Characterization and Purification of Glycosaminoglycans from Crude Biological Samples (original) (raw)
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Analytical biochemistry, 2014
We developed a method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would he...
A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides
Glycobiology, 2000
A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology, 4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to ∼0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di-and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sβ(1-3)GalNAc6S structure.
Glycobiology, 2006
Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d 4 (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.
Glycosaminoglycans and proteoglycans of normal and tumoral cartilages of humans and rats
Cancer research, 1979
Differences in the glycosaminoglycans and proteoglycans synthesized by "young," "adult," and tumoral chondrocytes are reported. Young cartilage and human chondrosarcoma contain chondroitin 4- and 6-sulfates, whereas adult human cartilage contains almost exclusively chondroitin 6-sulfate. High keratan sulfate content is reported in adult cartilage, whereas it is almost absent in young and tumoral cartilages. The electrophoretic pattern and keratan sulfate content in these proteoglycans from adult cartilage are clearly distinct from those of the young and tumoral cartilages. The high molecular weight is the distinguishing property of the glycosaminoglycan synthesized by tumoral chondrocytes.
Marine drugs, 2018
Chondroitin sulfate (CS) is a glycosaminoglycan actively researched for pharmaceutical, nutraceutical and tissue engineering applications. CS extracted from marine animals displays different features from common terrestrial sources, resulting in distinct properties, such as anti-viral and anti-metastatic. Therefore, exploration of undescribed marine species holds potential to expand the possibilities of currently-known CS. Accordingly, we have studied for the first time the production and characterization of CS from blackmouth catshark (), a shark species commonly discarded as by-catch. The process of CS purification consists of cartilage hydrolysis with alcalase, followed by two different chemical treatments and ending with membrane purification. All steps were optimized by response surface methodology. According to this, the best conditions for cartilage proteolysis were established at 52.9 °C and = 7.31. Subsequent purification by either alkaline treatment or hydroalcoholic alkal...
Journal of Biological Chemistry, 1997
Samples of aggrecan chondroitin sulfate, isolated from normal human knee cartilages of individuals from fetal to 72 years of age, were digested with chondroitin lyases. The products were analyzed by fluorescencebased anion exchange high performance liquid chromatography to separate and quantitate nonreducing terminal structures, in addition to internal unsaturated disaccharide products. The predominant terminal structures were the monosaccharides, GalNAc4S and GalNAc4,6S as they were present on 85-90% of all chains. The remaining chains terminated with the disaccharides GlcA1,3GalNAc4S and GlcA1,3GalNAc6S. Marked changes in the relative abundance of these terminals were identified in the transition from growth cartilage to adult articular cartilage. First, terminal GalNAc residues were almost exclusively 4-sulfated in aggrecan from fetal through 15 years of age, but were ϳ50% 4,6disulfated in aggrecans from adults (22-72 years of age). Second, the terminal disaccharide GlcA1,3GalNAc4S was on ϳ7% of chains on aggrecan from fetal through 15 years of age, but on only ϳ3% of chains on adult aggrecan. In contrast, the proportion of chains terminating in GlcA1,3GalNAc6S, ϳ9%, was unchanged from fetal to 72 years of age. This terminal disaccharide is proposed to be recognized by the widely used monoclonal antibody 3B3. However, chemical quantitation of the structure together with solid phase 3B3(؊) immunoassay of fetal and adult aggrecans showed that the content of the terminal disaccharide does not necessarily correlate with immunoreactivity of the proteoglycan, as chain density and presentation on the solid phase are critical factors for recognition of chain terminals by 3B3. The quantitative results obtained from chemical analyses of all nonreducing termini of aggrecan chondroitin sulfate chains revealed important changes in chain termination that occur when cellular activities are altered as adult articular cartilage is formed after removal of growth cartilage. These findings are discussed in relation to specific enzymatic steps that generate the nonreducing termini of chains in the biosynthesis pathway of chondroitin sulfate proteoglycans and their modulation in tissue development and pathology.
Histochemistry of glycosaminoglycans in cartilage ground substance
Histochemistry, 1986
The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A [, Arachis hypogaea, Ulex europaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding sites (positive staining for all leetins tested except H. pomatia) observed corresponded to areas positive for keratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.
Chicken cartilage (claw) is a waste of chicken cuts which are widely available in Indonesia. Cartilage part of chicken claw becomes a potential source of chondroitin sulfate (CS) and glucosamine (GS). This aim of this study was to determine the most optimal extraction methods of CS and GS from cartilage of chicken claw. Various types of extraction methods used in this study were taken from the extraction by using boiling water (2 and 2.5 hours), acetic acid (7 and 17 hours), as well as proteolysis by papain (24 and 48 hours). Parameters observed include chemical characteristics of powdered cartilage of chicken claw as well as CS and GS levels in powdered cartilage of chicken claw extract. The results showed that the levels of CS and GS of chicken claw cartilage powder were 2.17% and 13%, respectively. The highest GS level was obtained from the extraction with water treatment for 2.5 hours (8.1%). The treatment and duration of extraction will significantly affect the number of GS which was produced. The highest content of CS was obtained from the extraction with the enzyme treatment for 48 hours (2.47%). The best treatment is the extraction with water treatment for 2.5 hours which were the extracts with GS levels of 8.1% and 2.03% CS was selected through the analysis of multiple attribute.
Biochimica et Biophysica Acta (BBA) - General Subjects, 1978
The rate of metabolism of low-sulfated chondroitin 4-sulfate, a predominant glycosaminoglycan in blood, has been studied by administering intraperitoneally radioactive hexosamine and/or sulfate to rats. The biological half-life of the material was estimated to be 10-12 h, suggesting that the metabolic process of blood low-sulfated chondroitin sulfate is different from that of glycosaminoglycans in the tissue. Low-sulfated chondroitin sulfate (LSC), mainly in a form of proteoglycan, was found to be predominant in human serum and plasma, regardless of ages of subjects [1-3]. Blood from patients with Hurler's syndrome showed also LSC as a main acidic glycosaminoglycan, though the patients excreted a large amount of dermatan sulfate and heparan sulfate in urine [3]. Ubiquitous presence of LSC in the blood as the major acidic glycosaminoglycan suggested that metabolic process of LSC might be different from that of acidic glycosaminoglycans in tissues. Several investigators reported the metabolism of chondroitin sulfate and/or proteochondroitin sulfate after injecting the labeled materials into animals [4-7] ; however, no paper is available on the analysis of the metabolic rate of acidic glycosaminoglycans or proteoglycans produced by the animal itself which reflects the true physiological fate of the materials. The microanalytical method of acidic glycosaminoglycans, developed in our laboratory [8-10], enabled us to perform this direct analysis of acidic glycosaminoglycan metabolism. This paper describes incorporation of radioactive hexosamine and/or sulfate Abbreviations: LSC, low sulfated chondroitin sulfate; ADIOS, 2-acetamide-2-deoxy-3-O-03-D-gluco-4enepyranosyluxonic acid)-D-galactose; ADi4S, 2-acetarnide-2-deoxy-3-O-Q3-D-gluco-4-enepyranosyluxonic acid)-4-O-sulfo-D-galactose; ADi6S, 2-acetamide-2-deoxy-3-O-(~-D-gluco-4-enepyranosyltLronic acid)-6-Osulfo-D-galactose.