1,721,054 research outputs found
Microdetermination of chondroitin sulfate in normal and PXE-affected human plasma by HPLC and fluorophore-assisted carbohydrate electrophoresis (FACE)
No full textMicrodetermination of chondroitin sulfate in normal and PXE-affected human plasma by HPLC and fluorophore-assisted carbohydrate electrophoresis (FACE
Serum IgG responses to food antigens in the Italian population evaluated by highly sensitive and specific ELISA test
No full textUsing an optimized and validated ELISA method, we performed serum test for assaying the binding capacity of serum IgG to proteins extracted from approx. 160 different foods to investigate the reactivity of specific IgG antibodies in the Italian population composed of 6879 subjects (4551 females and 2328 males). 44 antigens showed an IgG response greater than 10% and only 14 aliments had an elevated reactivity greater than 20%, in particular, milk, from cow and goat, and several milk derivatives, along with egg albumen and yeasts. The IgG response to the high reactive food antigens depending on the age of the 6880 subjects was also analyzed. We demonstrated a high IgG response in a very large subject group to milk and milk derivatives, and egg albumen antigens, and we conclude that the validated ELISA test may be applied for the serum/plasma IgG antibody level determination as a useful indicator of adverse reactions to food and food hypersensitivity
Detection of submicrogram quantities of glycosaminoglycans on agarose gels by sequential staining with toluidine blue and Stains-All
No full textA sensitive method has been developed for the visualization of nonradiolabelled glycosaminoglycans resolved by agarose gel electrophoresis using staining with toluidine blue followed by Stains-All procedure. This method, which can detect as little as 10 ng of a single species, can be used to stain a few micrograms of a complex polysaccharide mixture. The combination of agarose gel electrophoresis and sequential toluidine blue/Stains-All staining can be applied to the analysis of all the complex glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate) and nonsulfated polyanions (i.e., hyaluronate, defructosylated capsular polysaccharide K4) as well as to comparisons of specificities of the glycosaminoglycan-degrading enzymes and the identification and quantification of the contaminations of other polysaccharides within glycosaminoglycan preparations with great sensitivity (about 0.1 %). Furthermore, this method can be used to stain low-molecular-mass fractions and oligosaccharides derived from the natural polyanions, such as heparin. This procedure may be particularly valuable in situations where the availability of glycosaminoglycan is very limited
Composition of urinary glycosaminoglycans in a patient with pseudoxanthoma elasticum and familial Mediterranean fever
No full textPseudoxanthoma elasticum (PXE) is a genetic disorder whose gene (ABCC6) encodes a transmembrane transporter called ABCC6/MRP6 [1], and characterized by a connective tissue disorder with accumulation of ion precipitates within the elastic fibers of skin, eyes and the whole cardiovascular system, and by collagen fibril abnormalities and accumulation in the extracellular space of abnormal masses of materials containing proteoglycans and a series of other matrix molecules. Familial Mediterranean fever (FMF) is an autosomal recessive disease characterised by fever and polyserositis [2]. The disease is caused by a defect in the gene encoding pyrin that is effective in the inflammatory response of neutrophils and monocytes. The most important complication of FMF is the development of secondary amyloidosis. Changes on the composition and structure of urinary glycosaminoglycans (GAGs) have been suggested as clinical markers in various diseases, including different types of cancer [3] and PXE [4]. We report a case of a French patient affected by PXE and FMF with amyloidosis in which the composition of urinary GAGs was quantitatively and qualitatively evaluated
Purification and characterization of hyaluronic acid from the mollusc bivalve Mytilus galloprovincialis
No full textHyaluronan (hyaluronic acid, HA) was for the first time extracted, purified and characterized from the species of mollusc bivalve Mytilus galloprovincialis. HA was characterized by agarose-gel electrophoresis, C-13-NMR, HPLC and normal polarity capillary electrophoresis by evaluating the unsaturated disaccharide, DeltaDiHA (Delta-hexuronic acid-N-acetyl-glucosamine) after treatment with chondroitin ABC lyase, and by separating A-tetrasaccharide and A-hexasaccharide generated by the specific action of hyaluronate lyase from Streptomyces hyalurolyticus. The weight average molecular weight (M-w) was found to be about 200 kDa as determined by HPSEC. HA from M. galloprovincialis was not able to interact with aggrecan from bovine cartilage to form high molecular mass aggregate and also had a very low specific viscosity, but it showed the same capacity to inhibit cell proliferation (50 mug per 10(3) human fibroblasts inhibit cell proliferation by about 50%) than high molecular mass HA. HA of M. galloprovincialis could have a physiological role in the regulation of cell functions. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved
Capillary electrophoresis of Biomolecules. Methods and Protocols.
