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The chemical modification of glycosaminoglycan structure by oxygen-derived species in vitro
Abstract
The effect of reactive oxygen species (ROS) on the chemical structure of glycosaminoglycans (GAG) was studied in order to consider their role in connective tissue damage during an inflammatory disease state such as periodontal disease. GAG were exposed to a radical generating system for 1 h and analysed by gel filtration for fragmentation and chemically with respect to uronic acid, hexosamine and sulfate content. Non-sulfated GAG, hyaluronan and chondroitin, were most susceptible to depolymerisation and chemical modification of uronic acid and hexosamine residues by ROS. Depolymerisation and chemical modification of sulfated GAG, chondroitin 4-sulfate, dermatan sulfate and heparan sulfate was significantly less than for non-sulfated GAG. The highly sulfated GAG heparin showed minimal depolymerisation by ROS, but uronic acid residues were readily modified. Analysis of the ROS-exposed residues suggests that uronic acid is capable of degrading to a 3-carbon aldehyde, malondialdehyde. Chondroitin sulfate exposed to ROS resulted in marginal desulfation. The results suggest that the presence of sulfate on the GAG chain may protect the molecule against ROS attack. However, chemical modification of GAG may affect proteoglycan function and be of importance in considering connective tissue destruction in a variety of pathological situations, including periodontal disease.
... Although sulphated glycosaminoglycan chains are not as susceptible to degradation by ROS as core proteins of proteoglycans, studies have investigated the effect of ROS exposure on the chondroitin 4-sulphate chains, suggesting such glycosaminoglycans undergo more selective ROS attack than their nonsulphated counterparts (Moseley et al, 1995(Moseley et al, , 1997. Detail relating to the mechanism of ROS attack suggests that radical generation is confined to sites on the D-glucuronic acid rings and the acetyl group of the N-acetylhexosamine rings which ultimately results in chain fragmentation (Volpi et al, 1995;Davies, 1996, 1998b) (Figure 2). ...
... Detail relating to the mechanism of ROS attack suggests that radical generation is confined to sites on the D-glucuronic acid rings and the acetyl group of the N-acetylhexosamine rings which ultimately results in chain fragmentation (Volpi et al, 1995;Davies, 1996, 1998b) (Figure 2). Of interest, comparative studies investigating the effect of ROS on a range of glycosaminoglycans have suggested that the highly sulphated glycosaminoglycan, heparin, to be even more resistant to depolymerisation and residue modification than the less sulphated chondroitin 4-sulphate, followed by the nonsulphated glycosaminoglycan, hyaluronan, as the most prone to depolymerisation (Moseley et al, 1995(Moseley et al, , 1997. Further, the nonsulphated hexuronic acid regions of the heparin molecule, primarily D-glucuronic acid, have been reported to be more susceptible to modification than the N-acetylglucosamine and N-sulphated D-glucosamine regions (Nagasawa et al, 1992;Vilar et al, 1997). ...
... Exposure to ROS resulted in a reduction in the specific viscosity and molecular size of these molecules. More recently, detailed investigations into the degradation of the glycosaminoglycans and proteoglycans associated with mineralised and non-mineralised periodontal tissues have been reported (Moseley et al, 1995(Moseley et al, , 1997(Moseley et al, , 1998. Within these in vitro studies, proteoglycans and glycosaminoglycans were exposed to a broad spectrum of ROS species of differing reactivity and over differing periods of time, in an attempt to relate the findings to the in vivo release of ROS by PMN during initial bursts and the more cumulative periods of disease activity. ...
Periodontal diseases are a group of chronic inflammatory conditions associated with the accumulation of bacterial plaque and the promotion of host inflammatory responses, resulting in extensive degradation of the periodontal extracellular matrix (ECM). Hyaluronan is one ECM component extensively degraded in such cases, while sulphated glycosaminoglycans (GAG) remain relatively intact. Host and bacterial enzymic activities have been proposed to be solely responsible for this degradation, although increasing evidence supports an additional role for polymorphonuclear leukocyte-derived reactive oxygen species (ROS).
... Although sulphated glycosaminoglycan chains are not as susceptible to degradation by ROS as core proteins of proteoglycans, studies have investigated the effect of ROS exposure on the chondroitin 4-sulphate chains, suggesting such glycosaminoglycans undergo more selective ROS attack than their nonsulphated counterparts (Moseley et al, 1995(Moseley et al, , 1997. Detail relating to the mechanism of ROS attack suggests that radical generation is confined to sites on the D-glucuronic acid rings and the acetyl group of the N-acetylhexosamine rings which ultimately results in chain fragmentation (Volpi et al, 1995;Davies, 1996, 1998b) (Figure 2). ...
... Detail relating to the mechanism of ROS attack suggests that radical generation is confined to sites on the D-glucuronic acid rings and the acetyl group of the N-acetylhexosamine rings which ultimately results in chain fragmentation (Volpi et al, 1995;Davies, 1996, 1998b) (Figure 2). Of interest, comparative studies investigating the effect of ROS on a range of glycosaminoglycans have suggested that the highly sulphated glycosaminoglycan, heparin, to be even more resistant to depolymerisation and residue modification than the less sulphated chondroitin 4-sulphate, followed by the nonsulphated glycosaminoglycan, hyaluronan, as the most prone to depolymerisation (Moseley et al, 1995(Moseley et al, , 1997. Further, the nonsulphated hexuronic acid regions of the heparin molecule, primarily D-glucuronic acid, have been reported to be more susceptible to modification than the N-acetylglucosamine and N-sulphated D-glucosamine regions (Nagasawa et al, 1992;Vilar et al, 1997). ...
... Exposure to ROS resulted in a reduction in the specific viscosity and molecular size of these molecules. More recently, detailed investigations into the degradation of the glycosaminoglycans and proteoglycans associated with mineralised and non-mineralised periodontal tissues have been reported (Moseley et al, 1995(Moseley et al, , 1997(Moseley et al, , 1998. Within these in vitro studies, proteoglycans and glycosaminoglycans were exposed to a broad spectrum of ROS species of differing reactivity and over differing periods of time, in an attempt to relate the findings to the in vivo release of ROS by PMN during initial bursts and the more cumulative periods of disease activity. ...
The pathological events leading to the destruction of the periodontium during inflammatory periodontal diseases are likely to represent complex interactions involving an imbalance in enzymic and non-enzymic degradative mechanisms. This paper aims to review the increasing body of evidence implicating reactive oxygen species (ROS), derived from many metabolic sources, in the pathogenesis of periodontal tissue destruction. ROS are generated predominantly by polymorphonuclear leukocytes (PMN) during an inflammatory response and are regarded as being highly destructive in nature. The detection of ROS oxidation products, the elevation of iron and copper ions, which catalyse the production of the most reactive radical species, and the identification of an imbalance in the oxidant/antioxidant activity within periodontal pockets, suggests a significant role for ROS in periodontal tissue destruction. In vitro studies have shown that ROS are capable of degrading a number of extracellular matrix components including proteoglycans, resulting in the modification of amino acid functional groups, leading to fragmentation of the core protein, whilst the constituent glycosaminoglycan chains undergo limited depolymerisation. The identification and characterisation of connective tissue metabolites in gingival crevicular fluid (GCF) resulting from the degradation of periodontal tissues, notably alveolar bone, provides further evidence for a role for ROS in tissue destruction associated with inflammatory periodontal diseases.
... A decrease in molecular mass upon exposure to % OH, generated in situ by reaction of hydrogen peroxide with transition metal ions (e.g. Cu 2+ or Fe 2+ ), has been reported for alginic acid (Smidsrød, Haug, & Larsen, 1965), glycosaminoglycans (Moseley, Waddington, Evans, Halliwell, & Embery, 1995), xanthan (Christensen, Myhr, & Smidsrød, 1996), scleroglucan (Hjerde, Stokke, Smidsrød, & Christensen, 1998), xyloglucan (Tabbı̀, Fry, & Bonomo, 2001), galactouronan (Elboutachfaiti, Delattre, Michaud, Courtois, & Courtois, 2008), and mannan (Čížová, Bystrický, & Bystrický, 2015;Machová, Čížová, & Bystrický, 2014). ...
... The presence of ionic functional groups can affect the susceptibility of polysaccharide to free-radical degradation. For example, only minor depolymerisation was observed in the case of moderately-sulphated glycosaminogylcans, namely chondroitin 4-sulphate, dermatan sulphate, and heparan sulphate, possessing 1-2 sulphate groups per disaccharide unit, while a highly-sulphated glycosaminoglycan heparin (3 sulphate groups per disaccharide unit) showed no depolymerisation at all (Moseley et al., 1995). Similarly, some protective effect against % OH degradation has been displayed by the carboxymethyl (CM) groups of chemically-modified cellulose (Wach et al., 2004), dextran, and mannan (Machová, Čížová et al., 2014). ...
Infection with Candida albicans can prove lethal in immuno-compromised patients. It is imperative to develop a vaccine against this common organism. The amphoteric derivatives of the mannan component of the Candida cell wall may present a prospective target for the development of such a vaccine; however, the radical processing by antigen-presenting cells of the immune system is not fully understood. In this work a set of tailor-made cationic and amphoteric derivatives of three different degrees of quaternization (DSQ 0.14–0.38) has been prepared by chemical modification of ultrasonically-treated mannan and three carboxymethylated mannan derivatives (DSCM 0.13–0.32). These were exposed to free-radical attack by [rad]OH, generated in situ by the Fenton reaction. Potential changes in composition, DSQ, and molar mass distribution due to free-radical degradation were monitored by elemental analysis, NMR and FTIR spectroscopies, and size exclusion chromatography. A protective effect of quaternization against [rad]OH degradation was found. Non-isothermal thermogravimetric analysis found that the thermal stability of this mannan was also improved by chemical modification.
... More recent studies by Moseley and co-workers have investigated the effects of a range of ROS on glycosaminoglycans and proteoglycans present in the soft and calcified tissues of the periodontium. Their findings demonstrated that all glycosaminoglycans undergo a variable degree of chain depolymerization and residue modification (especially in the presence of hydroxyl radicals) and that sulfated glycosaminoglycans were more resistant to ROS degradation than the non-sulfated glycosaminoglycan hyaluronan (282,283). Furthermore, it was shown that chondroitin sulfate proteoglycans from alveolar bone were particularly susceptible to damage by hydroxyl radicals, which caused degradation of both the core proteins and glycosaminoglycan chains (284). By contrast, hydrogen peroxide caused more selective damage with core proteins being more susceptible than glycosaminoglycan chains. ...
... Similarly, biochemical analysis of gingival proteoglycans in inflamed gingivae demonstrates a similar pattern of degradation of the core proteins with retention of relatively intact sulfated glycosaminoglycan chains (36,127,330). In contrast to its sulfated counterparts, the ubiquitous non-sulfated glycosaminoglycan hyaluronan, found to be most susceptible to ROS damage in vitro (282,283), is completely degraded in inflamed gingival tissue (36,127,330). Thus, the pattern of proteoglycan and glycosaminoglycan degradation seen in periodontitis reflects the in vitro data on the effects of ROS and is consistent with a role for oxidative damage to non-collagenous components of both the hard and soft tissues of the periodontium. ...
... Sulfated GAGs are more resistant than non-sulfated GAGs, such as hyaluronan, to ROS degradation in vitro. 23,24 Another important ECM component is type I collagen. Containing 1000 amino acids that form a triple helix, it resists nonspecific proteolytic attacks. ...
... ROS directly attack collagen, which results in fragmentation, and makes it more susceptible to breakdown by collagenase. 24,25 Superoxide anions and hydroxyl radicals are able to cleave collagen into small peptide fragments at the proline and hydroxyproline residues. 26 Furthermore, lipid peroxidation products (e.g., malondialdehyde) of ROS may interact with collagen and alter fibroblast functions. ...
Hypochlorous acid (HOCl) has both proinflammatory and anti-inflammatory properties, and seems to play an important role in the immune system. The regulation of normal flora contributes to periodontal health, and HOCl seems to have the ability to attack Gram-negative pathogens during periodontitis. Furthermore, high concentrations of HOCl promote healing by regulating cytokines and growth factors, killing pathogens through chlorination or oxidation, and modulating inflammation through the effects on nuclear factor κB and activator protein-1 of monocytes. After chlorination of taurine by HOCl, taurine chloramine is mostly an anti-inflammatory agent and enhances healing. Neither HOCl nor taurine chloramine are common in clinical applications owing to a lack of studies in animal and human models. Both compounds may be suitable as periodontal medication, as they are good antimicrobial agents, inflammation modulators, and healing promoters.
