Characterization Of Glycosaminoglycans Research Articles

Simultaneous determination of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan glycosaminoglycan disaccharides by high-performance liquid chromatography using a reverse-phase column with adamantyl groups

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.

Investigation of the Experimental Parameters of Ultraviolet Photodissociation for the Structural Characterization of Chondroitin Sulfate Glycosaminoglycan Isomers.

Glycosaminoglycans (GAGs) are linear polysaccharides that participate in a broad range of biological functions. Their incomplete biosynthesis pathway leads to nonuniform chains and complex mixtures. For this reason, the characterization of GAGs has been a difficult hurdle for the analytical community. Recently, ultraviolet photodissociation (UVPD) has emerged as a useful tool for determining sites of modification within a GAG chain. Here, we investigate the ability for UVPD to distinguish chondroitin sulfate epimers and the effects of UVPD experimental parameters on fragmentation efficiency. Chondroitin sulfate A (CS-A) and chondroitin sulfate B (CS-B), commonly referred to as dermatan sulfate (DS), differ only in C-5 uronic acid stereochemistry. This uronic acid difference can influence GAG-protein binding and therefore can alter the specific biological function of a GAG chain. Prior tandem mass spectrometry methods investigated for the elucidation of GAG structures also have difficulty differentiating 4-O from 6-O sulfation in chondroitin sulfate GAGs. Preliminary data using UVPD to characterize GAGs showed a promising ability to characterize 4-O sulfation in CS-A GAGs. Here, we look in depth at the capability of UVPD to distinguish chondroitin sulfate C-5 diastereomers and the role of key experimental parameters in making this distinction. Results using a 193 nm excimer laser and a 213 nm solid-state laser are compared for this study. The effect of precursor ionization state, the number of laser pulses (193 or 213 nm UVPD), and the use of the low-pressure versus high-pressure trap are investigated.

Characterization of Glycosaminoglycans in Gaping and Intact Connective Tissues of Farmed Atlantic Salmon (Salmo salar) Fillets by Mass Spectrometry.

In the Atlantic salmon (Salmo salar) aquaculture industry, gaping (the separation of muscle bundles from the connective tissue) is a major quality problem. This study characterized chondroitin sulfate (CS) and heparan sulfate (HS) in the connective tissue of intact and gaping salmon fillets from 30 salmon by mass spectrometry. Statistical difference was detected between gaping and intact tissues only when comparing pairwise samples from the same individual (n = 10). The gaping tissue had a lower content of monosulfated CS disaccharides (p = 0.027), and the relative distribution of CS disaccharides was significantly different (p < 0.05). The HS chains were short (average = 14.09, SD = 4.91), and the intact tissue seemed to have a more uniform HS chain structure compared to the gaping tissue. Time-series samples from the same individuals are recommended for future research to improve the understanding of reasons and implications of these differences.

Recent advances in glycosaminoglycan analysis by various mass spectrometry techniques.

This review summarizes progress in analysis of glycosaminoglycans using mass spectrometry (MS) approaches. The specific areas covered include analytical challenges, disaccharide analysis, top-down and bottom-up techniques, sequence analysis, and future perspectives. A brief outline of the complexity and heterogeneity of these unique saccharides and their analysis is provided along with examples of several recent studies. Unique problems and challenges in the characterization of glycosaminoglycans are discussed along with many of the analytical tools used in particular MS methods and the types of information provided. Advances in MS-related technologies have provided more sensitive and accurate detection and sequence analysis of this complex and chemically unique class of bioconjugates. Effective MS-based methods and automated data handling with bioinformatics tools have been developed for disaccharide analysis, top-down and bottom-up analysis, and sequencing studies of relatively short oligosaccharides. It is envisioned that further improvements in MS technologies along with bioinformatics methods will make sequencing studies of longer glycosaminoglycan chains easier and faster.

One-pot analysis of sulfated glycosaminoglycans.

