An improved method for the determination of galactosaminitol,glucosaminitol, glucosamine,and galactosamine on an amino acid analyzer

Existing methods for the separation and quantitation of galactosaminitol, glucosaminitol, glucosamine, and galactosamine with an amino acid analyzer have been improved substantially by (1) addition of 15% ($\text{v}\text{v}$) 1-propanol to the citrate-borate buffer, (2) utilization of a two-buffer system, (3) doubling the length of the ninhydrin reaction coil, and (4) inclusion of an internal standard, ε-amino-n-eaproic acid, in each sample. These modifications decrease the time for the separation of these compounds to 110 min and increase the sensitivity for hexosaminitols to 3–4 nmol, and that for hexosamines, to 2 nmol.     

Determination of amino sugars and amino acids in glycoconjugates using precolumn derivatization with o-phthalaldehyde.

This report examines the RP-HPLC separation of o-phthalaldehyde derivatives of amino acids, amino sugars, and amino sugar alcohols using either 2-mercaptoethanol or 3-mercaptopropionic acid. A method with pmol sensitivity for the analysis of N-acetylamino sugars of glycoconjugates was elaborated. Upon hydrolysis, amino sugars are reduced with borohydride. Automated precolumn derivatization and chromatographic conditions for the resulting hexosaminitols are the same as those used for the analysis of amino acids. The method has been tested with as little as 2 micrograms of bovine fetuin, with a glycopeptide from bromelain and with an oligosaccharide after periodate oxidation.     

HPLC analysis of hexosamine phosphates in biological samples.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.     

海南产螺旋藻粉蛋白质氨基酸

采用Tecator自动定氮仪和Waters PICO-TAG氨基酸自动分析仪测定了海南螺旋藻粉中蛋白质含量和17种氨基酸含量。结果显示海南螺旋藻粉中蛋白质含量达63.3%,总氨基酸含量达60.88%,其中必需氨基酸含量超过40%,比例平衡,符合FA0标准,可与国外产品媲美。     

Isocratic reverse-phase liquid chromatography assay for amino acid metabolic disorders using eluates of dried blood spots

A high-performance liquid chromatographic method for the simultaneous determination of the amino acids methionine, valine, tryptophan, phenylalanine, isoleucine, and leucine extracted from dried blood spots used in neonatal screening is described. The amino acids are eluted from a 3-mm filter paper disc of dried blood with an absolute ethanol:norleucine internal standard solution (1.5:1, v/v), derivatized with o-phthalaldehyde prior to injection, and separated on a C-18 reverse-phase column with subsequent fluorescent detection. The analysis time is under 9 min at the described sample dilution and the assay is linear from 15 to 300 mumols/liter for five of the amino acids and from 15 to 500 mumols/liter for valine. The interrun coefficients of variation are less than 10% (except for tryptophan) and the analytical recoveries exceed 85%. Results from patient samples correlate well with those from a Waters Pico-Tag amino acid analysis system and no apparent interferences were encountered. The rapid analysis time and the specificity of the assay will facilitate the presumptive diagnosis of the inherited amino acidopathies phenylketonuria, maple syrup urine disease, and homocystinuria/methioninemia as well as monitoring blood levels of diagnosed patients.     

Novel mannitol-containing oligosaccharides obtained by mild alkaline borohydride treatment of a chondroitin sulfate proteoglycan from brain.

Mannitol-containing oligosaccharides have been isolated from a rat brain proteoglycan after mild alkaline borohydride treatment under conditions which prevent "peeling." Their structural properties were studied by gas-liquid chromatography-mass spectrometry of disaccharides as their trimethylsilylated and permethylated derivatives, methylation, analysis, specific degradations, and CrO3 oxidation. The following components were identified: Gal(beta 1 leads to 4) [Fuc(alpha 1 leads to 3)]GlcNAc(beta 1 leads to 3)Manol,GlcNAc(beta 1 leads to 3)Manol, and Manol. Evidence was also obtained for the occurrence of a sialylated oligosaccharide and another (possibly sulfated) acidic oligosaccharide, both having the sequence GlcNAc(beta 1 leads to 3)Manol at their proximal ends. These mannitol-containing oligosaccharides constitute a novel group of alkali-labile oligosaccharides in mammalian glycoconjugates. The origin of the oligosaccharides and the possible occurrence of a carbohydrate-peptide linkage involving mannose are discussed.     

