Expression and distribution of N-acetyl and N-glycolylneuraminic acids in secreted and cell-associated glycoconjugates by two human osteosarcoma cell lines

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the dominant sialic acids (Sia) in mammals usually found in the non-reducing terminal of oligosaccharide side chains in glycoproteins and glycolipids. Their expression and distribution pattern have been correlated both with the malignant phenotype and tumor grade of human cancers. The aim of the present study was to determine by reversed-phase HPLC method the amounts of Neu5Ac and Neu5Gc as well as their distribution among the culture media and cell surface of MG-63 and Saos-2 human osteosarcoma cell lines of high and low metastatic potential. It was determined that MG-63 cells produce up to 5-fold more total sialic acid as compared with the Saos 2 cells. Neu5Ac accounts for ca 60% of the total sialic acids secreted by MG-63 cells, whereas Neu5Gc is the predominant sialic acid present on the MG-63 cell membrane. Saos 2 cells secrete considerable amounts of Neu5Ac to culture media. The obtained data indicate that the human osteosarcoma cells express both forms of Sia-containing glycoconjugates; the differences in the amounts of each of the two major Sia types and their distribution may be related to their differences in morphology and/or metastatic potentials.     

Determination of sialic acids in infant formula by liquid chromatography tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method using pneumatically assisted electrospray ionization (LC-ESI-MS/MS) was developed for determination of N-acetylneuramic acid and N-glycolylneuramic acid in infant formula. Reconstituted samples were hydrolysed in dilute sulfuric acid and deproteinized with acetonitrile. The extract was analysed directly without further clean-up by hydrophilic interaction chromatography. The substances were detected in negative ion mode and matrix matched standards were used for calibration. The relative intra-laboratory reproducibility standard deviation was better than 6% for both substances. An R2 of 0.985 was obtained by comparison with a classical colorimetric assay based on reaction with resorcinol. The developed method is expected to be applied for accurate routine analysis of infant formulas.     

Mass spectrometric analysis of the glycosphingolipid‐derived glycans from miniature pig endothelial cells and islets: identification of NeuGc epitope in pig islets

Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell‐to‐cell or cell‐to‐protein interactions. In this study, qualitative and quantitative analyses of GSL‐derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL‐derived glycans from the endothelial cells and islets, respectively, were characterized by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS) and collision‐induced fragmentation using positive‐ion electrospray ionization (ESI) ion‐trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α‐Gal‐terminated GSL was not detected but NeuGc‐terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL‐derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI‐TOF MS. The structural information of the GSL‐derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Identification of N-linked glycosylation sites in human nephrin using mass spectrometry

Nephrin is a type-1 transmembrane glycoprotein and the first identified principal component of the glomerular filtration barrier. Ten potential asparagine (N)-linked glycosylation sites have been predicted within the ectodomain of nephrin. However, it is not known which of these potential sites are indeed glycosylated and what type of glycans are involved. In this work, we have identified the terminal sugar residues on the ectodomain of human nephrin and utilized a straightforward and reliable mass spectrometry-based approach to selectively identify which of the ten predicted sites are glycosylated. Purified recombinant nephrin was subjected to peptide-N-glycosidase F (PNGase F) to enzymatically remove all the N-linked glycans. Since PNGase F is an amidase, the asparagine residues from which the glycans have been removed are deaminated to aspartic acid residues, resulting in an increase in the peptide mass with 1 mass unit. Following trypsin digestion, deglycosylated tryptic peptides were selectively identified by MALDI-TOF MS and their sequence was confirmed by tandem TOF/TOF. The 1 Da increase in peptide mass for each asparagine-to-aspartic acid conversion, along with preferential cleavage of the amide bond carboxyl-terminal to aspartic acid residues in peptides where the charge is immobilized by an arginine residue, was used as a diagnostic signature to identify the glycosylated peptides. Thus, nine of ten potential glycosylation sites in nephrin were experimentally proven to be modified by N-linked glycosylation.     

An HPLC method for the determination of selected amino acids in human embryo culture medium

A method for the determination of selected amino acids in culture medium using HPLC with fluorescence detection is described. Twenty hours after intra‐cytoplasmic sperm injection, one randomly selected zygote was transferred to the culture medium. After incubation (72 h after fertilization), the culture medium in which the embryo was incubated and blank medium was immediately stored at −80°C. Filtered medium samples were derivatized with ortho ‐phthalaldehyde (naphthalene‐2,3‐dicarboxaldehyde), forming highly fluorescent amino acids derivatives. Reverse‐phase columns (LichroCART, Purospher STAR RP_18e or Ascentis Express C₁₈) were used for the separation. The derivatives were analyzed by gradient elution with a mobile phase containing ethanol and sodium dihydrogen phosphate. The analytical performance of this method is satisfactory for all amino acids; the intra‐assay coefficients of variation were <10% and quantitative recoveries were between 95.5 and 104.4%. Changes in the levels of selected amino acids before and after human embryo cultivation were observed. After embryo incubation, the levels of all amino acids in the medium were increased, apart from aspartate and asparagine. After the cultivation of some embryos, amino acids which were not part of the medium were detected. Low amino acids turnover was observed in some embryos.     