Routine isolation, estimation, and characterization of glycosaminoglycans (GAGs) is quite challenging. This is compounded by the fact that the analysis is technique-intensive and more often there will be a limitation on the quantity of GAGs available for various structural, functional and biological studies. In such a scenario, the sample which can be made available for estimation and elucidation of disaccharide composition and species composition as well remains a challenge. In the present study, we have determined the feasibility where isolated sulfated GAGs (sGAG) that is estimated by metachromasia is recovered for further analysis. sGAG-DMMB complex formed after estimation of sGAG by DMMB dye-binding assay was decomplexed and sGAGs were recovered. Recovered sGAGs were analysed by cellulose acetate membrane electrophoresis and taken up for disaccharide composition analysis by HPLC after fluorescent labelling. Good recovery of sGAGs after metachromasia was observed in all samples of varying levels of purity by this protocol. Further analysis using cellulose acetate membrane electrophoresis showed good separation between species of sGAGs namely chondroitin/dermatan sulfate and heparan sulfate, with comparatively lesser interference from hyaluronic acid, a non-sulfated GAG. Analysis of recovered sGAGs, specifically heparan sulfate by HPLC showed characteristic disaccharide composition akin to that of GAG obtained by the conventional protocol. Thus, in the present paper, we show that sGAG can be recovered in comparatively purer form after routine estimation and can be used for further analysis thus saving up on the precious sample.

RNA Contaminates Glycosaminoglycans Extracted from Cells and Tissues.

Glycosaminoglycans (GAGs) are linear negatively charged polysaccharides and important components of extracellular matrices and cell surface glycan layers such as the endothelial glycocalyx. The GAG family includes sulfated heparin, heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate, and non-sulfated hyaluronan. Because relative expression of GAGs is dependent on cell-type and niche, isolating GAGs from cell cultures and tissues may provide insight into cell- and tissue-specific GAG structure and functions. In our objective to obtain structural information about the GAGs expressed on a specialized mouse glomerular endothelial cell culture (mGEnC-1) we adapted a recently published GAG isolation protocol, based on cell lysis, proteinase K and DNase I digestion. Analysis of the GAGs contributing to the mGEnC-1 glycocalyx indicated a large HS and a minor CS content on barium acetate gel. However, isolated GAGs appeared resistant to enzymatic digestion by heparinases. We found that these GAG extracts were heavily contaminated with RNA, which co-migrated with HS in barium acetate gel electrophoresis and interfered with 1,9-dimethylmethylene blue (DMMB) assays, resulting in an overestimation of GAG yields. We hypothesized that RNA may be contaminating GAG extracts from other cell cultures and possibly tissue, and therefore investigated potential RNA contaminations in GAG extracts from two additional cell lines, human umbilical vein endothelial cells and retinal pigmental epithelial cells, and mouse kidney, liver, spleen and heart tissue. GAG extracts from all examined cell lines and tissues contained varying amounts of contaminating RNA, which interfered with GAG quantification using DMMB assays and characterization of GAGs by barium acetate gel electrophoresis. We therefore recommend routinely evaluating the RNA content of GAG extracts and propose a robust protocol for GAG isolation that includes an RNA digestion step.

Preparation and Characterization of Glycosaminoglycan Chemokine Coreceptors.

Interactions between chemokines and glycosaminoglycans (GAGs) are crucial for the physiological and pathophysiological activities of chemokines. GAGs are therefore commonly designated as chemokine coreceptors which are deeply involved in the chemokine-signaling network. Studying the interaction of chemokines with GAGs is therefore a major prerequisite to fully understand the biological function of chemokines. GAGs are, however, a very complex class of biomacromolecules which cannot be produced by conventional recombinant methods and which, if purchased from commercial suppliers, are often not subjected to rigorous quality control and therefore frequently differ in batch characteristics. This naturally impacts chemokine-GAG interaction studies. In order to standardize the quality of our GAG ligands, we have therefore established protocols for the preparation and characterization of GAGs from various cells and tissues, for which we give practical examples relating to the major GAG classes heparin, heparan sulfate, and chondroitin sulfate. We will also outline robust and sensitive protocols for chemokine-GAG interaction studies. By this means, a better and more common understanding of the involvement of GAGs in chemokine-signaling networks can be envisaged.