... This could be explained by resistance to the hydroxyl radical attack due to the presence of sulfates (7.29%) in ALCM (fucoidan contamination) [10]. Moseley and chemical modification compared to non-sulfated GAG, suggesting that the presence of sulfate could protect molecules against reactive oxygen species (ROS) hydrolysis [42]. It has been reported that sulfate content has an effect on scavenging-generated hydroxyl radicals [43]. ...
Alginates extracted from two Moroccan brown seaweeds and their derivatives were
investigated for their ability to induce phenolic metabolism in the roots and leaves of tomato seedlings. Sodium alginates (ALSM and ALCM) were extracted from the brown seaweeds Sargassum muticum and Cystoseira myriophylloides, respectively. Low-molecular-weight alginates (OASM and OACM) were obtained after radical hydrolysis of the native alginates. Elicitation was carried out by foliar spraying 20 mL of aqueous solutions (1 g/L) on 45-day-old tomato seedlings. Elicitor capacities
were evaluated by monitoring phenylalanine ammonia-lyase (PAL) activity, polyphenols, and lignin production in the roots and leaves after 0, 12, 24, 48, and 72 h of treatment. The molecular weights (Mw) of the different fractions were 202 kDa for ALSM, 76 kDa for ALCM, 19 kDa for OACM, and 3 kDa for OASM. FTIR analysis revealed that the structures of OACM and OASM did not change after oxidative degradation of the native alginates. These molecules showed their differential capacity to induce natural defenses in tomato seedlings by increasing PAL activity and through the accumulation of polyphenol and lignin content in the leaves and roots. The oxidative alginates (OASM and OACM) exhibited an effective induction of the key enzyme of phenolic metabolism (PAL) compared to the alginate polymers (ALSM and ALCM). These results suggest that low-molecular-weight alginates
may be good candidates for stimulating the natural defenses of plants.
... The presence of sulfates contamination in ALCM could be the factor influencing the degradation process. Moseley et al., have suggested that the presence of sulfate could protect molecules against ROS hydrolysis [55]. Under the same conditions, oligomers produced by the degradation of alginate extracted from B. bifurcata, exhibited an M w of 5.5 kDa and 2 ≤ DP ≤ 24 after 6 h of treatment and 3.5 kDa after 10 h [26], which are different from values found in this study. ...
This study aims to assess for the first time the ability of marine polysaccharides and their derivatives to stimulate natural defenses in olive tree leaves. Alginates, ALSM, and ALCM were isolated from the brown algae Sargassum muticum and Cystoseira myriophylloides, respectively. The OASM and OACM fractions were obtained after radical depolymerization of ALSM and ALCM, respectively. Three sulfated polysaccharides, arabinogalactans (AGB and AGP) and fucoidans (FUCCM) were extracted from the green alga Codium decorticatum and the brown seaweed C. myriophylloides, respectively. The extraction yield of different extracts was in the range of 0.82–32% and the average molecular weight (Mw) varied from 3000 Da to 2173 kDa. The M/G ratios of ALSM and ALCM were 0.87 and 1.12, respectively. FUCCM contained 53% of fucose and 12.83% of sulfates. The AGB and AGP fractions were characterized by the presence of a high degree of sulfation and protein (12–23% (w/w)) and were composed mainly of galactose, glucose, and arabinose. The aqueous saccharide solutions were applied to the leaf discs of the olive tree at 0.5 g/L, 1 g/L, and 2 g/L, for 24 h. These molecules triggered defense responses, by showing a differential capacity to induce the activity of the phenylalanine and tyrosine ammonia-lyase (TAL and PAL), polyphenols, and lignin contents in the leaves of the olive tree. Alginates and their derivatives as well as arabinogalactans exhibited an important induction of TAL activity compared to the PAL. The sulfated polysaccharides were more effective compared to the unsulfated polysaccharides (alginates) which were active at a low concentration. The optimum concentration for most of the studied elicitors was 2 g/L. These results suggest the valorization of these molecules derived from marine biomass as inducers of natural defenses of the olive tree to protect against phytopathogens in the context of sustainable development.
... 18 The degradation of HA, on the contrary, is increased because of increased expression and activity of the HA-degrading enzyme hyaluronidase-2 (HYAL-2) in OA synovium. 19,20 ROS are another agents which contribute to degradation of HA 21,22 and other components of cartilage ECM 23,24 in OA joints. In a healthy joint, ROS are produced by chondrocytes, synoviocytes, and immune cells and are involved in intracellular signaling. ...
Osteoarthritis (OA) is one of the most common musculoskeletal disorders in the world. OA is often associated with the loss of viscoelastic and tribological properties of synovial fluid (SF) due to degradation of hyaluronic acid (HA) by reactive oxygen species (ROS) and hyaluronidases. Viscosupplementation is one of the ways how to effectively restore SF functions. However, current viscosupplementation products provide only temporal therapeutic effect because of short biological half‐life. In this article we describe a novel device for viscosupplementation (NV) based on the cross‐linked tyramine derivative of HA, chondroitin sulfate (CS), and high molecular weight HA by online determination of viscoelastic properties loss during degradation by ROS and hyaluronidase. Rheological and tribological properties of developed viscosupplement were compared with HA solutions with different molecular weights in the range 500–2000 kDa, which are currently commonly used as medical devices for viscosupplementation treatment. Moreover, based on clinical practice and scientific literature all samples were also diluted by model OA SF in the ratio 1:1 (vol/vol) to better predict final properties after injection to the joint. The observed results confirmed that NV exhibits appropriate rheological properties (viscosity, elastic, and viscous moduli) comparable with healthy SF and maintain them during degradation for a significantly longer time than HA solutions with molecular weight in the range 500–2000 kDa and cross‐linked material without CS.
... Several studies have, for instance, shown acute degradation of the endothelial glycocalyx during heart surgery with cardiopulmonary bypass (CPB) [34,35]. Hyperoxia during CPB causes degradation of glycocalyx proteoglycans and aggravates ischemia/reperfusion injury of different organs [36,37]. Standardized clinical circumstances during heart surgery on CPB could be a possible approach to investigate the influence of altered partial pressures of oxygen on endothelial glycan structures and their impact on outcome. ...
Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of α2-6 sialylated structures in response to hyperoxia.
... The laccase/TEMPO method has additionally been used to oxidize arabinoxylan, as well as guar galactomannan and konjac glucomannan, however, it has not yet been applied to GAGs [143]. The effect of reactive oxygen species on a range of GAGs were also investigated, but the consequences included depolymerization, de-sulfation, and oxidation [144]. ...
The linear anionic class of polysaccharides, glycosaminoglycans (GAGs), are critical throughout the animal kingdom for developmental processes and the maintenance of healthy tissues. They are also of interest as a means of influencing biochemical processes. One member of the GAG family, heparin, is exploited globally as a major anticoagulant pharmaceutical and there is a growing interest in the potential of other GAGs for diverse applications ranging from skin care to the treatment of neurodegenerative conditions, and from the treatment and prevention of microbial infection to biotechnology. To realize the potential of GAGs, however, it is necessary to develop effective tools that are able to exploit the chemical manipulations to which GAGs are susceptible. Here, the current knowledge concerning the chemical modification of GAGs, one of the principal approaches for the study of the structure-function relationships in these molecules, is reviewed. Some additional methods that were applied successfully to the analysis and/or processing of other carbohydrates, but which could be suitable in GAG chemistry, are also discussed.
... In vitro exposure of ROS (superoxide and hydroxyl radicals) to the eGCX promotes fragmentation of GAGs and loss of some of its components. Previous studies have demonstrated that hyaluronan and chondroitin sulfate are the most susceptible to depolymerization and chemical modifications by ROS (Halliwell, 1978;Greenwald and Moy, 1980;Bartold et al., 1984;Moseley et al., 1995Moseley et al., , 1997Lipowsky and Lescanic, 2013;Singh et al., 2013). Intact eGCX has the capability to quench free radicals by having binding sites for anti-oxidant enzymes like xanthine oxidoreductase (Adachi et al., 1993) and endothelial superoxide dismutase (eSOD) (Becker et al., 1994). ...
Expressed on the endothelial cell (EC) surface of blood vessels, the glycocalyx (GCX), a mixture of carbohydrates attached to proteins, regulates the access of cells and molecules in the blood to the endothelium. Besides protecting endothelial barrier integrity, the dynamic microstructure of the GCX confers remarkable functions including mechanotransduction and control of vascular tone. Recently, a novel perspective has emerged supporting the pleiotropic roles of the endothelial GCX (eGCX) in cardiovascular health and disease. Because eGCX degradation occurs in certain pathological states, the circulating levels of eGCX degradation products have been recognized to have diagnostic or prognostic values. Beyond their biomarker roles, certain eGCX fragments serve as pathogenic factors in disease progression. Pharmacological interventions that attenuate eGCX degradation or restore its integrity have been sought. This review provides our current understanding of eGCX structure and function across the microvasculature in different organs. We also discuss disease or injury states, such as infection, sepsis and trauma, where eGCX dysfunction contributes to severe inflammatory vasculopathy.
... • NO 2 and • OH radicals resulting from the decomposition of ONOOhave also been shown to react with GAGs such as hyaluronan, which is degraded by ROS/RNS-mediated scission of the polymeric chains [62][63][64]. Numerous other reports have also confirmed the susceptibility of hyaluronan, as well as other GAGs such as chondroitin sulfate and heparan sulfate, to ROS/RNS-mediated degradation [65][66][67][68][69][70][71][72]. Furthermore, experiments going back decades have demonstrated that superoxide can react with and fragment collagen, while other ROS/RNS-mediated modifications are capable of rendering collagen more susceptible to proteolysis [73][74][75]. ...
The transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is widely recognized as a master regulator of the cellular stress response by facilitating the transcription of cytoprotective genes. As such, the Nrf2 pathway is critical in guarding the cell from the harmful effects of excessive reactive oxygen species/reactive nitrogen species (ROS/RNS) and in maintaining cellular redox balance. While excessive ROS/RNS are harmful to the cell, physiological levels of ROS/RNS play important roles in regulating numerous signaling pathways important for normal cellular function, including the synthesis of extracellular matrix (ECM). Recent advances have underscored the importance of ROS/RNS, and by extension, factors that influence redox-balance such as Nrf2, in regulating ECM production and deposition. In addition to reducing the oxidative burden in the cell, the discovery that Nrf2 can also directly target genes that regulate and form the ECM has cemented it as a multifaceted player in the regulation of ECM proteins, and provides new insight into its potential usefulness as a target for treating ECM-related pathologies.
... Ces espèces très réactives contribuent à l'inhibition de la synthèse des constituants matriciels et à leur dégradation (AH, PO, collagènes). Ces effets s'exercent soit directement [Moseley, 1995;Panasyuk, 1994], soit indirectement, à la suite de l'activation de collagénases latentes [Murrel, 1995] et de l'inactivation de leurs inhibiteurs naturels, les TIMP [Shabani, 1998]. Les radicaux libres sont également soupçonnés de moduler l'expression de certains gènes, tels que celui de l'agrécane, ou encore ceux de certaines collagénases. ...
... Hyperoxia during CPB is best defined as a PaO2 in excess of 185 mmHg (60)(61)(62)(63)(64)(65). The presence of hyperoxia during CPB induces increased levels of ROS free radicals, which subsequently cause the depolymerization of the glycosaminoglycan chains of glycocalyx proteoglycans such as heparan sulfate, chondroitin sulfate, and hyaluronic acid (66)(67)(68). Randomly increasing the FiO2-100% during CPB as a theoretical attempt to address GME will subsequently degrade/damage the glomerular EGL and do little to prevent the destructive nature of GME. As an example, the damage of glomerular endothelial cells by hyperoxia can result in the presence of proteinuria from potential kidney injury (66). ...