Determination of mono- and dichloroacetic acids in betaine media by liquid chromatography

A simple and sensitive method has been developed for the analysis of residue amounts of chloroacetic acids in betaine samples based on derivatization by 1-naphthylamine (NA). The derivatized compounds are analyzed by reverse phase high performance liquid chromatography using methanol and water as mobile phase in the ratio of 32/68 (v/v) and phenyl column and PDA detection at 222 nm. The detection limits (LOD) of monochloroacetic acid (MCA) and dichloroacetic acid (DCA) are 0.1 and 0.15 μg mL⁻¹, respectively. The limits of quantification (LOQ) and the linear dynamic ranges (LDR) of MCA are found to be 1 and 1-400 μg mL⁻¹, respectively, and for DCA are found to be 3 and 3-400 μg mL⁻¹, respectively. The precision at the 5 ppm level for MCA and DCA are about 3% and 2%, (n = 5), respectively. The average recovery for MCA and DCA spiked to betaine samples are 98% and 97%, respectively.     

Determination of tricarboxylic acid cycle acids and other related substances in cultured mammalian cells by gradient ion-exchange chromatography with suppressed conductivity detection

An ion-exchange chromatography method was established for simultaneously analyzing the tricarboxylic acid (TCA) cycle acids and other related substances in cultured mammalian cells, including citrate, cis-aconitate, isocitrate, alpha-ketoglutarate, succinate, malate, fumarate, oxaloacetate, trans-aconitate, phosphate, lactate and pyruvate. A Dionex 600 ion chromatograph with an ion suppressor and a conductivity detector, and an IonPac AS11-HC analytical column were employed. An NaOH gradient elution containing 13.5% methanol contributed to sufficient separation of target substances. The stability of carboxylic acids was investigated and oxaloacetate was found to be extremely unstable especially at pH 3. TCA cycle acids and other related substances in Chinese hamster ovary (CHO) cells were separated completely, and lactate, malate, phosphate, citrate and cis-aconitate were quantified due to their higher concentrations. In the quantification of the five substances, detection limits (S/N=3) ranged from 0.12 to 0.48 microM, the correlation coefficients from 0.9982 to 1.0000 in their linear ranges of concentration, and the recoveries from 87 to 95%. The metabolic status of CHO cells was analyzed on the basis of the intracellular concentrations of TCA cycle acids.     

Trace enrichment of phenylcarboxylic acids from a model biological fluid and serum of human blood

The study was focused on the development of a solid‐phase extraction protocol for seven phenylcarboxylic acids from albumin solutions by using unmodified hyper‐cross‐linked polystyrene restricted access materials with crosslinking degrees varying from 100 to 400% (four of the acids are known to be markers of sepsis). The breakthrough volume of the most hydrophilic 3,4‐dihydroxybenzoic acid rises as the sorbent bridging extent grows. Inversely, the breakthrough volume of the most hydrophobic 3‐phenylpropionic acid was found to decrease considerably when the degree of crosslinking exceeds 200%. This unusual pattern is because of the superposition of two opposite tendencies. Increasing substitution extent of phenyls facilitates their π‐π‐interactions with polar compounds whereas rising density of the network reduces the accessibility of sorption sites to all solutes. Mini‐cartridges containing 30 mg of an optimal sorbent take up the acids completely and reversibly, the recoveries being close to 100% even in the presence of high concentrations of albumin. By coupling the developed solid‐phase extraction with high‐performance liquid chromatography and diode array detection technique, we managed to determine quantitatively phenylcarboxylic acids in the serum of a healthy patient blood, and the recoveries varied from 93 to 100% while the limit of quantification was (4–9) × 10^?7 M.     

Simultaneous determination of amino acids and carbohydrates in culture media of Clostridium thermocellum by valve-switching ion chromatography

An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5–4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum.     

Determination of lignans,phenolic acids and antioxidant capacity in transformed hairy root culture of Linum usitatissimum

Hairy root culture is a promising alternative method for the production of secondary metabolites. In this study, transformed root of Linum usitatissimum was established using Agrobacterium rhizogenes A4 strain from root cultures for lignans, phenolic acids and antioxidant capacity determination. Total lignin content (secoisolariciresinol diglucoside, secoisolariciresinol and matairesinol) was 55.5% higher in transformed root cultures than in the non-transformed root culture. Secoisolariciresinol was detected in higher concentration (2.107 μmol/g DM) in the transformed root culture than non-transformed culture (1.099 μmol/g DM). Secoisolariciresinol diglucoside and matairesinol were exclusively detected in the transformed root culture, but were not found in the non-transformed root culture. The overall production of phenolic acids in transformed roots was approximately 3.5 times higher than that of the corresponding non-transformed culture. Free radical scavenging DPPH˙ and ABTS˙⁺ assays showed 2.9-fold and 1.76-fold higher anti-oxidant activity in transformed root culture as compared to non-transformed.     