ANTICOAGULANT POTENTIAL OF MARINE POLYCHAETE (NEREIS SPECIES)

There has been an accelerated rate of mortality and morbidity stemming from various chronic diseases worldwide over recent years. Anticoagulants play a pivotal role as agents for the prevention and treatment of thromboemb olic disorder. Glycosaminoglycans (GAGs) are linear polysaccharides found in the extracellular matrix and biological fluids of animals where they interact with hundreds of proteins and perform a variety of critical roles. The present study reports the isol ation and characterization of glycosaminoglycans from marine polychaete ( Nereis sp). The isolated glycosaminoglycans were quantified as 12 g/kg and 0.83 mg/kg from the crude and purified samples respectively. Polychaete showed the anticoagulant activity of the 58 USP units/mg from crude; whereas purified samples showed 114 USP units/mg. The structural characterization of anticoagulant GAG was carried out by Fourier Transform Infrared spectroscopy (FTIR) by comparing with that of the standard GAG. The result s of this study could pave the way for further research and exploration of GAGs from marine

The Low Level Laser Therapy Effect on the Remodeling of Bone Extracellular Matrix

The low level laser therapy (LLLT) has been used as an option to accelerate the regeneration of bone tissue. In this study, both femurs of male Wistar rats (30 animals) were injured with a drill and the effect of LLLT using a laser diode (100 mW at 660 nm) in the bone matrix on the left paw measured. LLLT effect on the healing bone tissue matrix was evaluated by a combination of immunohistochemical histomorphometry, confocal immunofluorescence microscopy and isolation and characterization of glycosaminoglycans. Histomorphometric analysis showed that LLLT increased bone matrix and showing more organized. Alcian Blue and PAS staining seems to suggest differential glycosaminoglycans and glycoproteins. The data showed increased expression of chondroitin sulfate and hyaluronic acid, after reduction as the LLLT and mature bone, resembling the expression of osteonectin and biglycan. The difference in expression of siblings (DMP-1, OPN and BSP) is in accordance with the repair accelerated bone formation after the application of LLLT as compared with control. The expression of osteonectin and osteocalcin supports their role in bone mineralization protein, indicating that LLLT accelerates this process. The overall data show that LLLT bone changes dynamic array, shortening the time period involved in the bone repair.

Characterization of Glycosaminoglycans by15N NMR Spectroscopy and in Vivo Isotopic Labeling

Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of (1)H and (13)C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on (1)H-(15)N two-dimensional NMR experiments is described. The (15)N- and (1)H-chemical shifts of amide signals from (15)N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6-, or 4,6-) and easily distinguishable from those of DS. The amide signals from residual (15)N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of (15)N despite its low percentage. However enrichment of the (15)N-content in GAGs using metabolic incorporation from (15)N-glutamine added to cell culture media is also demonstrated and used to distinguish metabolic states in different cell types.

Quantification of Heparan Sulfate Disaccharides Using Ion-Pairing Reversed-Phase Microflow High-Performance Liquid Chromatography with Electrospray Ionization Trap Mass Spectrometry

The glycosaminoglycan (GAG) family of biomacromolecules is composed acidic and linear chains of repeating disaccharide units. Quantitative disaccharide composition analysis is essential for the study and characterization of GAGs. Heparan sulfate and heparin consist of multiple disaccharide units and can be well-separated by ion-pairing reversed-phase microflow high-performance liquid chromatography (IPRP-Mf-HPLC). Each disaccharide can be detected and its mass confirmed by electrospray ionization mass spectrometry (ESI-MS). Isotopically enriched disaccharides were prepared chemoenzymatically from a uniformly (13)C,(15)N-labeled N-acetylheparosan (-GlcA(1-->4)GlcNAc-) obtained from the fermentation of E. coli K5. These isotopically enriched disaccharides have identical HPLC retention times and mass spectra as their unlabeled counterparts and were used in liquid chromatography-mass spectrometry (LC-MS) as internal standards. The ratio of intensities between each pair of enriched and nonenriched disaccharides showed a linear relationship as a function of concentration. With the use of these calibration curves, the amount of each disaccharide (> or = 2 ng/disaccharide) could be quantified in four heparan sulfate samples analyzed by this method.