On the outer surface of a human cell there is a dense layer of complex carbohydrates called glycocalyx, also referred to as glycans or the sugar coating on the cell surface, which is composed of a complex array of oligosaccharide and polysaccharide glucose chains that are covalently bonded to proteoglycans and lipids bound to the cell membrane surface. Studies of an intact endothelial glycocalyx layer (EGL) have revealed a number of critical functions that relate the importance of this protective layer to vascular integrity and permeability. These functions include the following: stabilization and maintenance of the vascular endothelium, an active reservoir of essential plasma proteins (i.e., albumin, antithrombin, heparan sulfate, and antioxidants), a buffer zone between the blood (formed elements) and the surface of the endothelium, and a mechanotransducer to detect changes in shear stress that facilitate vascular tone. There have been numerous review articles about the structure and function of endothelial glycocalyx over the past two decades, yet there still remains a significant knowledge gap in the perfusion literature around the importance of EGL. Perioperative fluid management and gaseous microemboli can both contribute to the damage/degradation of endothelial glycocalyx. A damaged EGL can result in systemic and myocardial edema, platelet and leukocyte adhesion, fluid extravasation, and contributes to microvascular perfusion heterogeneity. Knowledge of the importance of endothelial glycocalyx will enable clinicians to have a better understanding of the impact of gaseous microbubbles, hyperoxia, and ischemic reperfusion injury during cardiac surgery. The purpose of this article is to provide an in depth review of the EGL and how this protective barrier impacts the microcirculation, fluid homeostasis, inflammation, and edema during cardiac surgery.
... The major ROS that reacts with polysaccharides is the hydroxyl radical. However, hyaluronic acid, a component of connective tissue in mammals, has been shown to be oxidatively depolymerized by H 2 O 2 and Fe 2+ in vitro [62]. (8) and (69)). ...
Mammalian mitochondria have a 16.5 kb genome encoding for 13 polypeptides, 2 rRNAs, and 22 tRNAs essential for mitochondrial function. Mutations and deletions in mitochondrial DNA (mtDNA) are implicated in some hereditary diseases as well as aging, cancer and neurodegeneration. Here, we examine the role of DNA ligase in mtDNA maintenance, and the role of mtDNA damage inflicted by hydrogen peroxide (H2O2) or methyl methanesulfonate (MMS) in mitochondrial dysfunction. We establish that mitochondrial DNA ligase activity is essential for survival, persistent mtDNA damage is not sufficient to induce rapid mtDNA loss and mitochondrial dysfunction. We removed and replaced the normal mitochondrial DNA ligase III with different forms of mitochondrially-targeted DNA ligase, and found that mitochondrial DNA ligase activity is necessary for cellular survival and that any type of DNA ligase activity in the mitochondria is sufficient to maintain mtDNA integrity and copy number. To study the relationship between mtDNA integrity and mitochondrial function, we treated cells with H2O2 or with the alkylating agent MMS, both of which resulted in persistent mtDNA lesions. However, only the H2O2-treated cells showed mtDNA loss and mitochondrial dysfunction by 8 hours post-treatment, indicating that persistent mtDNA damage does not necessarily cause a rapid loss of mtDNA or mitochondrial function. These data suggest that oxidants are more efficient than alkylating agents at driving mtDNA loss and mitochondrial dysfunction.
We then addressed the cause of H2O2-induced loss of mtDNA 8 hours following treatment. We hypothesized that this loss of mtDNA is dependent upon mitochondrial fission and mitophagy. In order to test this hypothesis, we treated cells with the fission inhibitor mdivi-1. Mdivi-1 protected mtDNA against oxidative-induced mtDNA damage but not MMS-induced mtDNA damage. Because mdivi-1 is thought to act through inhibition of Drp1, we performed siRNA-mediated knockdown (KD) of Drp1 and observed that the knockdown did not recapitulate mdivi-1 treatment in protecting against H2O2-induced mtDNA damage. Furthermore, treating Drp1 KD cells with mdivi-1 still showed the protective effects of mdivi-1 on mtDNA damage. These results suggest that the mdivi-1 mediated protection of oxidant-induced mtDNA damage may be independent of its role in inhibiting mitochondrial fission.
... LMW-HA distributes highly in the gingival tissues of patients at an initial phase of periodontitis [28]. The pathological accumulation of LMW-HA is suggested to be due to various degrading mechanisms such as depolymerization with reactive oxygen species [29] and aceleration of LMW-HA synthesis [30]. In addition, a number of mammalian hyaluronidases (HAase) capable of enzymatically degrading hyaluronan have recently been shown to be associated with inflammation [31]. ...
... The role of heparanase in extravasation has been demonstrated more specifically by Edovitsky et al, who locally administrated siRNA to target endothelial heparanase, resulting in decreased delayed-type hypersensitivity inflammatory response in vivo (102). Apart from heparanase, leukocytes can also produce reactive oxygen species (ROS), which can cause HS depolymerization (103)(104)(105). However, to our knowledge the importance of ROS-induced HS degradation in leukocyte extravasation has not been shown to date. ...
Heparan sulfate proteoglycans (HSPGs) are glycoconjugates that are implicated in various biological processes including development, inflammation and repair, which is based on their capacity to bind and present several proteins via their carbohydrate side chains (glycosaminoglycans; GAGs). Well-known HSPGs include the family of syndecans and glypicans, which are expressed on the plasma membrane and perlecan, agrin and collagen type XVIII, which are present in basement membranes. In this review, we provide an overview of the current knowledge on the role and regulation of HSPGs in leukocyte extravasation. In the non-inflamed endothelial glycocalyx HSPGs are anti-adhesive, and there are several indications that active regulation of HSPG core protein expression and/or GAG modification occurs upon inflammation. We address the current evidence for the role of HSPGs in leukocyte extravasation through interaction with the leukocyte adhesion molecule L-selectin, chemokines and other binding partners. Finally, a number of possibilities to use HSPGs as therapeutics or targets in anti-inflammatory strategies are discussed.
... While the microglias are scanning the CNS, they are supposed to check if all the cells in their vicinity are healthy, otherwise microglia will start to phagocytose. Under pathological conditions such as inflammation or cancer, it is known that reactive oxygen damage the cellular glycocalyx [26][27][28][29][30]. An intact glycocalyx is essential for normal cellular function and any alteration of the glycocalyx might induce immediate responses of the microglia [23]. ...
Microglia has emerged not only as an essential inflammatory cell but also as a major player in the development of the adult brain. Microglia phagocytize extra-numerical synapses during postnatal development, maintain and strengthen the remaining subset of synapses, remodel synaptic circuits and clearing apoptotic newborn neurons. Thereby, microglia plays a crucial role for the establishment, plasticity and function of adult neural circuits. In addition to the key role in normal brain function, any imbalance in microglia activity has been associated with neurodegenerative diseases. Microglial cells respond rapidly to smallest pathological changes, this being a vital aspect in many tissue scaring and the local confinement of focal lesions. It is assumed that the high motility of microglial cells represents an important requirement to fulfill the numerous functions. In this review will highlight the role of microglial motility in the healthy and the injured brain, and discuss how impairment of microglia motility can affect normal brain function.
... Changes in the surface chemistry of nanoceria have also been reported to provide tuneable responses in terms of protein adsorption and cellular uptake [24]. The present study sought to functionalise nanoceria with heparin to investigate the activity of these particles in a biological environment as both heparin and nanoceria exhibit antioxidant properties protecting cells from free-radical induced damage [2,25,26]. ...
Cerium oxide nanoparticles (nanoceria) are effective in scavenging intracellular reactive oxygen species (ROS). In this study nanoceria synthesized by flame spray pyrolysis (dXRD = 12 nm) were functionalised with heparin via an organosilane linker, 3-aminopropyltriethoxysilane. Nanoceria were functionalised with approximately 130 heparin molecules per nanoparticle as determined by thermo gravimetric analysis. Heparin functionalised nanoceria were more effectively internalised by the human monocyte cell line, U937, and U937 cells that had been activated with phorbol 12 myristate 13-acetate (PMA) than bare nanoceria. The heparin functionalised nanoceria were also more effective in scavenging ROS than nanoceria in both activated and unactivated U937 cells. Heparin coupled nanoceria were found to be biologically active due to their ability to bind fibroblast growth factor 2 and signal through FGF receptor 1. Additionally, the heparin-coupled nanoceria, once internalised by the cells, were found to be degraded by 48 h. Together these data demonstrated that heparin enhanced the biological properties of nanoceria in terms of cellular uptake and ROS scavenging, while the nanoceria themselves were more effective at delivering heparin intracellularly than exposing cells to heparin in solution.
... ROS are well known to depolymerise and damage the structure of HS, CS and HA GAG [27]. Non-sulphated GAG, HA appears to be particularly susceptible to damage from ROS [28]. Loss of sulphation from GAG chains particularly HS is a known feature in diabetes and is linked to chronic hyperglycemia [29]. ...
Reactive oxygen species (ROS) play a key role in the pathogenesis of proteinuria in glomerular diseases like diabetic nephropathy. Glomerular endothelial cell (GEnC) glycocalyx covers the luminal aspect of the glomerular capillary wall and makes an important contribution to the glomerular barrier. ROS are known to depolymerise glycosaminoglycan (GAG) chains of proteoglycans, which are crucial for the barrier function of GEnC glycocalyx. The aim of this study is to investigate the direct effects of ROS on the structure and function of GEnC glycocalyx using conditionally immortalised human GEnC. ROS were generated by exogenous hydrogen peroxide. Biosynthesis and cleavage of GAG chains was analyzed by radiolabelling (S and H-glucosamine). GAG chains were quantified on GEnC surface and in the cell supernatant using liquid chromatography and immunofluorescence techniques. Barrier properties were estimated by measuring trans-endothelial passage of albumin. ROS caused a significant loss of WGA lectin and heparan sulphate staining from the surface of GEnC. This lead to an increase in trans-endothelial albumin passage. The latter could be inhibited by catalase and superoxide dismutase. The effect of ROS on GEnC was not mediated via the GAG biosynthetic pathway. Quantification of radiolabelled GAG fractions in the supernatant confirmed that ROS directly caused shedding of HS GAG. This finding is clinically relevant and suggests a mechanism by which ROS may cause proteinuria in clinical conditions associated with high oxidative stress.
... Moreover, under pathological conditions including inflammation and cancer ROS are produced causing oxidative damage to the glycocalyx. The degradation of glycosaminoglycans (Moseley et al., 1995(Moseley et al., , 1997 or N-linked oligosaccharides at N-acetylglucosamine residues (Eguchi et al., 2002) on exposure to oxidative stress has been demonstrated. Furthermore, high concentration of soluble sialic acids in serum as a sign for the removal of the sialic acid cap were measured during inflammatory processes (Goswami et al., 2003;Painbeni et al., 1997). ...
Microglia sense intact or lesioned cells of the central nervous system (CNS) and respond accordingly. To fulfill this task, microglia express a whole set of recognition receptors. Fc receptors and DAP12 (TYROBP)-associated receptors such as microglial triggering receptor expressed on myeloid cells-2 (TREM2) and the complement receptor-3 (CR3, CD11b/CD18) trigger the immunoreceptor tyrosine-based activation motif (ITAM)-signaling cascade, resulting in microglial activation, migration, and phagocytosis. Those receptors are counter-regulated by immunoreceptor tyrosine-based inhibition motif (ITIM)-signaling receptors, such as sialic acid-binding immunoglobulin superfamily lectins (Siglecs). Siglecs recognize the sialic acid cap of healthy neurons thus leading to an ITIM signaling that turns down microglial immune responses and phagocytosis. In contrast, desialylated neuronal processes are phagocytosed by microglial CR3 signaling via an adaptor protein containing an ITAM. Thus, the aberrant terminal glycosylation of neuronal surface glycoproteins and glycolipids could serve as a flag for microglia, which display a multitude of diverse carbohydrate-binding receptors that monitor the neuronal physical condition and respond via their ITIM- or ITAM-signaling cascade accordingly. © 2012 Wiley Periodicals, Inc.
... Together, these observations suggest that ROS play a critical role in mediating high glucose-induced HPR1 production. It should be noted that ROS are also capable of directly shedding off heparan sulphate side chains by heparan sulphate depolymerisation [38,39]. Our experiment in Fig. 3 showed that ROS at higher concentrations led to complete loss of heparan sulphate on the cell surface, but did not proportionately increase HPR1 production in conditioned media or HPR1 gene expression. ...
