Studies on the generation of hydrogen peroxide during some non-enzymic reactions changing the hyaluronic acid molecule

The effect of catalase on non-enzymic-induced changes in the conformation of hyaluronic acid in a vitreous humour preparation was measured using viscometry. Ascorbate, heavy metal ions, riboflavin or EDTA all lowered the viscosity of hyaluronic acid solutions. These effects could be prevented by the addition of catalase. This suggested that H₂O₂ is produced by these compounds and that the resulting change in conformation of hyaluronic acid may be due to peroxyl and hydroxyl attack by the free radicals thus generated.     

The role of superoxide and hydroxyl radicals in the degradation of hyaluronic acid induced by metal ions and by ascorbic acid

Purified commercial hyaluronic acid contains significant amounts of iron. Addition of Fe2+ to solutions of it causes depolymerization, which is inhibited by catalase and scavengers of the hydroxyl radical (. OH) but not by superoxide dismutase. Fe3+ is ineffective. Ascorbic acid also depolymerizes hyaluronic acid, apparently because it can reduce Fe3+ in the reaction mixtures to Fe2+. Ascorbate-induced depolymerization is inhibited by the specific iron chelator desferrioxamine, by catalase, and by scavengers of the hydroxyl radical. The relevance of these observations to rheumatoid arthritis and inflammatory joint diseases is discussed.     

Hyaluronic acid synthesis in articular cartilage: an inhibition by hydrogen peroxide

The synthesis of hyaluronic acid by bovine articular cartilage in culture was inhibited after treatment with xanthine oxidase and hypoxanthine. Through the use of catalase, superoxide dismutase and the specific iron chelator diethylenetriaminepentaacetic acid, the active species responsible for inhibition was shown to be hydrogen peroxide. Hydrogen peroxide generated by glucose oxidase was also inhibitory. Some recovery of hyaluronic synthesis was evident after a further period of culturing. Proteoglycan synthesis was inhibited in parallel with hyaluronic acid synthesis.     

Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.     

Hyaluronic Acid Degradation by Ascorbic Acid and Influence of Iron

The effects of ascorbic acid, iron and ADP on hyaluronic acid, a compound present in inflamed joints, were investigated in an in vitro system. Ascorbic acid induces degradation of hyaluronic acid which increased in the presence of FeCl, and which is additionally stimulated by ADP chelated ferric ions. The hyaluronic acid degrading reactions induced by the Fe-III/ADP/ascorbic acid system were inhibited by catalase and formate to various extents whereas the presence of superoxide dismutase did not exert any inhibitory effect. Desferrioxamine, a specific iron chelator, completely inhibited hyaluronic acid depolymerisation by ascorbic acid as well as in combination with FeCl₃ or FeCl₃/ADP, respectively. We suggest that the ultimate hyaluronic acid degrading species is OH', generated via the Fe-III/ADP catalysed Haber Weiss reaction. There is also an indication for the involvement of perferryl or/and ferryl species in the degradation process.     

Retinal oxidative stress induced by high intraocular pressure

Glaucoma is an optic neuropathy in which retinal ganglion cells die probably through an apoptotic process. Apoptosis is known to involve free radicals in several systems including the retina. In this context, the aim of the present work was to analyze retinal oxidative damage in rats with glaucoma induced by the chronic injection of hyaluronic acid in the eye anterior chamber. The results showed a significant decrease in total retinal superoxide dismutase and catalase activities after 6 and 3 weeks of treatment with hyaluronic acid, respectively. Also, although GPX activity increased after 10 weeks of ocular hypertension, GSH levels significantly decreased at 6 weeks of treatment with hyaluronic acid. Moreover, retinal lipid peroxidation significantly increased in a time-of-hypertension-dependent manner. On the other hand, a significant decrease in both diurnal and nocturnal retinal melatonin content was detected at 3, 6, or 10 weeks of treatment with hyaluronic acid. The present results suggest that retinal oxidative stress may be involved in glaucomatous cell death. Thus, manipulation of intracellular redox status using antioxidants may be a new therapeutic tool to prevent glaucomatous neurodegeneration.     