Modulation of aggrecan and ADAMTS expression in ovine tendinopathy induced by altered strain

To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model. A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase-polymerase chain reaction. Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased. The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Glycosaminoglycans in Hydra magnipapillata (Hydrozoa, Cnidaria): demonstration of chondroitin in the developing nematocyst, the sting organelle, and structural characterization of glycosaminoglycans

The hydrozoan is the simplest organism whose movements are governed by the neuromuscular system, and its de novo morphogenesis can be easily induced by the removal of body parts. These features make the hydrozoan an excellent model for studying the regeneration of tissues in vivo, especially in the nervous system. Although glycosaminoglycans (GAGs) and proteoglycans (PGs) have been implicated in the signaling functions of various growth factors and play critical roles in the development of the central nervous system, the isolation and characterization of GAGs from hydrozoans have never been reported. Here, we characterized GAGs of Hydra magnipapillata. Immunostaining using anti-GAG antibodies showed chondroitin or chondroitin sulfate (CS) in the developing nematocyst, which is a sting organelle specific to cnidarians. The CS-PGs might furnish an environment for assembling nematocyst components, and might themselves be components of nematocysts. Therefore, GAGs were isolated from Hydra and their structural features were examined. A considerable amount of CS, three orders of magnitude less heparan sulfate (HS), but no hyaluronan were found, as in Caenorhabditis elegans. Analysis of the disaccharide composition of HS revealed glucosamine 2-N-sulfation, glucosamine 6-O-sulfation, and uronate 2-O-sulfation. CS contains not only nonsulfated and 4-O-sulfated N-acetylgalactosamine (GalNAc) but also 6-O-sulfated GalNAc. The average molecular size of CS and HS was 110 and 10 kDa, respectively. It has also been established here that CS chains are synthesized on the core protein through the ubiquitous linkage region tetrasaccharide, suggesting that indispensable functions of the linkage region in the synthesis of GAGs have been conserved during evolution.

Characterization of glycosaminoglycans in tubular epithelial cells: Calcium oxalate and oxalate ions effects

The interaction between tubular epithelial cells and calcium oxalate crystals or oxalate ions is a very precarious event in the lithogenesis. Urine contains ions, glycoproteins and glycosaminoglycans that inhibit the crystallization process and may protect the kidney against lithogenesis. We examined the effect of oxalate ions and calcium oxalate crystals upon the synthesis of glycosaminoglycans in distal [Madin-Darby canine kidney (MDCK)] and proximal (LLC-PK1) tubular cell lines. Glycosaminoglycan synthesis was analyzed by metabolic labeling with (35)S-sulfate and enzymatic digestion with specific mucopolysaccharidases. Cell death was assessed by fluorescent dyes and crystal endocytosis was analised by flow cytometry. The main glycosaminoglycans synthesized by both cells were chondroitin sulfate and heparan sulfate most of them secreted to the culture medium or present at cellular surface. Exposition of MDCK cells to oxalate ions increased apoptosis rate and the incorporation of (35)S-sulfate in chondroitin sulfate and heparan sulfate, while calcium oxalate crystals were endocyted by LLC-PK1, induced necrotic cell death, and increased (35)S-sulfate incorporation in glycosaminoglycans. These effects seem to be specific and due to increased biosynthesis, since hydroxyapatite and other carboxylic acid did not induced cellular death or glycosaminoglycan synthesis and no changes in sulfation degree or molecular weight of glycosaminoglycans could be detected. Thapsigargin inhibited the glycosaminoglycan synthesis induced by calcium oxalate in LLC-PK1, suggesting that this effect was sensitive to the increase in cytosolic calcium. Tubular cells may increase the synthesis of glycosaminoglycans to protect from the toxic insult of calcium oxalate crystals and oxalate ions, what could partially limit the lithogenesis.