The content of heparan sulphate is reduced in the endothelium under hyperglycaemic conditions and may contribute to the pathogenesis of atherosclerosis. Heparanase-1 (HPR1) specifically degrades heparan sulphate proteoglycans. We therefore sought to determine whether: (1) heparan sulphate reduction in endothelial cells is due to increased HPR1 production through increased reactive oxygen species (ROS) production; and (2) HPR1 production is increased in vivo in endothelial cells under hyperglycaemic and/or atherosclerotic conditions.
HPR1 mRNA and protein levels in endothelial cells were analysed by RT-PCR and Western blot or HPR1 enzymatic activity assay, respectively. Cell surface heparan sulphate levels were analysed by FACS. HPR1 in the artery from control rats and a rat model of diabetes, and from patients under hyperglycaemic and/or atherosclerotic conditions was immunohistochemically examined.
High-glucose-induced HPR1 production and heparan sulphate degradation in three human endothelial cell lines, both of which were blocked by ROS scavengers, glutathione and N-acetylcysteine. Exogenous H(2)O(2) induced HPR1 production, subsequently leading to decreased cell surface heparan sulphate levels. HPR1 content was significantly increased in endothelial cells in the arterial walls of a rat model of diabetes. Clinical studies revealed that HPR1 production was increased in endothelial cells under hyperglycaemic conditions, and in endothelial cells and macrophages in atherosclerotic lesions.
Hyperglycaemia induces HPR1 production and heparan sulphate degradation in endothelial cells through ROS. HPR1 production is increased in endothelial cells from a rat model of diabetes, and in macrophages in the atherosclerotic lesions of diabetic and non-diabetic patients. Increased HPR1 production may contribute to the pathogenesis and progression of atherosclerosis.
... The radicals at carbons which form glycosidic bonds will undergo a β-scission reaction resulting in the breakdown of polysaccharide chains (Gilbert et al. 1981; Hawkins and Davies 1996; Rees et al. 2008). Notably, sulfated polysaccharides are shown more resistant to hydroxyl radical attack (Moseley et al. 1995). Peroxynitrite has been shown to degrade hyaluronic acid (HA) and chondroitin sulfate via a hydroxyl radical-like mechanism (Li et al. 1997; Al-Assaf et al. 2003; Kennett and Davies 2007). ...
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced and are tightly regulated to maintain a redox balance (or homeostasis) together with antioxidants (e.g. superoxide dismutase and glutathione) under normal physiological circumstances. These ROS/RNS have been shown to be critical for various biological events including signal transduction, aging, apoptosis, and development. Despite the known beneficial effects, an overproduction of ROS/RNS in the cases of receptor-mediated stimulation and disease-induced oxidative stress can inflict severe tissue damage. In particular, these ROS/RNS are capable of degrading macromolecules including proteins, lipids and nucleic acids as well as polysaccharides, and presumably lead to their dysfunction. The purpose of this review is to highlight (1) chemical mechanisms related to cell-free and cell-based depolymerization of polysaccharides initiated by individual oxidative species; (2) the effect of ROS/RNS-mediated depolymerization on the successive cleavage of the glycosidic linkage of polysaccharides by glycoside hydrolases; and (3) the potential biological outcome of ROS/RNS-mediated depolymerization of polysaccharides.
Skin ailments can also be treated by other semi-solid preparations like ointment, paste or cream but it has certain demerits like less Spreadability coefficient, distressing to sticky nature. Skin is the best for topical drug delivery as it is readily accessible. There are distinctive semi-solid preparations that are utilized for topical applications. The most influential advantage of topical drug delivery system is the direct application at the site of action to produce its action for prolong period of time. Pharmaceutical gel is one of the basic drug delivery systems. The pharmaceutical gels are preferred over its stability and better application properties. It is less greasy and can easily be washed out from site of application. Pharmaceutical gels form a three dimensional matrix system. The essential network of gel should be a combination of solvent and gelling agents. The gelling agent acts as a dispersion medium in which the therapeutic agents are embedded or weaved equitably. This chapter highlights the fundamental preferences of gel details over its morphological and rheological properties, formulation considerations and analytical and stability aspects.
Reactive oxygen species (ROS) overproduction and oxidative stress are increasingly being implicated in the extracellular matrix (ECM) degradation associated with chronic inflammatory conditions, such as periodontal diseases. The present study investigated the effects of ROS exposure on the proteoglycans of gingival tissues, utilizing an in vitro model system comprised of supra-physiological oxidant concentrations, to ascertain whether gingival proteoglycan modification and degradation by ROS contributed to the underlying mechanisms of ECM destruction during active gingivitis. Proteoglycans were purified from ovine gingival tissues and exposed to increasing H2O2 concentrations or a hydroxyl radical (·OH) flux for 1 h or 24 h, and ROS effects on proteoglycan core proteins and sulfated glycosaminoglycan (GAG) chains were assessed. ROS were capable of degrading gingival proteoglycans, with ·OH species inducing greater degradative effects than H2O2 alone. Degradative effects were particularly manifested as amino acid modification, core protein cleavage, and GAG chain depolymerization. Proteoglycan core proteins were more susceptible to degradation than GAG chains with H2O2 alone, although core proteins and GAG chains were both extensively degraded by ·OH species. Proteoglycan exposure to ·OH species for 24 h induced significant core protein amino acid modification, with decreases in glutamate, proline, isoleucine, and leucine; and concomitant increases in serine, glycine, and alanine residues. As clinical reports have previously highlighted proteoglycan core protein degradation during chronic gingivitis, whereas their sulfated GAG chains remain relatively intact, these findings potentially provide further evidence to implicate ROS in the pathogenesis of active gingivitis, complementing the enzymic mechanisms of periodontal tissue destruction already established.
Chondroitin sulfate (CS) is a sulfated glycosaminoglycan with diverse biological activities, which are influenced by molecular weight (Mw) and sulfation pattern. In the present work, we take advantage of the characteristic high Mw of fish CS (51-70 kDa) to obtain lower Mw fragments with hyaluronidase and chondroitinase ABC. With this aim, we present a pseudo-mechanistic model capable of reproducing the decrease in Mw of CS from five different fish species over 24 h at four enzyme to substrate ratios. The fitting parameters of the model for each species allow to establish conditions of reaction to produce CS of the desired Mw. Furthermore, the main features of the sulfation pattern of fish CS remain in the depolymerized fragments, highlighting the feasibility of the proposed approach.
The glycosaminoglycan hyaluronan (HA) is a key component of the microenvironment surrounding cells. In healthy tissues, HA molecules have extremely high molecular mass and consequently large hydrodynamic volumes. Tethered to the cell surface by clustered receptor proteins, HA molecules crowd each other, as well as other macromolecular species. This leads to severe nonideality in physical properties of the biomatrix, because steric exclusion leads to an increase in effective concentration of the macromolecules. The excluded volume depends on both polymer concentration and hydrodynamic volume/molecular mass. The biomechanical properties of the extracellular matrix, tissue hydration, receptor clustering, and receptor-ligand interactions are strongly affected by the presence of HA and by its molecular mass. In inflammation, reactive oxygen and nitrogen species fragment the HA chains. Depending on the rate of chain degradation relative to the rates of new synthesis and removal of damaged chains, short fragments of the HA molecules can be present at significant levels. Not only are the physical properties of the extracellular matrix affected, but the HA fragments decluster their primary receptors and act as endogenous danger signals. Bioanalytical methods to isolate and quantify HA fragments have been developed to determine profiles of HA content and size in healthy and diseased biological fluids and tissues. These methods have potential use in medical diagnostic tests. Therapeutic agents that modulate signaling by HA fragments show promise in wound healing and tissue repair without fibrosis.
Osteoarthritis (OA) is a chronic degenerative joint disorder characterized by morphological, biochemical, molecular, and biomechanical changes in all joint tissues. Despite the heterogenic pathogenesis, it has been described that the imbalance between production of reactive oxygen species and cellular scavenging mechanisms—or oxidative stress—is critical for disease progression. Thus, the production of ROS not only destroys cartilage but also amplifies the inflammatory process that helps to perpetuate disease. On the other hand, since estrogens play an important role in preserving homeostasis of articular tissues, they could also act protecting the joint tissues against oxidative stress. In agreement, it has been shown that estrogen deficiency results in an increased oxidation load eventually leading to DNA damage. Moreover, the total antioxidant capacity could be restored with the administration of 17β-estradiol, indicating that estrogens might buffer the impact of oxidative stress. Estrogens represent an interesting approach for the treatment of OA, with favorable direct effects on the chondrocyte metabolism and antioxidative properties.
To explore new biopolymer-degrading methods that are simple, specific, and effective, a cupric ion and hydrogen peroxide system was investigated for its ability to oxidatively fragment N-acetylglucosamine-containing polysaccharides, exemplified by hyaluronic acid. Treating hyaluronic acid with cupric ion and hydrogen peroxide led to depolymerization, whose extent depended on the concentrations of cupric ion and peroxide. Fragmented hyaluronic acids with defined size were obtained, indicating an apparent region-specificity of the reaction. The effect was attributed to a specific cupric ion binding to the N-acetylglucosamines, a cupric ion activation of hydrogen peroxide to form reactive oxygen species, and restricted oxidative cleavage of the glycosidic bonds. Such region-specificity might render the simple chemical depolymerization method useful for targeted biodegradation applications.
In the healthy retina, microglial cells represent a self-renewing population of innate immune cells, which constantly survey their microenvironment. Equipped with receptors, a microglial cell detects subtle cellular damage and rapidly responds with activation, migration, and increased phagocytic activity. While the involvement of microglial cells has been well characterized in monogenic retinal disorders, it is still unclear how they contribute to the onset of retinal aging disorders including age-related macular degeneration (AMD). There is evidence, that microglial activation is not solely a secondary manifestation of retinal tissue damage in age-related disorders. Thus, work in the aging rodent and human retina suggests that long-lived and genetically predisposed microglia transform into a dystrophic state, with loss of neuroprotective functions. In this concept, malfunction of aging microglia can trigger a chronic low-grade inflammatory environment that favors the onset and progression of retinal degeneration.
Significance:
Inflammatory diseases (such as arthritis) of the extracellular matrix (ECM) are of considerable socioeconomic significance. There is clear evidence that reactive oxygen species (ROS) and nitrogen species released by, for instance, neutrophils contribute to the degradation of the ECM. Here we will focus on the ROS-induced degradation of the glycosaminoglycans, one important component of the ECM.
Recent advances:
The recently developed "anti-TNF-α" therapy is primarily directed against neutrophilic granulocytes that are powerful sources of ROS. Therefore, a more detailed look into the mechanisms of the reactions of these ROS is reasonable.
Critical issues:
Since both enzymes and ROS contribute to the pathogenesis of inflammatory diseases, it is very difficult to estimate the contributions of the individual species in a complex biological environment. This particularly applies as many products are not stable but only transient products that decompose in a time-dependent manner. Thus, the development of suitable analytical methods as well as the establishment of useful biomarkers is a challenging aspect.
Future directions:
If the mechanisms of ECM destruction are understood in more detail, then the development of suitable drugs to treat inflammatory diseases will be hopefully much more successful.
Extracellular oxygen concentrations affect cellular metabolism and influence tissue
function. Detection methods for these extracellular oxygen concentrations currently have
poor spatial resolution and are frequently invasive. Fluorescence Lifetime Imaging
Microscopy (FLIM) offers a non-invasive method for quantifying local oxygen
concentrations. However, existing FLIM methods also show limited spatial resolution >1 μm and low time-resolved accuracy and precision, due to widefield time-gate.