NMR analysis of novel ganglioside GM4 analogues containing de‐N‐acetyl and lactamized sialic acid: probes for searching new ligand structures for human L‐selectin

Detailed analysis of the ¹H and ¹³C NMR spectra of two novel ganglioside GM4 analogues de‐N‐acetyl sialyl GM4 ( 1 ) and cyclic sialyl GM4 ( 2 ), which contain de‐N‐acetyl and lactamized sialic acid, respectively, instead of the usual N‐acetylneuraminic acid, was carried out. The combination of NMR data, such as cyclization shifts, coupling pattern, intraresidual NOEs and the appearance of NH proton, provided the ^{5, 2}B conformation for 2 . Moreover, the conformation of a glycosidic bond connecting the Neu and Gal residues was determined by some interresidual NOEs in both 1 and 2 . Copyright © 2002 John Wiley & Sons, Ltd.     

HPLC method for comparative study on tissue distribution in rat after oral administration of salvianolic acid B and phenolic acids from Salvia miltiorrhiza

A sensitive and selective high-performance liquid chromatography method was developed and validated to determine the prototype of salvianolic acid B and the metabolites of phenolic acids (protocatechuic acid, vanillic acid and ferulic acid) in rat tissues after oral administration of total phenolic acids and salvianolic acid B extracted from the roots of Salvia miltiorrhiza, respectively. The tissue samples were treated with a simple liquid-liquid extraction prior to HPLC. Analysis of the extract was performed on a reverse-phase C(18) column with a mobile phase consisting of acetonitrile and 0.05% trifluoracetic acid. The calibration curves for the four phenolic acids were linear in the given concentration ranges. The intra-day and inter-day relative standard deviations in the measurement of quality control samples were less than 10% and the accuracies were in the range of 88-115%. The average recoveries of all the tissues ranged from 78.0 to 111.8%. This method was successfully applied to evaluate the distribution of the four phenolic acids in rat tissues after oral administration of total phenolic acids of Salvia miltiorrhiza or salvianolic acid B and the possible metabolic pathway was illustrated.     

Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine-conjugated bile acids in human serum

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H]+. The collision-induced dissociation of the molecular ion produced fragment ions at [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+. The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N-CO, O-CO, and C-OCO, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M+H]+ --> [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+, 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was <15% at broad linear dynamic ranges (0.0244-25 nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.     

Determination of kynurenic acid levels in rat brain microdialysis samples and changes in kynurenic acid levels induced by N‐acetylaspartic acid

The levels of kynurenic acid, an endogenous antagonist of α₇ nicotinic acetylcholine and N‐methyl‐D ‐aspartate receptors, were measured in microdialysis samples obtained from the prefrontal cortices of rats using column‐switching high‐performance liquid chromatography with fluorescence detection. When the perfusate was constantly infused at a rate of 1.0 μ/min, the in vitro recovery of kynurenic acid through the dialysis membrane was approximately 20.4%, and the precision was within 1.31%. Endogenous kynurenic acid in the microdialysis sample was clearly detected using column‐switching high‐performance liquid chromatography. As an application study, N‐acetyl‐L ‐aspartic acid, an endogenous metabolite and precursor of N‐acetyl‐L ‐aspartyl‐L ‐glutamic acid, which is an agonist of metabotropic glutamate receptors, was infused for 120 min through the microdialysis probe. The kynurenic acid level significantly increased during the infusion of N‐acetyl‐L ‐aspartic acid, suggesting that kynurenic acid might have some association with N‐acetyl‐L ‐aspartic acid in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of low-molecular-weight organic acids and chlorinated acid herbicides in environmental water by a portable CE system with contactless conductivity detection

This report describes a method to simultaneously determine 11 low-molecular-weight (LMW) organic acids and 16 chlorinated acid herbicides within a single run by a portable CE system with contactless conductivity detection (CCD) in a poly(vinyl alcohol) (PVA)-coated capillary. Under the optimized condition, the LODs of CE-CCD ranged from 0.056 to 0.270 ppm, which were better than for indirect UV (IUV) detection of the 11 LMW organic acids or UV detection of the 16 chlorinated acid herbicides. Combined with an on-line field-amplified sample stacking (FASS) procedure, sensitivity enhancement of 632- to 1078-fold was achieved, with satisfactory reproducibility (RSDs of migration times less than 2.2%, and RSDs of peak areas less than 5.1%). The FASS-CE-CCD method was successfully applied to determine the two groups of acidic pollutants in two kinds of environmental water samples. The portable CE-CCD system shows advantages such as simplicity, cost effectiveness, and miniaturization. Therefore, the method presented in this report has great potential for onsite analysis of various pollutants at the trace level.     