Isolation, purification and characterization of glycosaminoglycans in the fluids of the mollusc Anodonta cygnea

The isolation, purification and characterization of Anodonta cygnea haemolymph, and extrapallial fluid glycosaminoglycans (Gags), which have high calcium affinity, were carried out in order to better understand the process of nacreous shell biomineralization. Our results show the existence of two different Gags with similar contents in the two fluids, throughout the year, but with significant seasonal variations for both. In the winter months, we identified by electrophoresis, only one kind of Gag chain (hyaluronic acid) while two different Gags (hyaluronic acid and heparan sulphate-like) from both fluids of A. cygnea were found in summer months. Quantification showed the total Gag fractions of both fluids in average, at their highest concentration (79.8 mg/L) in the highest calcification summer period. In contrast, the period of the year with the lowest concentration of total Gags (54.6 mg/L) occurred during the winter when calcification is reduced. This significant decrease between seasons is correlated mainly with the sulphated fraction, being 37.1 mg/L in the summer while only 9.2 mg/L in the winter haemolymph. The present data suggest that a heparan sulphate-like Gag has a relevant role in the biomineralization mechanisms acting as the calcium carbonate nucleator in the shell.

Isolation and characterization of glycosaminoglycans from bovine follicular fluid and their effect on sperm capacitation

The majority of published studies have reported the use of commercial heparin to capacitate bovine sperm. However, heparin is not present in the female genital tract fluids. In this study, we purified large amounts of glycosaminoglycans (GAGs) from bovine follicular fluid (FF), characterized them and determined their potential to capacitate sperm. FF-GAGs were isolated by protease digestion, lipid extraction, and by different precipitation conditions and then purified by ion exchange chromatography. Two GAGs, heparan sulfate and chondroitin sulfate B, were present in FF. To determine the capacitation potential of FF-GAGs, bovine ejaculated sperm were incubated 5 hr with or without 12 or 24 microg/ml of each of the FF-GAG fractions or with heparin (12 microg/ml). The purified FF-GAGs and heparin did not stimulate sperm acrosome reaction (AR), but stimulated sperm capacitation. Fractions 1 and 2 (heparan sulfate) were more active to promote capacitation (stimulated up to 3.2-fold) than fractions 3 and 4 (mostly chondroitin sulfate B). Fractions 3 and 4 stimulated capacitation two times more than the control (without FF-GAGs or heparin). When the heparan sulfate impurity was removed from fractions 3 and 4 by acid hydrolysis, the capacitation-promoting activity associated with these fractions did not change significantly. When 24 microg/ml of fraction 1 or 2 were used, the percentage of sperm capacitation observed was similar to the capacitation with 12 microg/ml of heparin. Our results also indicated that the FF-GAGs interact strongly with the BSP proteins. Therefore, it is concluded that heparan sulfate is the GAG that is the most potent capacitating factor present in bovine FF.

Characterization of glycosaminoglycans during tooth development and mineralization in the axolotl, Ambystoma mexicanum

Glycosaminoglycans (GAGs) involved in the formation of the teeth of Ambystoma mexicanum were located and characterized with the cuprolinic blue (CB) staining method and transmission electron microscopy (TEM). Glycosaminoglycan–cuprolinic blue precipitates (GAGCB) were found in different compartments of the mineralizing tissue. Various populations of elongated GAGCB could be discriminated both according to their size and their preferential distribution in the extracellular matrix (ECM). GAGCB populations that differ in their composition could be attributed not only to the compartments of the ECM but also to different zones and to different tooth types (early-larval and transformed). Larger precipitates were only observed within the dentine matrix of the shaft of the early-larval tooth. The composition of the populations differed significantly between the regions of the transformed tooth: pedicel, shaft and dividing zone. In later stages of tooth formation, small-sized GAGCBs were seen as intracellular deposits in the ameloblasts. It is concluded that the composition of GAGCB populations seems to play a role in the mineralization processes during tooth development in A. mexicanum and influence qualitative characteristics of the mineral in different tooth types and zones, and it is suggested that GAGs might be resorbed by the enamel epithelium during the late phase of enamel formation.