Formation of methyl ester of 2-methylglyceric acid from thymine glycol residues: a convenient new method for determining radiation damage to DNA

Thymine glycol residues in DNA or thymidine were converted to methyl 2-methylglycerate by reaction with alkaline borohydride followed by methanolic HCl. The product was labeled either from [3H]DNA or from [3H]borohydride and was followed by cochromatography with authentic 14C-labeled material. Following acid hydrolysis, the identity of 2-methylglyceric acid was confirmed by high-resolution mass spectrometry, NMR, IR, and elemental analysis. Treatment of DNA or thymidine with X-irradiation, with H2O2 and Fe2+, with H2O2, Cu2+, and ascorbate, and with H2O2 and ultraviolet light, permanganate, or sonication all produced methyl 2-methylglycerate in varying amounts after alkaline borohydride and methanolic HCl, whereas untreated DNA did not. The data indicate that certain oxidants including hydroxyl radicals generated by chemical means or from radiolysis of water convert thymine residues to thymine glycols in DNA, which can be determined as methyl 2-methylglycerate.     

The effect of alkaline borohydride treatment onN-linked carbohydrates of glycoproteins

The effects of treatments of the glycoprotein ribonuclease-B, the proteins ribonuclease-A and myoglobin, and the glyco-amino acid GlcNAc(1-N) Asn with alkalil alkaline sodium borohydride, and aqueous sodium borohydride were systematically studied as a function of the concentration of the reagents, the temperature, and the length of the treatment. High-field¹H-NMR spectroscopy, chromatographic methods and amino-acid analysis were used to characterize products of the treatments of the various compounds. Our results indicate that mild alkaline borohydride treatment, as well as aqueous borohydride treatment alone, is capable of extensively degrading polypeptides and of partially releasing theN-linked glycans from ribonuclease-B. Initially, glycopeptides are produced, the peptide portion of which consists of several amino acids, which are further hydrolyzed to yield a mixture of glyco-asparagines and oligosaccharide-alditols in the ratio of 4:1. Strong alkaline borohydride treatment of ribonuclease-B is capable of completely releasing theN-linked carbohydrates as oligosaccharide-alditols.Abbreviation RNase ribonuclease     

Quantitation of oligosaccharides released by the β-elimination reaction

A quantitative micromethod has been described for monitoring the rate and extent of the β-elimination reaction as applied to O-glycosyl-glycoproteins utilizing alkaline tritiated borohydride. The procedure simultaneously labels the released oligosaccharides by their reduction to the corresponding tritiated alditols. The alkaline tritiated borohydride treatment also results in the labeling of the protein moiety of the glycoprotein and this can be quantitatively separated from the carbohydrate moiety on a cation exchange resin; the carbohydrate moiety is not adsorbed, while the protein moiety is adsorbed and then eluted with HCl. The radioactivity in the aqueous eluate of the resin is therefore a direct measure of the amount of oligosaccharides released by the β-elimination reaction. The sensitivity of the method is dependent on the specific activity of the tritiated sodium borohydride used. The stoichiometry of the reaction has been established by the use of N-acetylgalactosaminyl-O-glycoproteins, demonstrating that at the completion of the β-elimination reaction: (a) none of the radioactivity attributable to the protein moiety contaminates the carbohydrate moiety, (b) all the carbohydrate components of the glycoprotein are found in the aqueous eluate from the cationic exchange resin, (c) all the radioactivity in this aqueous eluate is associated with the sugar known to be at the reducing end of the oligosaccharide chain bound to serine or threonine of the glycoprotein (in the examples discussed, N-acetylgalactosamine), and (d) there is no additional hydrolysis of the oligosaccharide chains during the processing.     