No full textCapillary electrophoresis (CE) is a relatively new separation technique suitable for handling small amounts of sample very important in bioanalytical research and in various clinical, diagnostic, genetic, and forensic applications. CE offers several similarities to HPLC (high performance liquid chromatography) such as ease of use, high resolution, speed, on-line detection, and full automation capability. CE encompasses a family of related separation techniques that use narrow-bore fused-silica capillaries to separate a complex array of large and small molecules. High electric field strengths are used to separate molecules with differences in charge, size and hydrophobic properties. CE may be utilized according to several separation techniques:
1. Capillary Zone Electrophoresis (CZE) is the simplest form of CE where the separation mechanism is based on differences in the charge-to-mass ratio of the analytes.
2. Capillary Gel Electrophoresis (CGE) is the adaptation of traditional gel electrophoresis into the capillary by using soluble polymers to create a replaceable molecular sieve allowing size separations.
3. Capillary Isoelectric Focusing (CIEF) allows amphoteric molecules, proteins, to be separated in a pH gradient generated between the cathode and anode.
4. Isotachophoresis (ITP) is a focusing technique based on the migration of compounds between leading and terminating electrolytes.
5. Micellar Electrokinetic Capillary Chromatography (MECC or MEKC) is a mode of separation in which surfactants are added to the buffer solution at concentrations that form micelles. This technique is useful to resolve both charged and neutral compounds.
6. Micro Emulsion Electrokinetic Chromatography (MEEKC) is a technique in which solute partition takes place between moving oil droplets and the aqueous buffer. This allows the separation of both aqueous and water-insoluble compounds
7. Non-Aqueous Capillary Electrophoresis (NACE) involves the separation of analytes in non aqueous media that allow additional selectivity options in methods development. It is valuable for separations of water-insoluble compounds and for hyphenation with MS detection.
8. Capillary Electrochromatography (CEC) is a hybrid separation method that couples the high separation efficiency of CZE with HPLC and uses an electric field rather than hydraulic pressure to propel the mobile phase through a packed bed.
Due to its high resolving power and sensitivity, CE has been applied in the analysis of simple and complex (macro)molecules providing concentration and structural characterization data essential for understanding their biological functions. Although CE technology may be applied to many different types of research, it has gained its reputation from the study of molecules that have traditionally been difficult to separate. In general, CE should be considered first when dealing with highly polar, charged analytes. In fact, CE excels in the analysis of ions when rapid results are desired, and has become the predominant technique for the analysis of both basic and chiral pharmaceuticals. This technology is replacing traditional electrophoresis for the characterization and analysis of macromolecules such as nucleic acids, proteins, carbohydrates, and promises to be a valuable tool in tackling the characterization challenges posed by proteome-wide analysis and DNA sequencing and genotyping.
This Volume on the capillary electrophoresis of Biomolecules provides the reader with the latest break-throughs and improvements in CE and CE techniques applied to several classes of bio(macro)molecules, in particular simple and complex carbohydrates (polysaccharides), aminoacids, peptides and proteins, enzymes, and nucleic acids. Along with practical procedures, reviews discussing CE applications related to bio(macro)molecules are also included.
As the editor of this Volume, I would like to thank all the contributors for their articles, able to provide a better understanding of the analytical phenomena related to CE and by widening the scope of their possible applications. Acknowledgement is due to humana Press Editors for their assistance in bringing this issue to publication
Two analytical approaches to the evaluation of chondroitin sulfate in european food supplements.
