced by Penicillium chrysogenum

Two distinct extracellular alpha-L-arabinofuranosidases (AFases; EC 3.2.1.55) were purified from the culture filtrate of Penicillium chrysogenum 31B. The molecular masses of the enzymes were estimated to be 79 kDa (AFQ1) and 52 kDa (AFS1) by SDS-PAGE. Both enzymes had their highest activities at 50 degrees C and were stable up to 50 degrees C. Enzyme activities of AFQ1 and AFS1 were highest at pH 4.0 to 6.5 and pH 3.3 to 5.0, respectively. Addition of 10 mg/ml arabinose to the reaction mixture decreased the AFS1 activity but hardly affected AFQ1. Both enzymes displayed broad substrate specificities; they released arabinose from branched arabinan, debranched arabinan, arabinoxylan, arabinogalactan, and arabino-oligosaccharides. AFS1 also showed low activity towards p-nitrophenyl-beta-D-xylopyranoside. An exo-arabinanase, which catalyzes the release of arabinobiose from linear arabinan at the nonreducing terminus, acted synergistically with both enzymes to produce L-arabinose from branched arabinan.     

Determination of amino acids in rat brain microdialysate with 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate as labeling reagent by high performance liquid chromatographic fluorescence detection and mass spectrometric identification

A high performance liquid chromatography method for the determination of 20 amino acids (AAs), using 1,2,5,6-dibenzocarbazole-9-ethyl chloroformate (DBCEOC-Cl) as a novel fluorescent labeling reagent, has been developed and applied for the analysis of AAs in rat brain microdialysate. The simultaneous separation of 20 AA derivatives was achieved on a Hypersil BDS C(18) column with gradient elution. And the identification of AA derivatives was carried out by on-line electrospray ionization mass spectrometry in positive ion mode. The AA derivatives were detected with excitation and emission at 300 nm and 395 nm, respectively. Excellent linear responses were observed with coefficients of >0.9988. The detection limits ranged from 0.217 to 4.75 nmol/L, at a signal-to-noise ratio of 3. The intra-day and inter-day precision for each AA was <3.2% and <4.3%, respectively. The mean recoveries for all AAs studied were in the range of 92.5-105.6%. Good compositional data could be obtained from as little as 15 μL of microdialysate. Facile DBCEOC-Cl derivatization coupled with high performance liquid chromatography and fluorescence detection allowed the development of a highly sensitive method for the quantitative analysis of trace levels of AAs from microdialysate.