This study describes a new optimised approach using FLIM to quantity
extracellular oxygen concentration with high accuracy ( ±7 μmol/kg) and spatial resolution
( ≅ 0.3 μm). An oxygen sensitive fluorescent dye, tris(2,2 ′-bipyridyl)ruthenium(II) chloride
hexahydrate [Ru(bipy)3]+2, was excited with a multi-photon laser and fluorescence lifetime
was measured using time-correlated single photon counting (TCSPC). The system was fully
calibrated with optimised techniques developed for avoiding artefacts associated with
photon pile-up and phototoxicity, whilst maximising spatial and temporal resolution. An
extended imaging protocol (1800 sec) showed no phototoxic effects on cells at dye
concentrations of <0.4 mM. Extracellular spatial oxygen gradients were identified around
isolated chondrocytes, seeded in three-dimensional agarose gel. The technique was
validated by regulating oxygen cellular consumption and thus confirming that the oxygen
gradient was governed by cellular consumption. The technique identified a subpopulation
of cells exhibiting statistically significant spatial oxygen gradients at the cell perihery. The
subpopulation was shown to be significantly larger in cell diameter correlating with what
that expected from chondrocytes in the deep zone. This technique provides an exciting
opportunity to non-invasively quantify pericellular spatial oxygen gradients from within
three-dimensional cellular constructs without prior manipulation of the cells. Thus by
examining cellular metabolisms it will advance our understanding of the optimal cellular
environment for tissue engineering and regenerative medicine.
Accumulation and fragmentation of hyaluronic (HA) accompanies the inflammatory changes in the periodontium and gingival crevicular fluid are involved in periodontitis, but the mechanism for this is unknown. Recently, three human hyaluronan-synthase (HAS1, 2, and 3) genes have been cloned and characterised as synthesising hyaluronans of different molecular weights. Both HAS1 and HAS2 synthesise high molecular-weight HA, whereas HAS3 produces lower molecular weight HA. In the present study the regulation of HAS genes by cytokines in cultured human periodontal ligament (PDL) cells was investigated using a novel real-time fluorescence polymerase chain reaction detection system. Human PDL cells derived from premolars were cultured with or without tumour necrosing factor (TNF)-α (1–100 ng/ml), interlukin (IL)-1β (0.1–10 ng/ml) and interferon (IFN)-γ (1–100 ng/ml). Expression of HAS mRNA was assessed in cultured cells treated with these cytokines for 0–24 h. The expression of HAS2 mRNA was enhanced about 4.5- and 2.2-fold at maximum after 3-h stimulation with 10 ng/ml TNF-α and 1 ng/ml IL-1β, respectively, whereas IFN-γ exerted little effect on HAS2 or HAS3 mRNA expression during the experiment. Expression of HAS3 mRNA was increased by about 14- and 10-fold after 3-h stimulation with 10 ng/ml TNF-α and 1 ng/ml IL-1β, respectively. These results suggest that TNF-α and IL-1β regulate HAS expression, and consequently may result in an accumulation of HA and an increase in HA of a lower molecular-weight.
Microglia are the resident immune cells of the central nervous system (CNS) and perform typical scavenging and innate immune functions. Their capacity to eliminate extracellular aggregates and apoptotic neural material without inflammation is crucial for brain tissue homeostasis and repair. To fulfill these tasks, microglia express a whole set of recognition receptors including toll-like (TLRs), carbohydrate-binding, Fc, complement and cytokine receptors. Receptors recognizing carbohydrate structures are strongly involved in microglial repair function. Carbohydrate-binding receptors can be divided into two major subgroups: the sulfated glycosaminoglycan (SGAG)-binding receptors and the lectins (Siglecs, galectins, selectins). SGAG-binding receptors recognize anionic structural motifs within extended SGAG chains. Siglecs bind to the sialic acid cap of the intact glycocalyx. Other lectin family members such as galectins recognize lactosamine units typically exposed after alteration of the glycocalyx. Dependent on the type of microglial carbohydrate-binding receptors that are stimulated, either a pro-inflammatory cytotoxic or an anti-inflammatory repair-promoting response is evoked. The carbohydrate-binding receptors are also crucial in regulating microglial function such as phagocytosis during neurodegenerative or neuroinflammatory processes. A balance between microglial carbohydrate-binding receptor signaling via an immunoreceptor tyrosine-based activation motif or an immunoreceptor tyrosine-based inhibitory motif is required to polarize microglial cells appropriately so that they create a microenvironment permissive for neural regenerative events.
Due to an increasing life expectance, osteoarthritis (OA) is one of the most common chronic diseases. Although strong efforts have been made to regenerate degenerated joint cartilage, OA is a progressive and irreversible disease up to date. Among other factors the dysbalance between free radical burden and cellular scavenging mechanisms defined as oxidative stress is a relevant part of OA pathogenesis. Here, only little data are available about the mediation and interaction between different joint compartments. The article provides a review of the current literature regarding the influence of oxidative stress on cellular aging, senescence and apoptosis in different joint compartments (cartilage, synovial tissue and subchondral bone). Free radical exposure is known to promote cellular senescence and apoptosis. Radical oxygen species (ROS) involvement in inflammation, fibrosis control and pain nociception has been proven. The data from literature indicates a link between free radical burden and OA pathogenesis mediating local tissue reactions between the joint compartments. Hence, oxidative stress is likely not only to promote cartilage destruction but also to be involved in inflammative transformation, promoting the transition from clinically silent cartilage destruction to apparent OA. ROS induced by exogenous factors such as overload, trauma, local intraarticular lesion and consecutive synovial inflammation cause cartilage degradation. In the affected joint, free radicals mediate disease progression. The interrelationship between oxidative stress and OA etiology might provide a novel approach to the comprehension and therefore modification of disease progression and symptom control.
Osteoarthritis (OA) is one of the most common chronic diseases, with increasing importance due to increased life expectancy. On a cellular level, the pathophysiology of joint function impairment and ultimate destruction associated with OA remains poorly understood. Free radicals are highly reactive molecules involved in both normal intracellular signal transduction and degenerative cellular processes. An imbalance between the free radical burden and cellular scavenging mechanisms, defined as oxidative stress, has been identified as a relevant factor in OA pathogenesis. This literature review elucidates the involvement of nitrosative and oxidative stress in cellular ageing in joints, cell senescence, and apoptosis. Free radical exposure is known to promote cellular senescence and apoptosis, and the involvement of radical oxygen species (ROS) in inflammation, fibrosis control, and pain nociception has been proven. A relatively novel approach to OA pathophysiology considers the joint to be a dynamic system consisting of 3, continuously interacting compartments, cartilage, synovial tissue, and subchondral bone. Current knowledge concerning free radical involvement in paracrine signalling in OA is reviewed. The interrelationship between oxidative imbalances and OA pathophysiology may provide a novel approach to the comprehension, and therefore modification, of OA disease progression and symptom control.
Synovial fluid is a approximately 0.15% (w/v) aqueous solution of hyaluronic acid (HA), a polysaccharide consisting of alternating units of GlcA and GlcNAc. In synovial fluid of patients suffering from rheumatoid arthritis, HA is thought to be degraded either by radicals generated by Fenton chemistry (Fe2+/H2O2) or by NaOCl generated by myeloperoxidase. We investigated the course of model reactions of these two reactants in physiological buffer with HA, and with the corresponding monomers GlcA and GlcNAc. meso-Tartaric acid, arabinuronic acid, arabinaric acid and glucaric acid were identified by GC-MS as oxidation products of glucuronic acid. When GlcNAc was oxidised, erythronic acid, arabinonic acid, 2-acetamido-2-deoxy-gluconic acid, glyceric acid, erythrose and arabinose were formed. NaOCl oxidation of HA yielded meso-tartaric acid; in addition, arabinaric acid and glucaric acid were obtained by oxidation with Fe2+/H2O2. These results indicate that oxidative degradation of HA proceeds primarily at glucuronic acid residues. meso-Tartaric acid may be a useful biomarker of hyaluronate oxidation since it is produced by both NaOCl and Fenton chemistry.
The glomerular basement membrane (GBM) is a kind of net that remains in a state of dynamic equilibrium. Heparan sulfate proteoglycans (HSPGs) are among its most important components. There are much data indicating the significance of these proteoglycans in protecting proteins such as albumins from penetrating to the urine, although some new data indicate that loss of proteoglycans does not always lead to proteinuria. Heparanase is an enzyme which cleaves beta 1,4 D: -glucuronic bonds in sugar groups of HSPGs. Thus it is supposed that heparanase may have an important role in the pathogenesis of proteinuria. Increased heparanase expression and activity in the course of many glomerular diseases was observed. The most widely documented is the significance of heparanase in the pathogenesis of diabetic nephropathy. Moreover, heparanase acts as a signaling molecule and may influence the concentrations of active growth factors in the GBM. It is being investigated whether heparanase inhibition may cause decreased proteinuria. The heparanase inhibitor PI-88 (phosphomannopentaose sulfate) was effective as an antiproteinuric drug in an experimental model of membranous nephropathy. Nevertheless, this drug is burdened by some toxicity, so further investigations should be considered.
A novel biopolymer-based antioxidant, chitosan conjugated with gallic acid (chitosan galloylate, chitosan-GA), is proposed. Electron paramagnetic resonance (EPR) demonstrates a wide range of antioxidant activity for chitosan-GA as evidenced from its reactions with oxidizing free radicals, that is, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), horseradish peroxidase (HRP)-H(2)O(2), carbon-centered alkyl radicals, and hydroxyl radicals. The EPR spectrum of the radical formed on chitosan-GA was attributed to the semiquinone radical of the gallate moiety. The stoichiometry and effective concentration (EC(50)) of the DPPH free radical with chitosan-GA show that the radical scavenging capacity is maintained even after thermal treatment at 100 degrees C for an hour. Although the degree of substitution of GA on chitosan was about 15%, its antioxidant capacity, that is, the reaction with carbon-centered and hydroxyl radicals, is comparable to that of GA.
Glycosaminoglycans (long-chain polysaccharides) are major components of the extracellular matrix, glycocalyx, and synovial fluid. These materials provide strength and elasticity to tissues and play a key role in regulating cell behavior. Modifications to these materials have been linked to multiple human pathologies. Although modification may occur via both enzymatic and nonenzymatic mechanisms, there is considerable evidence for oxidant-mediated matrix damage. Peroxynitrite (ONOO(-)/ONOOH) is a potential mediator of such damage, as elevated levels of this oxidant are likely to be present at sites of inflammation. In this study we demonstrate that hyaluronan and chondroitin sulfate are extensively depolymerized by HO(.) and CO3(.-), but not NO2(.), which may be formed from peroxynitrite. Polymer fragmentation is shown to be dependent on the radical flux, to be O2-independent, and to occur in a site-selective manner as indicated by the detection of disaccharide fragments. EPR spin trapping experiments with polymers, oligomers, and component monosaccharides, including 13C-labeled materials, have provided evidence for the formation of specific carbon-centered sugar-derived radicals. The time course of formation of these radicals is consistent with these species being involved in polymer fragmentation.
The molecular events occurring at the interface between titanium and connective tissue were investigated in order to help explain the unique biocompatible properties of titanium implants and their successful osseointegration into bone tissue. In this study the influence of commercially pure titanium and titanium peroxy gels on the breakdown of the connective tissue component and serum derived factor, hyaluronan, by reactive oxygen species (ROS), produced during the insertion of an implant in vivo, was examined. Hyaluronan breakdown was monitored in vitro in the presence of a hydroxyl radical flux, generated in the presence and absence of titanium powder and discs. Parallel studies examined the breakdown of hyaluronan by hydroxyl radicals in the presence of a titanium peroxy gel, prepared by incubation of the titanium powder or discs in concentrated hydrogen peroxide. The hyaluronan degradation products were separated according to their hydrodynamic size by gel exclusion chromatography. Similarly, experiments were also performed examining the degradation of 2-deoxy-D-ribose by a hydroxyl radical flux in order to demonstrate the detrimental potential of the hydroxyl radicals and to provide a measure of the effectiveness of titanium and titanium peroxy gels as scavengers of ROS. Titanium reduced the harmful effects of the hydroxyl radicals on the breakdown of hyaluronan, presumably acting as a scavenger for the reactive species, possibly by absorbing them into its surface oxide layer, which spontaneously forms on the surface. In contrast, the formation of a titanium peroxy gel from the titanium powder or on the surface of titanium discs enhanced breakdown of both the hyaluronan chains and 2-deoxy-D-ribose. The implications of these findings with regards to the biocompatible nature of the titanium and the ability of these implants to successfully osseointegrate are discussed.