Characterization of glycosaminoglycans by capillary electrophoresis.

Glycosaminoglycans are linear, sulfated polysaccharides that are found in virtually all animal tissues. With the exception of hyaluronic acid, glycosaminoglycans are biosynthesized as proteoglycans in which one or more glycosaminoglycan chains are attached to a core protein (). Chondroitin and dermatan sulfates are comprised of alternating 1→3, 1→4 linked N-acetyl-d-galactosamine and hexuronic acid (either d-glucuronic or l-iduronic acid) residues. These saccharide residues can be O-sulfonated at various positions on both sugar residues. Heparin and heparan sulfate are structurally related and are comprised of a hexuronic acid residue (either l-iduronic or d-glucuronic acid) 1→4 linked to a D-glucosamine (either N-sulfo or N-acetyl-d-glucosamine) residue. Both saccharide residues can be O-sulfonated at a variety of different positions, making these glycosaminoglycans structurally complex. Hyaluronic acid contains a simple repeating structure of 1→3, 1→4 linked N-acetyl-d-glucosamine and d-glucuronic acid residues. Hyaluronic acid is an unusual glycosaminoglycan as it is not sulfonated and can have a very high molecular weight, up to 2 × 106 Da compared to 1–3 × 105 for the other glycosaminoglycans. Keratan sulfate is a glycosaminoglycan that contains no uronic acid and is comprised of a repeating 1→3, 1→4 linked, O-sulfonated N-acetyl-d-glu-cosamine and d-galactose residues.

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs) – chondroitin sulfate (CS) and dermatan sulfate (DS) – were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.

An accumulation of proteoglycans in scarred fascia.

A little is known about proteoglycan (PG) changes, occuring in the course of scarring of tissues another than skin. The aim of present study was biochemical characterization of glycosaminoglycans (GAGs) and proteoglycans (PGs) of normal and scarred fascia. Samples of normal fascia lata were taken at autopsy from 23 individuals and samples of scarred fascia lata were removed from 23 patients at reoperations for femoral fracture. The obtained tissues were divided into two samples: first of them was submitted to GAG isolation and the second one to PG isolation. GAGs were extracted by extensive papain digestion followed by the fractionation using cetylpyridinium chloride. In order to qualitative and quantitative characterization GAGs were submitted to electrophoresis on cellulose acetate before and after treatment with enzymes, specifically depolymerizing some kinds of GAGs. PGs were extracted using 4 M guanidine HCl followed by purification by forming complexes with Alcian blue. PGs were submitted to gel permeation chromatography on Sepharose 4B. In order to obtain core proteins PGs were depolymerized with chondroitinase ABC. The purified PGs and their core proteins were separated with sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE). It was found that total GAGs content was significantly elevated in scarred fascia. Both types of fascia contained chondroitin-, dermatan- and heparan sulphates and hyaluronic acid. Dermatan sulphates (DS) were the predominant GAGs of normal and scarred fascia. The contents of all GAG types were increased in scarred fascia. Both types of fascia contained two kinds of dermatan sulphate proteoglycans (DSPGs); first being similar to biglycan and the second one similar to decorin, as it was judged by molecular weight of their native molecules and core proteins as well as type of GAG components. Densitometric analysis showed that decorin is a predominant DSPG in both fascia types, but in scarred tissue the ratio of biglycan to decorin is considerably higher. Moreover, in scarred fascia a large chondroitin sulphate proteoglycan (CSPG) was also observed. The obtained results have shown that the scar formation is accompanied by quantitative and qualitative alterations in GAGs/PGs resembling those observed in hypertrophic skin scars. The biochemical modification of the scarred fascia lata may partly explain the clinically manifested damage to biomechanical properties of this tissue.