Characterization of the O-linked oligosaccharide structures on P-selectin glycoprotein ligand-1 (PSGL-1)

P-selectin glypoprotein ligand-1, PSGL-1, a specific ligand for P-, E-, and L-selectin, was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin and platelet P-selectin- or E-selectin receptor globulin-agarose chromatography. The O-linked oligosaccharides on the ligand were released by mild alkaline sodium borohydride treatment and analyzed by a combination of ion-exchange, size exclusion, lectin, and paper chromatography, together with specific exoglycosidase treatments and chemical modifications. Approximately 91% of the radioactivity released from PSGL-1 was recovered in five O-linked glycans: GalNAc (approximately 4% of the total structures), Gal, 3GalNAc (36%), and Gal, 3GalNAc substituted with one (45%), two (6 %), or three (3%) N-acetyllactosamine repeat units. None of these structures contained fucose, and the majority were substituted with at least one sialic acid. The N-acetyllactosmine-containing structures appeared to be core 2. The remaining 9% of the radioactivity recovered in O-linked oligosaccharides from PSGL-1, eluted in two peaks at 11.8 and 10.2 glucose units, on size-exclusion chromatography. Results from lectin chromatography and chemical and enzymatic degradation experiments suggest that the major portion of the radioactivity in these peaks is associated with sialylated N-acetyllactosamine-type oligosaccharides, substituted with fucose at the penultimate residue in the nonreducing end. Since both sialic acid and fucose reportedly are crucial requirements for selectin binding, these results suggest that only a minor portion, approximately 4.5%, of the O-linked oligosaccharides on PSGL-1 are involved in the interaction with the selectins.     

Sialoglycopeptides from bovine milk fat globule membrane.

Milk fat globule membrane was shown to contain sialic acid, all of which could be released without disruption of the fat globule. Sialoglycopeptides were cleaved from the surface of intact fat globules by Pronase and fractionated on Sephadex G-50. Further fractionation of the major sialoglycopeptide peak on DEAE-Sephadex gave two groups of sialoglycopeptides eluted with 0.1 M NaCl (Group A) and 0.5 M NaCl (Group B), respectively. Refractionation gave a major sialoglycopeptide from each of the two groups together with a total of three minor sialoglycopeptides. All five sialoglycopeptides eluted as single peaks using shallow salt gradients on DEAE-Sephadex and contained a hydrophilic peptide chain together with galactose, mannose, N-acetylgalactosamine, N-acetylglucosamine, and sialic acid. Glycopeptides of Group A but not Group B contained fucose. The major sialoglycopeptide of Group B released 35% of its hexose and hexosamine on treatment with alkaline borohydride leaving a sialoglycopeptide which had reduced serine and threonine and elevated alanine levels and in addition contained 2-aminobutyric acid. An oligosaccharide fraction containing N-acetylgalactosaminitol, galactose and sialic acid in a molar ratio of 1:1:2 was partially characterised from the clevage mixture. The major sialoglycopeptide of Group A had a more complex carbohydrate structure and showed no released carbohydrate on treatment with alkaline borohydride. The sialoglycopeptides of milk fat globule membrane show many similarities with those of erythrocyte membrane and have a potential use in comparative and structural studies.     

In vitro labeling of the sialic acid moiety of glycoconjugates with carbon-14

Labeling of sialoglycoproteins with carbon-14 in vitro was performed by reacting the aldehyde groups, generated by mild periodate oxidation of the terminal sialyl groups, with 14C-labeled sodium cyanide to produce the labeled cyanohydrin derivatives (Kiliani reaction). Labeling with tritium was carried out by reduction of the aldehyde groups generated on the sialyl residues with 3H-labeled sodium borohydride following standard procedures. The behavior of both types of labeled specimens of fetuin and ovine submaxillary mucin, individually and in mixtures, was investigated by gel-filtration chromatography, gel electrophoresis, and cesium bromide gradient ultracentrifugation. The labeled sialyl residues were subjected to partial characterization: color yield with the resorcinol and thiobarbituric acid reagents, behavior on ion-exchange chromatography, and susceptibility to mild acid and enzymatic hydrolyses. In addition to these model glycoproteins, this procedure was also utilized to label the sialoglycoproteins present in human tracheobronchial secretions collected from normal subjects and patients with chronic bronchitis. The potential uses of this approach for comparative studies of normal and pathological sialoglycoconjugates available in minute amounts is described. The extension of this approach to the labeling of the galactosyl and N-acetylgalactosaminyl moieties of glycoconjugates following treatment with galactose oxidase is outlined.     