Treatment of high-molecular-weight hyaluronan (HA) with peroxynitrite at neutral pH (ONOO-/ONOOH) results in altered mobility on agarose gel electrophoresis, as well as reduced limiting viscosity number. Both effects are consistent with a reduction in HA molecular weight. HA is protected from peroxynitrite attack to varying extents by addition of alternate target molecules. Thiourea is extremely effective as a protective agent, dimethyl sulfoxide is moderately effective, while sodium benzoate and mannitol are slightly effective. A similar pattern of protection is observed when HA is degraded by hydroxyl radical generated by a metal ion/hydrogen peroxide system. On the basis of these observations, peroxynitrite is proposed to have hydroxyl radical-like activity in degrading HA.
Reactive oxygen species (ROS) are produced and released into the extracellular spaces in numerous diseases and contribute to development and progression, for example, of inflammatory diseases, proteinuria, and tumor invasion. However, little is known about ROS-induced chemical changes of interstitial matrix proteins and their consequences for the integrity of the matrix meshwork. As basement membranes and other matrices are highly cross-linked and complex, the relatively simple matrix produced by Engelbreth-Holm-Swarm (EHS) sarcoma, and proteins isolated therefrom, were incubated in vitro with defined concentrations of ROS that were generated by the Fenton or xanthine oxidase/xanthine reactions. This resulted in two counter-current effects. Although up to approximately 15% of the EHS matrix proteins were released into the supernatant in a ROS dose-response relationship, the residual insoluble matrix was partially cross-linked by ROS. Matrix proteins released into the supernatants were examined by rotary shadowing, quantitative sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting, and fluorospectrometry for loss of tryptophans and formation of bityrosine residues. At relatively low ROS concentrations, selective liberation of morphologically intact laminin/entactin was found that, however, failed to reassociate and showed oxidative damage of its tryptophan residues. At higher ROS concentrations, laminin and entactin were progressively disintegrated, partially fragmented, and eventually completely degraded. At this point oligomers of type IV collagen predominated in the supernatant, and proteoglycans were not encountered at any concentration of ROS. Similar gradual molecular changes were also obtained when fractions of isolated soluble EHS matrix proteins were incubated with graded concentrations of ROS. In these experiments, the formation of covalently linked oligomers and aggregates paralleled the ROS-dependent formation of cross-linking bityrosine groups. ROS scavengers pinpointed to the hydroxyl radical as the most damaging radical species. Protease inhibitor experiments suggested that degradation of matrix proteins was caused primarily by the direct action of ROS and not by proteolysis by potentially contaminating proteases. Collectively, these results provide evidence that EHS matrix proteins show differential sensitivity to ROS-induced damage in a reproducible, sequential pattern, in the order entactin > laminin > type IV collagen, and that ROS cause partial dissociation and cross-linking of the EHS matrix.
Heparan sulfate, the polysaccharide side chain of heparan sulfate proteoglycan, is important for the permselective properties of the glomerular basement membrane. In this report, we show a role for hydroxyl radicals in heparan sulfate degradation and an enhanced glomerular basement membrane permeability. First, in enzyme-linked immunosorbent assay, exposure of coated heparan sulfate (proteoglycan) to reactive oxygen species resulted in a +/-50% decrease of binding of a monoclonal antibody against heparan sulfate, whereas binding of an antibody against the core protein remained unaltered. Second, on polyacrylamide gel electrophoresis, the molecular weight of heparan sulfate exposed to radicals was reduced which indicates depolymerization. Both in enzyme-linked immunosorbent assay and gel electrophoresis, hydroxyl radicals are instrumental for heparan sulfate degradation as shown by the addition of various radical scavengers. Third, in an experimental model for human nephrotic syndrome (Adriamycin nephropathy in rats), glomerular basement membrane staining of two recently described anti-heparan sulfate antibodies (JM403 and KJ865) was reduced by 24 and 43%. Treatment of Adriamycin-exposed rats with the hydroxyl radical scavenger dimethylthiourea both reduced albuminuria by 37% (p < 0.01) and partly prevented loss of heparan sulfate staining by 53% (JM403) and 39% (KJ865) (p < 0.03). In contrast to the heparan sulfate side chains, the core protein expression and the extent of glycanation did not change in Adriamycin nephropathy. We conclude that glomerular basement membrane heparan sulfate is susceptible to depolymerization by hydroxyl radicals leading to loss of glomerular basement membrane integrity and albuminuria.
The effect of reactive oxygen species (ROS), generated by in vitro stimulation of isolated PMN upon the main GAG components of mineralised and non-mineralised connective tissues was investigated. PMN were isolated from whole blood and the production of the ROS superoxide (O2.-) and hydroxyl radicals (.OH) was stimulated by the addition of phorbol myristyl acetate (PMA) and PMA/FeCl3-EDTA chelate respectively and their production assessed over a 24 h period. The glycosaminoglycans (GAG), hyaluronan, chondroitin 4-sulphate and dermatan sulphate, were exposed to the ROS fluxes, incubated at 37 degrees C for 1 h and 24 h. GAG fragmentation was examined by gel exclusion chromatography and modification to hexuronic acid and hexosamine residues determined. Stimulation of PMN with PMA resulted in a burst of O2.- production for 1 h, which was sustained at a reduced level for 24 h. Fragmentation of GAG was observed for all GAG examined. Modification to the GAG was evident, with hyaluronan being more susceptible to loss of GAG residues than sulphated GAG. Modification of sugar residues increased with the incubation time and loss of the hexuronic acid residues was greater than loss of hexosamine residues. Addition of FeCl3-EDTA chelate, which led to the generation of .OH and was sustained over the 24 h period, demonstrated similar trends of GAG modification although increased degradation and loss of hexosamine and hexuronic acid were observed. GAG chains are constituents of PGs and their modification is likely to affect the function of these macromolecules and be of importance in considering the pathogenesis of inflammatory diseases, including periodontal diseases.
Reactive oxygen species (ROS) are being increasingly implicated in the connective tissue degradation associated with chronic inflammatory conditions, such as periodontal disease. The present study investigated the effects of ROS on the proteoglycans (PG) of alveolar bone which are important structural components within the periodontium. PG were isolated from ovine alveolar bone and exposed to increasing concentrations of hydrogen peroxide (H2O2) or to a hydroxyl radical (.OH) flux for 1 h or 24 h, and the degradation products examined for depolymerisation and chemical modification of the PG structure. ROS were demonstrated to be capable of degrading alveolar bone PG in vitro, the .OH species resulting in greater modification than H2O2. The degradative effects observed included cleavage of the protein core and depolymerisation of the GAG chains. The core proteins were more susceptible to degradation than the GAG chains in the presence of H2O2 alone, although both the core proteins and the GAG chains were extensively degraded in the presence of a .OH flux for both 1 h and 24 h. Exposure of the PG to .OH for 24 h resulted in significant modification to the amino acid composition with decreases in the proportion of leucine and the complete loss of proline, tyrosine and phenylalanine evident. The results highlight the potential role of ROS as an important mechanism in considering the pathology of periodontal tissue destruction.
Using chondroitin as a substrate, a new type of exo-beta-glucuronidase (EC 3.2.1.31) from rabbit liver was purified using a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, gel filtration on Sephracryl S-300, affinity chromatography through heparin-Sepharose CL-6B, and preparative polyacrylamide gel electrophoresis. This enzyme acts only on non-sulfated glycosaminoglycans and their oligosaccharides and was shown to be quite different from exo-beta-glucuronidase, which does act on p-nitro-phenyl-beta-D-glucuronide with regard to the following properties. 1) Neither sulfated glycosaminoglycanoligosaccharides nor p-nitrophenyl-beta-D-glucuronide were substrates for the enzyme. 2) The molecular weight was found to be about 130,000 by gel filtration, compared with a molecular weight of 280,000-300,000 for beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 3) The enzyme showed maximal activity at pH 5.0, compared with an optimum pH of 4.5 for beta-glucuronidase, which acts on p-nitro-phenyl-beta-D-glucuronide. 4) The enzyme showed maximal activity in 0.075 M NaCl but no activity above 0.25 M NaCl. 5) The enzyme was inhibited strongly by compounds bearing a sulfate group. 6) The enzyme did not react with an antibody against beta-glucuronidase acting on p-nitrophenyl-D-glucuronide. It is suggested that the enzyme may be involved in the catabolism of glycosaminoglycans, acting especially on chondroitin after the desulfation reaction and/or hyaluronic acid, but showing little involvement with the detoxification system.
The oxidative reductive depolymerization (ORD) of hyaluronate has been investigated. A solution of hyaluronate (Mr 4.07 x 10(5] in phosphate buffer (pH 7.2) was incubated in the presence of Fe2+ for 24 h at 37 degrees C under an oxygen atmosphere to yield depolymerized hyaluronate (ORD fragments; an average Mr of 2,600). The ORD fragments contain 21 and 24% less hexosamine and uronic acid, respectively, but no olefinic linkage. They were exhaustively digested with chondroitinase AC-II. The resulting oligosaccharides and monosaccharides were separated by gel filtration and ion-exchange chromatography, and their structures were determined by proton and carbon-13 NMR, fast atom bombardment mass spectrometry, and chromatographic techniques combined with chemical modifications. The following structures derived from the reducing ends of the ORD fragments were identified: 4,5-unsaturated GlcA(beta 1----3)-N-acetyl-D-glucosaminic acid (where GlcA- represents glucuronosyl-) (21%), 4,5-unsaturated GlcA(beta 1----3)GlcNAc(beta 1----3)-D-arabo-pentauronic acid (24%), and N-acetyl-D-glucosamine (51%). The following structures derived from the nonreducing ends were identified: L-threo-tetro-dialdosyl-(1----3)GlcNAc (a tentative structure, 8%), N-acetylhyalobiuronic acid (20%), and N-acetyl-D-glucosamine (45%). The results indicate that the ORD reaction of hyaluronate proceeds essentially by random destruction of unit monosaccharides due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable glycosidic substituents.
The small proteoglycans (PG) of bone consist of two different molecular species: one containing one chondroitin sulfate chain (PG II) and the other, two chains (PG I). These two proteoglycans are found in many connective tissues and have Mr = 45,000 core proteins with clear differences in their NH2-terminal sequences. Using antisera produced against synthetic peptides derived from the human PG I and PG II NH2 termini, we have isolated several cDNA clones from a lambda gt11 expression library made against mRNA isolated from human bone-derived cells. The clones, which reacted with antisera to the PG II peptide, were sequenced and found to be identical with the PG II class of proteoglycan from human fibroblasts known as PG-40 or decorin. The clones reacting to the PG I antisera, however, had a unique sequence. The derived protein sequence of PG I showed sufficient homology with the PG II sequence (55% of the amino acids are identical, with most others involving chemically similar amino acid substitutions) to strongly suggest that the two proteins were the result of a gene duplication. PG II (decorin) contains one attached glycosaminoglycan chain, while PG I probably contains two chains. For this reason, we suggest that PG I be called biglycan. The biglycan protein sequence contains 368 residues (Mr = 42,510 for the complete sequence and Mr = 37,983 for the secreted form) that appears to consist predominantly of a series of 12 tandem repeats of 24 residues. The repeats are recognized by their conserved leucines (and leucine-like amino acids) in positions previously reported for a diverse collection of proteins (none of which is thought to be proteoglycans) including: two morphogenic proteins (toll and chaoptin) in the fruit fly; a yeast adenylate cyclase; and two human proteins, the von Willebrand Factor-binding platelet membrane protein, GPIb, and a rare serum protein, leucine-rich glycoprotein.
We have previously shown that treatment of neonatal human articular-cartilage proteoglycan aggregates with H2O2 results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid and in fragmentation of the link proteins [Roberts, Mort & Roughley (1987) Biochem. J. 247, 349-357]. We now show the following. (1) Hyaluronic acid in proteoglycan aggregates is also fragmented by treatment with H2O2. (2) Although H2O2 treatment results in loss of the ability of the proteoglycan subunits to interact with hyaluronic acid, the loss of this function is not attributable to substantial cleavage of the hyaluronic acid-binding region of the proteoglycan subunits. (3) In contrast, link proteins retain the ability to bind to hyaluronic acid following treatment with H2O2. (4) The interaction between the proteoglycan subunit and link protein is, however, abolished. (5) N-Terminal sequence analysis of the first eight residues of the major product of link protein resulting from H2O2 treatment revealed that cleavage occurred between residues 13 and 14, so that the new N-terminal amino acid is alanine. (6) In addition, a histidine (residue 16) is converted into alanine and an asparagine (residue 21) is converted into aspartate by the action of H2O2. (7) Rat link protein showed no cleavage or modifications in similar positions under identical conditions. (8) This species variation may be related to the different availability of histidine residues required for the co-ordination of the transition metal ion involved in hydroxyl-radical generation from H2O2. (9) Changes in function of these structural macromolecules as a result of the action of H2O2 may be consequences of both fragmentation and chemical modification.