No full textThe amount and quality of CS from several Czech Republic food supplement/nutraceutical preparations was determined. In order to quantify CS, two different analytical approaches were applied after their validation [see Volpi, N. & Maccari, F. (2008) Quantitative and qualitative evaluation of chondroitin sulfate in dietary supplements. Food Anal. Chem., 1, 195-204], specific and sensitive agarose-gel electrophoresis and SAX-HPLC determination of the constituent disaccharides after treatment with specific chondroitin lyases.The CS content in food supplement products were found to conform to the label specifications only in four of the ten analyzed samples. Four of the food supplement preparations were found to contain approx. 0-1% CS in comparison with 47, 17, 12 and 6% declared on the label. Two products were found to have approx. 30-45% of the declared CS, and one preparation was found to contain approx. 2% hyaluronic acid. SAX-HPLC separation of unsaturated disaccharides for the nutraceutical CS was also used to evaluate its quality and the possible origin. The CS contained in eight food supplements resulted to be of bovine or porcine origin, one from cartilagineous fishes and in one case it was not possible to determine the origin due to a very low CS content.On the basis of these analytical results, the quality of these ten Czech Republic food supplement formulations is poor and strict regulations for quality control should be mandatory in order to guarantee the manufacture of high quality products. Furthermore, specific and accurate analytical procedures should be enforced for the control of high quality products and applied by quality control laboratories to confirm the purity and label claim of CS in raw materials and nutraceuticals
Structural characterization of the skin glycosaminoglycans in patients with pseudoxanthoma elasticum
No full textBackground. Complex polysaccharides, glycosaminoglycans (GAGs), their amount and fine structure were determined in the skin (epidermis + dermis) of pseudoxanthoma elasticum (PXE)-affected patients in comparison with healthy subjects. Methods. Non-lesional skin GAGs were extracted and specifically determined by enzymatic treatment and HPLC separation.Results. Dermatan sulfate (DS) and hyaluronic acid (HA) were found the major GAG species with a DS percentage of approx. 20, a HA content of 58% and a chondroitin sulfate (CS) unsaturated 6-sulfated disaccharide amount of about 21%. Skin from PXE patients showed a similar HA percentage (61%), a corresponding DS content (22%) and no modification of the CS 6-sulfated disaccharide (16.7%). No changes of the total charge density and non-sulfated/sulfated GAGs ratio was noted along with no modification of the position of the sulfate groups (4s/6s) on the CS/DS backbone for PXE-affected subjects. However, a significant increase by about 88% (p<0.01) of the total amount of GAGs (HA+DS+CS) was found in PXE groups versus normal subjects. Conclusions. The altered metabolic processes produce in the skin of PXE-affected patients an increase in the total GAGs able to accumulate salts, in particular calcium ions, within the elastic fibers and to produce ion precipitates affecting the organization of matrix fibre
Characterization of a low-sulfated chondroitin sulfate isolated from the hemolymph of the freshwater snail Planorbarius corneus
No full textGlycosaminoglycans (GAGs) from the hemolymph of the freshwater snail Planorbarius corneus were recovered at about 0.9 μg/mL, being composed of a unique species characterized as chondroitin sulfate (CS) with a molecular mass of approx. 31,000 and having glucuronic acid as hexuronic acid. This macromolecule was determined to be composed of a low-sulfated polysaccharide made up of approx. 25% of the nonsulfated disaccharide, 17% of the 6-sulfated disaccharide, and about 58% of the 4-sulfated disaccharide, with a charge density value of 0.75 and a 4-sulfated/6-sulfated ratio of approx. 3.4. The data obtained suggest that the CS recovered in the Planorbarius corneus hemolymph is similar to the main human plasma polysaccharide and it may be generated as a main product of the catabolic processes
Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation
No full textWe describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGS) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 mug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGS was about 0.1-0.5 mug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGS and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization
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