High-performance liquid chromatographic determination of modafinil and its two metabolites in human plasma using solid-phase extraction

A simple procedure for the simultaneous determination of modafinil, its acid and sulfone metabolites in plasma is described. The assay involved an extraction of the drug, metabolites and internal standard from plasma with a solid-phase extraction using C₁₈ cartridges. These compounds were eluted by methanol. The extract was evaporated to dryness at 40°C under a gentle stream of nitrogen. The residue was redissolved in 250 μl of mobile-phase and a 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile-phase (26%, v/v acetonitrile in 0.05 M orthophosphoric acid buffer adjusted to pH 2.6) at a flow-rate of 1.1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 225 nm. Intra-day coefficients of variation ranged from 1.0 to 2.9% and inter-day coefficients from 0.9 to 6.1%. The limits of detection and quantitation of the assay were 0.01 μg/ml and 0.10 μg/ml respectively.     

High-resolution separation of disaccharide and oligosaccharide alditols from chondroitin sulphate, dermatan sulphate and hyaluronan using CarboPac PA1 chromatography

Recent literature indicates that specific glycosaminoglycanstructures are involved in various biological processes, suchas anticoagulation, growth factor activation and viral infection.The initial step in the structural analysis of glycosaminoglycansis a definitive compositional analysis of its characteristicdisaccharide repeat structures. Current chromatographic or electrophoreticprocedures may have limitations in analysing glycosaminoglycansamples that are in low abundance, contain novel structuresthat need to be further characterized, or are metabolicallylabelled from radioactive precursors as a result of biosyntheticexperiments. This study presents a new methodology for analysingdisaccharides and oligosaccharides derived from chondroitinsulphate, dermatan sulphate and hyaluronan that fulfils theabove criteria. The procedure involves the separation of reducedforms of these glycoconjugates on a CarboPac PA1 column usingalkaline eluants. This study adopted a strategy which uses specificenzymes to release these disaccharides from their glycosaminoglycanforms. A borohydride reduction reaction was modified to be compatiblewith the buffer conditions commonly used with these enzymesin order to quantitatively reduce the disaccharides to theiralditol forms (thereby stabilizing them to alkaline pH). Chromatographyconditions were established which separated all known disaccharidealditol structures from chondroitin sulphate, dermatan sulphateand hyaluronan with extremely high resolution in a single run.Integrated pulsed amperometry was compared to UV absorbancemeasurement at 232 nm as two sensitive methods for detectingthese reduced disaccharides; most of them could be routinelydetected in the range of 50–500 ng. Data are presentedapplying this method to quantify hyaluronan in a biologicalsample which contains {small tilde}5000 cells and only {smalltilde}10 ng of hyaluronan. Additional data are presented todemonstrate that this procedure will also separate oligosaccharidealditols derived from hyaluronan. borohydride reduction glycosaminoglycans integrated pulsed amperometry     

The effect of mild alkali and alkaline borohydride on the carbohydrate and peptide moieties of fetuin

In the light of recent reports, based on radioactive labelling studies, that substantial amounts of N-linked oligosaccharides are released from protein under the mild-alkaline borohydride degradation conditions that are usually used to release O-linked oligosaccharides, we have investigated by chemical methods the effects of alkali alone and alkaline borohydride on the carbohydrate and peptide moieties of fetuin. The chromatographic profiles on Sephadex G50 columns, of the hexose- and ninhydrin-positive components of the native and Pronase-treated glycoprotein have been compared with those obtained after treatment with mild alkali alone (0.05 M-NaOH, 50 degrees C, 16 h) or mild-alkaline borohydride (0.05 M-NaOH containing 1 M-NaBH4, 50 degrees C, 16 h). Composition and methylation analyses have been performed on carbohydrate-containing peaks and the following conclusions were drawn: mild alkali treatment alone liberated a minor hexose- and ninhydrin-positive component and mild-alkaline borohydride treatment gave a major hexose-containing peak: both of these co-chromatographed on a Sephadex G50 column with Pronase glycopeptides. The polypeptide backbone was totally broken down by the alkaline borohydride treatment. The presence of released N-linked chains after alkaline borohydride treatment was confirmed. However, from the carbohydrate composition it was calculated that no more than 10-20% of the N-linked chains were released from protein. The results of methylation analysis have raised the possibility that this release is in part due to cleavage of the chitobiosyl core.     