Two heparin families (SM- and FM- heparins) have been fractionated with barium by selective precipitation at different temperatures. Fragments were obtained from these two fractions by enzymic and chemical degradation. Some structural features and pharmacological activities of FM- and SM-heparins as well as the fragments are now reported. The fragments, prepared by ascorbate/H2O2 oxidation (M.W. 4,500) or heparitinase II (M.W. 5,500), are enriched with trisulfated disaccharide units whereas heparin (M.W.12,500) and FM-heparin (M.W. 10,200) contain also N-sulfated and N-acetylated disaccharides. The chemical and enzymic fragments show 1/3 to 1/5 of the anticoagulant activity of heparin by the USP and APTT methods. The LPL releasing activity is also low in the fragments as well as the AXa activity measured by the chromogenic method. On the other hand the AXa activity measured by the Yin and Wessler procedure is two times higher in the fragments when compared to heparin. The fragments resulting from heparin after heparitinase II degradation and ascorbate/H2O2 oxidation were rich in trisulfated disaccharide units whereas heparin and FM-heparin contained also measurable amounts of monosulfated and N-acetylated disaccharide units. The mechanism of ascorbate oxidation and the structural requirements for the pharmacological activities of heparin is discussed in view of these and other findings.
Enzymatically genierated superoxide radical. by reactitng with hydrogen peroxide to prduce the hydroxyl radical, depolymerized
puirified hyaluronic acid and bovine synovial flulid. Since phagocytizing polymorphonuclear leukocytes produce superoxide
radicals, this reac-tion is sutggested and shown to be quantitatively feasible as the ini vivo mechanism of synovial fluid
degradation in anl inflamed joint. Superoxide dismutase, and catalase protect synaovial fluid against such degradation in
vitro.
Proteoglycans deposited in the basal lamina of [14C] glucosamine-labeled normal and [3H]glucosamine-labeled transformed mouse mammary epithelial cells grown on type I-collagen gels, were extracted in 4 M guanidinium chloride and cofractionated over Sepharose CL 4B. The heparan sulfate chains carried by these proteoglycans were isolated by treatment with alkaline borohydride, protease K, chondroitinase ABC, and cetylpyridinium chloride precipitation. Heparan sulfate isolated from transformed cell cultures consistently eluted from DEAE-cellulose at lower salt concentrations and was of smaller apparent Mr when chromatographed over Sepharose CL 6B, than heparan sulfate of normal cell cultures. Experiments using doubly labeled cultures ([3H]glucosamine and [35S]sulfate) demonstrated an approximately 30% reduction in the sulfate/hexosamine ratio in heparan sulfate derived from transformed cultures. Both N- and O-sulfate were decreased. The decreased Mr and decreased sulfation of heparan sulfate upon transformation appear sufficient to explain the altered heparan sulfate/chondroitin sulfate ratios previously observed in these cells. These changes may have implications for the molecular interactions in which these proteoglycans are normally engaged during basal lamina assembly, and cause the poor basal lamina formation displayed by these transformed cells.
Superoxide anion, a highly reactive free radical, was generated in vitro using enriched purified xanthine oxidase. Collagen solutions exposed to superoxide radical failed to gel normally when heated to 37 degrees C. The magnitude of the inhibition of gelation was propotional to duration of exposure and to flux of superoxide. Since inhibition of collagen gelation reflects alteration of collagen biochemistry, and/or collagen degradation, it is suggested that the action of free radicals produced in vivo by leukocytes may adversely affect the structural or functional integrity of cartilage and adjacent joint structures.
Changes in the serum levels of immunoglobulin IgA, IgG, IgM, positive acute phase proteins and complement activity, as well as the lymphocyte subpopulations and the neutrophil leucocyte-related chemiluminescence in the blood of patients with apical granuloma as related to endodontic and surgical treatment were investigated. Measurements were performed on admission, and 7 days and 3 months after the treatment. Elevated IgM concentration, positive acute-phase protein levels and spontaneous whole-blood chemiluminescence were noted at admission. However, a significant decrease in the serum level of each of the six investigated acute-phase proteins, and in the spontaneous chemiluminescence of blood was observed during the 3-month follow-up period. The significant increase in serum complement activity following therapy suggests that complement fixation might have occurred in these patients. A significant increase in the ratio of early sheep erythrocyte rosette-forming lymphocytes was also observed. The results of this study provide evidence for complete recovery after elimination of local inflammation by proper endodontic treatment and apicectomy in patients with apical granuloma.
A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.
In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O2- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myristate acetate (PMA) decreased the O2-. formation in control GCF, while in CAP patients produced a marked enhancement of O2-. generation. The circulating PMN of control subjects did not show a spontaneous O2-. formation, differently from CAP patients. On the contrary, a similar O2-. production was measured when the circulating PMN were stimulated with PMA. Moreover, the antioxidant activity measured in 10 microliters of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O2- formation supported by a xanthine/xanthine oxidase system. Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O2- formation in respect to the intrinsic antioxidant property of GS.
The role of bacteria in the initiation of periodontitis is well-documented and the end result, destruction of the alveolar bone and periodontal connective tissue, is readily observed; but the events occurring between these two points in time remain obscure and are the focus of this paper. Bacteria induce tissue destruction indirectly by activating host defense cells, which in turn produce and release mediators that stimulate the effectors of connective tissue breakdown. Components of microbial plaque have the capacity to induce the initial infiltrate of inflammatory cells including lymphocytes, macrophages, and PMNs. Microbial components, especially lipopolysaccharide (LPS), have the capacity to activate macrophages to synthesize and secrete a wide array of molecules including the cytokines interleukin-1 (IL-1) and tumor-necrosis factor-alpha (TNF-alpha), prostaglandins, especially PGE2, and hydrolytic enzymes. Likewise, bacterial substances activate T lymphocytes and they produce IL-1 and lymphotoxin (LT), a molecule having properties very similar to TNF-alpha. These cytokines manifest potent proinflammatory and catabolic activities, and play key roles in periodontal tissue breakdown. They induce fibroblasts and macrophages to produce neutral metalloproteinases such as procollagenase and prostromelysin, the serine proteinase urokinase-type plasminogen activator (u-PA), tissue inhibitor of metalloproteinase (TIMP), and prostaglandins, u-PA converts plasminogen into plasmin, which can activate neutral metalloproteinase proenzymes, and these enzymes degrade the extracellular matrix components. TIMP inactivates the active enzymes and thereby blocks further tissue degradation. Several amplification and suppression mechanisms are involved in the process. While LPS activates macrophages to produce IL-1, IL-1 is autostimulatory and can therefore amplify and perpetuate its own production. Interferon-gamma (INF-gamma) suppresses autostimulation, but it enhances LPS-induced IL-1 production. PGE2 exerts a control over the whole process by suppressing production of both IL-1 and TNF-alpha. Furthermore, the activated cells produce an IL-1 receptor antagonist that binds to the IL-1 receptor but does not induce the biologic consequences of IL-1 binding. Other cytokines such as transforming growth factor-beta (TGF-beta) suppress production of metalloproteinases and u-PA. Thus the progression and extent of tissue degradation is likely to be determined in major part by relative concentrations and half-life of IL-1, TNF-alpha, and related cytokines, competing molecules such as the IL-1 receptor antagonist, and suppressive molecules such as TGF-beta and PGE2. These molecules control levels of latent and active metalloproteinase and u-PA, and the availability and concentration of TIMP determines the extent and duration of degradative activity.
Proteoglycans were extracted from EDTA-demineralized human alveolar bone under dissociative conditions using 4 M guanidinium chloride in the presence of protease inhibitors. The extract was further purified by anion-exchange chromatography on DEAE-Sephacel, using a step-wise salt gradient. The proteoglycan-rich fraction was analysed for carbohydrate, protein and amino acid composition and molecular size by SDS-PAGE. Glycosaminoglycan content was determined by cellulose acetate electrophoresis after proteolysis. The sulphate isomers of the glycosaminoglycans were confirmed by Fourier-transformed infra-red spectroscopy. Two chondroitin sulphate-proteoglycan species were identified with molecular weights of 79 and 55-65 kDa, respectively. The core proteins had molecular weights of 49 kDa for both proteoglycans, with the amino acid content rich in glycine, leucine, glutamate and aspartate. The chondroitin sulphate chains were mainly as the 4-sulphate isomer forms although low but detectable amounts of 6-sulphate isomer were also present.
The proteinase inhibitory ability of alpha 1 antitrypsin was measured in 23 samples of rheumatoid arthritis synovial fluid, eight osteoarthritic synovial fluids and nine normal control serum samples. For each sample a detailed kinetic analysis was performed with porcine pancreatic elastase as the target proteinase. Samples were stored for less than 24 hours at 4 degrees C before analysis, which does not significantly alter the proportion of inactive alpha 1 antitrypsin. In rheumatoid synovial fluid the elastase inhibitory ability was disproportionately depressed relative to the immunochemically determined concentrations of alpha 1 antitrypsin.
Uric acid is an end-product of purine metabolism in Man, and has been suggested to act as an antioxidant in vivo. Products of attack upon uric acid by various oxidants were measured by high performance liquid chromatography. Hypochlorous acid rapidly oxidized uric acid, forming allantoin, oxonic/oxaluric and parabanic acids, as well as several unidentified products. HOCl could oxidize all these products further. Hydrogen peroxide did not oxidize uric acid at detectable rates, although it rapidly oxidized oxonic acid and slowly oxidized allantoin and parabanic acids. Hydroxyl radicals generated by hypoxanthine/xanthine oxidase or Fe2(+)-EDTA/H2O2 systems also oxidized uric acid to allantoin, oxonic/oxaluric acid and traces of parabanic acid. Addition of ascorbic acid to the Fe2(+)-EDTA/H2O2 system did not increase formation of oxidation products from uric acid, possibly because ascorbic acid can 'repair' the radicals resulting from initial attack of hydroxyl radicals upon uric acid. Mixtures of methaemoglobin or metmyoglobin and H2O2 also oxidized uric acid: allantoin was the major product, but some parabanic and oxonic/oxaluric acids were also produced. Caeruloplasmin did not oxidize uric acid under physiological conditions, although simple copper (Cu2+) ions could, but this was prevented by albumin or histidine. The possibility of using oxidation products of uric acid, such as allantoin, as an index of oxidant generation in vivo in humans is discussed.
Gingival crevicular fluid is regarded as a promising medium for the detection of markers of periodontal diseases activity. The collection protocols are straight forward and non-invasive and can be performed at specific sites of interest in the periodontium. Because the fluid accumulates at the gingival margin, it will contain potential markers derived not only from the host tissues and serum but also the subgingival microbial plaque, and thus an extremely broad range of candidate molecules may be investigated. However, the ability to successfully describe indicators of current disease activity and predictors of future disease is dependent not only upon the choice of the biochemical marker but also on the accurate description of the health status of the sample sites using currently available clinical and radiographic methods. Areas of study which currently show the most promise involve the analysis of host enzyme activities directed against components of the extracellular matrix, the nature of the glycosaminoglycans released into the sulcus and the concentration in gingival crevicular fluid of certain mediators of the inflammatory process, most notably prostaglandin E2.
Patients with localized juvenile periodontitis (LJP) exhibit defective neutrophil functions to a variety of environmental and host stimuli. It is not clear, however, how many of the measurable functions are defective and whether individual patients exhibit single or multiple dysfunctions. The purpose of this study was to evaluate chemotaxis, phagocytosis, specific granule release and superoxide production in a group of 23 previously unreported LJP patients. Our results indicate that all 23 of these LJP patients exhibited chemotaxis depression to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and endotoxin-activated serum (EAS). Smaller groups from the 23 chemotactically defective LJP group were used to test other function due to inability to obtain sufficient quantities of blood. Fourteen of 14 LJP patients tested exhibited defective phagocytosis. Ten LJP patients were evaluated for specific granule release, and 14 LJP patients were evaluated for superoxide production. Both granule release and superoxide production were found to be normal in chemotactically defective LJP patients. Since both defective and normal responses noted in the same neutrophil populations are mediated by the same receptor, it is hypothesized that the cellular defect lies in a post receptor pathway.