Spectrophotometric Determination of Esomeprazole Magnesium in Commercial Tablets Using 5‐Sulfosalicylic Acid and N‐Bromosuccinimide

Two simple, sensitive and economical spectrophotometric methods have been developed for the determination of esomeprazole magnesium in commercial dosage forms. Method A is based on the reaction of esomeprazole magnesium with 5‐sulfosalicylic acid in methanol to form a yellow product, which absorbs maximally at 365 nm. Method B utilizes the reaction of esomeprazole magnesium with N‐bromosuccinimide in acetone‐chloroform medium to form α‐bromo derivative of the drug peaking at 380 nm. Under the optimized experimental conditions, Beer's law is obeyed in the concentration ranges of 2‐48 and 10‐100 μg mL^?1 with molar absorptivity of 2.11 × 10⁴ and 4.57 × 10⁴L mol^?1 cm^?1 for methods A and B, respectively. The limits of detection for methods A and B are 0.35 and 0.46 μg mL^?1, respectively. No interference was observed from excipients commonly present in tablet formulations. Methods A and B are successfully applied to the commercial tablets for the estimation of esomeprazole magnesium with good accuracy and precision. The results compare favorably with the reference spectrophotometric method indicating no significant difference between the methods compared.     

Analysis of amino acids in individual human erythrocytes by capillary electrophoresis with electroporation for intracellular derivatization and laser-induced fluorescence detection

A method for monitoring amino acids in single erythrocytes is described. For intracellular derivatization, reagent fluorescein isothiocyanate (FITC) was introduced into living cells by electroporation. For an 8 microm erythrocyte, the analytes were diluted by a factor of only 1.6. After completion of the derivatization reaction, a single cell was injected into the separation capillary tip and lysed there. The derivatized amino acids were separated by capillary electrophoresis, followed by laser-induced fluorescence detection. Nine amino acids were quantitatively determined, with amounts of amino acids ranging from 3.8-32 amol/single cell.     

Determination of genomic N3‐methylthymidine in human cancer cells treated with nitrosamines using capillary electrophoresis with laser‐induced fluorescence

Methylating substances alter DNA by forming N3‐methylthymidine (N3mT), a mutagenic base modification. To develop a sensitive analytical method for the detection of N3mT in DNA based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF), we synthesized the N3mT‐3’‐phosphate as a chemical standard. The limit of detection was 1.9 amol of N3mT, which corresponds to one molecule of N3mT per 1000 normal nucleotides or 0.1%. With this method, we demonstrated that the carcinogenic nitrosamine N’‐nitrosonornicotine (NNN) induced N3mT in the human lung cancer cell line A549. Treatment with NNN also caused an elevated degree of 5‐hydroxymethylcytidine (5hmdC) in DNA, while the methylation degree (i.e. 5‐methylcytidine; 5mdC) stayed constant. According to our data, NNN could, via yet unknown mechanisms, play a role in the formation of N3mT as well as 5hmdC. In this study we have developed a new sensitive analytical method using CE‐LIF for the simultaneous detection of the three DNA modifications, 5mdC, 5hmdC and N3mT.     

Determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid by derivatization followed by high‐performance liquid chromatography with ultraviolet detection

Methanesulfonic acid is routinely used in pharmaceuticals but can contain potentially genotoxic impurities such as methyl methanesulfonate and ethyl methanesulfonate. The aim of this study was to develop a simple high‐performance liquid chromatography with ultraviolet detection method for determining methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid. Samples (250 mg) in water/acetonitrile (200 μL) were first combined with 10.0 mol/L sodium hydroxide solution (270 μL). Then they were mixed with 2.0 mg/mL N ,N‐diethyldithiocarbamate (500 μL), diluted to 5 mL with N ,N‐dimethylacetamide and allowed to react at 80°C for 1 h. The derivatives were analyzed using gradient high‐performance liquid chromatography with ultraviolet detection (277 nm) and structurally elucidated by liquid chromatography with mass spectrometry. With acetonitrile/5 mmol/L ammonium acetate solution as the eluent and 1 mL/min as the flow rate on a C18 column, the derivatives were eluted at 10.6 and 14.8 min. Good linearity (correlation coefficients > 0.999) and low limits of quantitation (0.6 ppm) were obtained. The recoveries were in the range of 80–115% with relative standard deviation < 5.0%. Finally, the established method was successfully used for the determination of methyl methanesulfonate and ethyl methanesulfonate in methanesulfonic acid.     

On-capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser-induced fluorescence detection: application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.