Isocratic high-performance liquid chromatographic determination of thiacetazone by direct injection of plasma into an internal surface reversed-phase column

Thi report describes the determination of thiacetazone in human and rat plasma by direct-injection high-performance liquid chromatography (HPLC). Plasma filtrate (50 μl) was injected directly into the internal surface reversed-phase (ISRP) mixed-functional phenyl column (Capcell Pak, 50×4.6 mm, 5 μm) and eluted with an aqueous mobile phase containing 7.5% acetonitrile at a flow-rate of 1 ml/min. With UV detection at 322 nm, thiacetazone eluted at 11.0 min whereas endogenous interferences eluted before 5 min. The lower detection limit for a 50-μl sample at a signal-to-noise ratio of 5 was 63 ng/ml, which was several hundred fold lower than its cytotoxic concentrations determined from in vitro cell line studies. At a concentration range of 0.17 to 2.7 μg/ml, the recovery of thiacetazone was 98.0±4.4% (mean±S.D.). The intra- and inter-day coefficients of variation were 3.0±1.4% and 4.2±2.1%, respectively. This method was successfully applied to study the pharmacokinetics of thiacetazone in rats. The direct injection method is simple, requires small sample volume and does not require sample extraction, internal standard, or gradient elution.     

Simultaneous determination of in total 17 opium alkaloids and opioids in blood and urine by fast liquid chromatography-diode-array detection-fluorescence detection,after solid-phase extraction

A fast liquid chromatographic method with tandem diode array-fluorescence detection for the simultaneous determination of in total 17 opium alkaloids and opioids is presented. Blank blood and urine samples (1 ml) were spiked with different concentrations of a standard mixture, as well as with the internal standard, butorphanol (2000 ng/ml). After solid-phase extraction, based on weak cation exchange (Bond Elut CBA SPE columns), the extracts were examined by HPLC-DAD-FL. By using a "high-speed" phenyl column (53 x 7.0 mm I.D., particle size 3 microm) eluted with a gradient system (A: water-methanol (90:10, v/v), B: methanol, both containing 25 mM triethylammoniumformate (pH(A) = 4.5)) all compounds could be baseline separated within 12 min. The method was validated and its applicability was demonstrated by the analysis of real-time forensic cases.     

A fluorometric assay for the quantitation of aminosugars.

A method is presented for the quantitative determination of aminosugars in glycoproteins. Glycoproteins are acid hydrolyzed and reacted with the fluorogenic reagent 1-dimethylaminonaphthalene-5-sulfonyl chloride. Following cellulose thin layer electrophoresis in pyridine/acetic acid (pH 4.4), 5 h at 500 V, appropriate areas of the plate are scraped and the dansyl hexosamines are eluted with 95% ethanol. Aliquots are then applied to a silica gel tlc plate and subjected to ascending thin layer chromatography in cyclohexane/ethylacetate/ethanol (6/4/3). After spraying the plate with triethanolamine/isopropanol ($\text{1}\text{4}$), fluorescent intensities are measured by in situ scanning. The aminosugar content of the glycoprotein is determined from a curve generated from a series of standards run concurrently on each plate. The method clearly resolves glucosamine from galactosamine, and is sensitive for the detection of aminosugars in the subnanomole range.     

High-performance liquid chromatographic determination of cotinine in urine in isocratic mode

A simple procedure for the determination of cotinine, major metabolite of nicotine in urine, is described. The assay involved a liquid–liquid extraction with dichloromethane in alkaline environment. The extract was dried at ambient temperature under a gentle stream of nitrogen. The residue was dissolved in 300 μl of mobile phase and 30 μl aliquot was injected via an automatic sampler into the liquid chromatograph and eluted with the mobile phase (10–9%, v/v methanol and acetonitrile, respectively in potassium dihydrogenphosphate buffer adjusted to pH 3.4) at a flow rate of 1 ml/min on a C₈ Symmetry cartridge column (5 μm, 150 mm×3.9 mm, Waters) at 25°C. The eluate was detected at 260 nm. Internal standard was 2-phenylimidazole. Sensitive and specific, this technique was performed to test urine of diabetic patients (smokers and non-smokers) admitted in an endocrinology service. Urinary cotinine seems to be a better marker of smoking status than thiocyanates.