Proteoglycans have been isolated and analysed from extracts of normal and chronically inflamed human gingiva in order to determine the effects of chronic inflammation on these important soft connective tissue extracellular macromolecules. The uronic acid content of glycosaminoglycans isolated by papain digestion of normal and inflamed gingiva did not differ significantly. Likewise, electrophoretic analysis revealed that the content of hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate was similar. The sulfated glycosaminoglycans from both sources eluted from a Sepharose C1-6B column with a Kav of 0.45 (approximate Mr 25,000). However, hyaluronic acid from normal gingiva was predominantly of a large size eluting in the void volume of a Sepharose. CL-6B column, while that isolated form inflamed tissue was mostly a small molecular weight species which eluted in the included volume of a Sepharose CL-6B column. Using dissociative conditions, intact proteoglycans could be more readily extracted from inflamed tissues (90% of the total tissue uronic acid) than from normal tissues where only 80% of the total tissue uronic acid was extractable. Even though DEAE-Sephacel ion-exchange chromatography revealed no differences in charge between normal and inflamed gingival proteoglycans, Sepharose CL-4B chromatography revealed more molecular size polydispersity in samples from inflamed tissue than from normal tissue. Taken together, these results indicate that while hyaluronic acid is depolymerized in inflamed tissue, no evidence of sulfated glycosaminoglycan degradation was found. Therefore, the most likely cause for disruption to the molecular integrity of the proteoglycans is via proteolytic alteration to the proteoglycan core protein.
In 12 patients with juvenile periodontitis (JP), stimulation of peripheral polymorphonuclear neutrophils (PMN) by opsonized bacteria showed increased release of free oxygen radicals and of elastase activity in relation to that of pair-matched healthy controls. The elastase increase was not associated with higher intracellular content of elastase, since the measurement of homogenized PMN cells did not differ between the patient group and the control group; nor did the spontaneous elastase activity of nonstimulated cells differ. Release of elastase by contaminating lymphocytes and platelets could be excluded, since no elastase activity from these cells or interference by these cells could be demonstrated in the assay system used. When formyl-methionyl-leucyl-phenylalanine was used as a stimulant, the chemiluminescence in the patient PMN cells did not differ with statistical significance from their pair-matched controls. The increased release of free oxygen radicals and of elastase seems to be a characteristic of the PMN cells in juvenile periodontitis, indicating hyperactive cells with possible pathogenic effects.
Proteoglycans were extracted with 4 M guanidinium chloride at 6 degrees C and purified by ion-exchange chromatography and precipitation with cetyl-pyridinium chloride. Chromatography on Sepharose CL-4B under dissociating conditions separated larger (PG1) and smaller (PG2) proteoglycans. Gingival PG2, by virtue of its amino-acid composition and the exclusive presence of L-iduronate-rich dermatan sulphate, was a proteodermatan sulphate (PDS) with a similar molecular weight to periodontal-ligament PDS. Reaction with four monoclonal antibodies to bovine skin PDS confirmed the relationship between these small proteoglycans and that of skin. Their glycoprotein cores, liberated by digestion with chondroitinase ABC, were similar in size (mol. wt = 55,000 by SDS-gel electrophoresis). Pulp PG2 had a small amount of PDS but the main component contained D-glucuronate-rich sulphated galactosaminoglycans. Similar galactosaminoglycans, which included chondroitin sulphate, characterized the larger proteoglycans of gingiva and pulp; significant amounts of L-iduronic acid-rich dermatan sulphate or heparan sulphate were not present.
This report describes a microprocedure that may be used for direct measurement of proteoglycans and glycosaminoglycans, after chromatographic elution with chaotropic reagents. The assay is based on the ability of the sulfated glycosaminoglycans to bind to the cationic dye, dimethylmethylene blue, in solution. Inclusion of guanidinium chloride (0.24 M) in the assay resulted in a stable dye-proteoglycan interaction, but eliminated the interference of other anionic macromolecules such as DNA. The assay is rapid, sensitive, and reproducible and therefore useful for processing several samples. Finally, the procedure can be used for quantitative determination of several types of proteoglycans and glycosaminoglycans.
The effect of a chemically induced oxygen-derived free radical flux has been studied on extracted porcine gingival hyaluronic acid and proteoglycans as well as on cryostat sections of porcine gingivae. The result of the free radical producing system on hyaluronic acid and the proteoglycans was one of reduction of specific viscosity and molecular size. When frozen sections were subjected to the same oxygen-derived free radical flux and subsequently stained with Alcian blue, a noticeable decrease in staining intensity was observed when compared to control sections. These results are considered to reflect the in vitro capacity of oxygen-derived free radicals to depolymerize the two major non-fibrous extracellular macromolecules of gingivae. It is postulated that such a mechanism may be responsible, at least in part, for the destruction of gingival proteoglycans and hyaluronic acid observed in inflammatory periodontal disease.
To investigate possible mechanisms of hyaluronic acid depolymerization, superoxide anion and other secondary oxygen-derived free radicals were generated in vitro and allowed to act upon a hyaluronate substrate. Superoxide, generated either enzymatically with xanthine oxidase or by stimulation of polymorphonuclear leukocytes, reduced the viscosity of hyaluronate solutions dramatically while the chromatographic profiles of the glycosaminoglycan shifted toward lower molecular weights. Superoxide-treated hyaluronate also became susceptible to further degradation by beta-N-acetylglucosaminidase A. Experiments with scavengers of various toxic oxygen-derived free radicals clearly implicated these reactants as mediators of hyaluronate depolymerization. Generation of superoxide by leukocytes in vivo may account for the loss of synovial fluid viscosity that accompanies inflammatory joint disease.
The proteoglycans characterized were those isolated from the calcified matrix of mature bovine bone [Franzén & Heinegård (1984) Biochem. J. 224, 47-58]. The average molecular mass of the bone proteoglycan is 74 600 Da, determined by sedimentation-equilibrium centrifugation in 4M-guanidinium chloride. Its sedimentation coefficient (s0(20),w) is 3.04 S. The apparent Mr of its core protein is 46 000, estimated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chondroitinase ABC-digested proteoglycan. A more likely molecular mass of the core protein is 30 000 Da, as calculated from the molecular mass and the protein content (40%) of the proteoglycan. The bone proteoglycan contains one or probably two chondroitin sulphate chains each with a molecular mass (weight-average) of 33 700 Da and several oligosaccharides both of the N-glycosidically and the O-glycosidically linked type. Antibodies against the homogeneous bone proteoglycans were raised in rabbits. An e.l.i.s.a. (enzyme-linked immunosorbent assay) method was developed that allowed specific quantification of bone proteoglycans at nanogram levels. The specificity of the antibodies was tested by using the e.l.i.s.a. method. The bone proteoglycan showed partial cross-reactivity with the small proteoglycan of cartilage. The antibodies were used to localize immunoreactivity of bone proteoglycans by indirect immunofluorescence in frozen sections of foetal bovine epiphysial growth plate. The fluorescence was entirely found in the primary spongiosa, and no fluorescence was found among the hypertrophied chondrocytes or in the region of provisional calcification.
Transmission infrared spectroscopy has been used to study some aspects of the mechanism of binding of chondroitin-4-sulphate to hydroxyapatite. The disappearance of absorption maxima characteristic of the covalently bound sulphate groups indicates that these groups may be involved in bridging the chondroitin sulphate molecule to the crystal lattice of the hydroxyapatite. The technique may be valuable, both in studying the formation of organic films on tooth surfaces and the mechanisms of endogenous mineralisation.
A proteoglycan purified from 4 M-guanidinium chloride extracts of bovine periodontal ligament closely resembled that of bovine skin, except for a rather lower protein content and a higher molecular weight (120 000 compared with about 90 000) by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The latter difference was explained by the molecular weights (29 000 and 16 000) of the respective dermatan sulphate components, each of which was rich in L-iduronate (about 75% of the total hexuronate). Significant amounts of other glycosaminoglycans did not occur in these proteoglycans, which were homogenous on gel chromatography and agarose/polyacrylamide-gel electrophoresis. Polydispersity was observed in sedimentation equilibrium experiments, but proteolysis or self-association of the proteodermatan sulphates may have affected these results. Ligament proteoglycans that were almost completely extracted with 0.1 M-NaCl contained less protein of a completely different amino acid composition than the proteodermatan sulphates. They were heterogeneous in size but generally smaller than cartilage proteoglycans and L-iduronate was a component, comprising about 7% of the total hexuronate of the sulphated galactosaminoglycan chains. The latter consisted of two fractions differing in molecular weight, but a dermatan sulphate with a high L-iduronate content was not present. These proteoglycans had some resemblance to D-glucuronate-rich proteoglycans of other non-cartilaginous tissues. Such compounds, however, are difficult to categorize at present.
The effect of reactive oxygen species (ROS) generated by a xanthine oxidase hypoxanthine system (mainly H2O2) on proteoglycan (PG) metabolism and structure was investigated in vitro, using cell monolayers of cultured rabbit articular chondrocytes and purified resident and newly synthesized proteoglycans. It was shown that ROS generated in this system frequently stimulate (at low concentrations), and consistently inhibit (at higher concentrations), the incorporation of 35SO4 and 3H-glucosamine into PG molecules synthesized by cultured chondrocytes. The inhibition of isotopes' incorporation at higher enzyme concentrations was suppressed completely by heating xanthine oxidase and allopurinol with superoxide dismutase (SOD) and catalase. ROS at high concentration also inhibited 3H-uridine incorporation but had no effect on 35SO4 and 3H-uridine uptake by the cells. They also alter hyaluronan (HA) and PG monomers by fragmenting the core protein moiety and destroying the hyaluronic acid binding region. Altered PG monomers do not interact with HA to form complexes, but fragmented HA still retain a significant PG monomer-binding capacity. PG-HA complexes are easily and irreversibly destroyed by ROS. These results suggest that ROS may at low fluxes stimulate PG-synthesis under physiological conditions and alter cartilage metabolism and structure in conditions where they are overproduced, such as in rheumatoid arthritis, and in hemochromatosis and other iron storage diseases.
The respiratory burst activity in peripheral neutrophils from nine patients with localized juvenile periodontitis (LJP) and age- and sex-matched healthy controls was studied by measuring the intensity of luminol-enhanced chemiluminescence (CL) induced by unopsonized and three differently opsonized zymosan particles, formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). The neutrophils from LJP patients showed in general more intense CL with all activators than did their controls. Particularly, the CL response induced by unopsonized zymosan particles and FMLP were significantly higher (p < 0.05 and 0.001). When comparisons were made between female LJP patients (n = 6) and matched controls, also serum-opsonized and IgG-opsonized zymosan particles produced CL was significantly increased (p < 0.05). In order to determine whether the elevated CL responses to zymosan particles were due to altered levels of the interacting receptors on neutrophil surface, an immunofluorescence analysis of the expression of IgG-Fc-receptors (FcR) and complement receptors (CR) was performed with flow cytometry. No significant difference in the expression of FcRII, FcRIII, CR1 and CR3 was detected in LJP group compared to controls. Since the elevated CL responses can not be explained by changes in receptor numbers it is hypothesized that the increased respiratory burst activity in LJP may be caused by altered post receptor signalling pathway.
A modification of Dische's carbazole reaction for uronic acid in the presence of borate is described. The advantages of the procedure are: 1.(1) There is an approximately twofold increase of sensitivity. The OD is a linear function of concentration between 4 and 40 μg/ml.2.(2) Maximum color develops immediately.3.(3) The color is stable for at least 16 hr.4.(4) There is greater reproducibility and reduction of interference by chloride ion and oxidants.It has been found possible to distinguish between heparin, heparin derivatives, and other polyuronides of connective tissue by comparing the effect of chlorides on the color yield in both procedures.
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