Homogentisic acid autoxidation and oxygen radical generation: implications for the etiology of alkaptonuric arthritis

The metabolic disorder, alkaptonuria, is distinguished by elevated serum levels of 2,5-dihydroxyphenylacetic acid (homogentisic acid), pigmentation of cartilage and connective tissue and, ultimately, the development of inflammatory arthritis. Oxygen radical generation during homogentisic acid autoxidation was characterized in vitro to assess the likelihood that oxygen radicals act as molecular agents of alkaptonuric arthritis in vivo. For homogentisic acid autoxidized at physiological pH and above, yielding superoxide (O2-)2 and hydrogen peroxide (H2O2), the homogentisic acid autoxidation rate was oxygen dependent, proportional to homogentisic acid concentration, temperature dependent and pH dependent. Formation of the oxidized product, benzoquinoneacetic acid was inhibited by the reducing agents, NADH, reduced glutathione, and ascorbic acid and accelerated by SOD and manganese-pyrophosphate. Manganese stimulated autoxidation was suppressed by diethylenetriaminepentaacetic acid (DTPA). Homogentisic acid autoxidation stimulated a rapid cooxidation of ascorbic acid at pH 7.45. Hydrogen peroxide was among the products of cooxidation. The combination of homogentisic acid and Fe3+-EDTA stimulated hydroxyl radical (OH.) formation estimated by salicylate hydroxylation. Ferric iron was required for the reaction and Fe3+-EDTA was a better catalyst than either free Fe3+ or Fe3+-DTPA. SOD accelerated OH. production by homogentisic acid as did H2O2, and catalase reversed much of the stimulation by SOD. Catalase alone, and the hydroxyl radical scavengers, thiourea and sodium formate, suppressed salicylate hydroxylation. Homogentisic acid and Fe3+-EDTA also stimulated the degradation of hyaluronic acid, the chief viscous element of synovial fluid. Hyaluronic acid depolymerization was time dependent and proportional to the homogentisic acid concentration up to 100 microM. The level of degradation observed was comparable to that obtained with ascorbic acid at equivalent concentrations. The hydroxyl radical was an active intermediate in depolymerization. Thus, catalase and the hydroxyl radical scavengers, thiourea and dimethyl sulfoxide, almost completely suppressed the depolymerization reaction. The ability of homogentisic acid to generate O2-, H2O2 and OH. through autoxidation and the degradation of hyaluronic acid by homogentisic acid-mediated by OH. production suggests that oxygen radicals play a significant role in the etiology of alkaptonuric arthritis.     

Depolymerization of Hyaluronic Acid by D-Fructose 6-Phosphate

Depolymerization of hyaluronic acid obtained from Streptococcus zooepidemicus by D-fructose 6-phosphate was investigated for characterization of reducing sugar-mediated degradation of biopolymers under physiological conditions. The extent of depolymerization was monitored by the decrease of viscosity of a reaction mixture containing 1.0% hyaluronic acid, D-fructose 6-phosphate, and 1.0 × 10^?2 mM of Cu²⁺ in phosphate buffer, pH 7.4. It was found that the depolymerization of hyaluronic acid was dependent on the concentration of the reducing sugar and was specifically accelerated by the presence of Cu²⁺. The reaction was found to be significantly inhibited by catalase, superoxide dismutase (SOD), 1,2-dihy­ droxybenzene 3,5-disulfonic acid (Tiron), and chelating agents such as EDTA and diethylene triamine penta­ acetic acid (DETAPAC), although the inhibition by SOD was low. Almost the same depolymerization rates were observed in hyaluronic acid preparations of different molecular weight (1.1 × 10⁶, 8.8 × 10⁵, and 6.8 × 10⁵). The rates, however, were different for hyaluronic acids obtained from S. zooepidemicus, rooster comb, and umbilical cord. It was concluded that depolymerization of the polysaccharide was caused by active oxygen species generated by the autoxidation of D-fructose 6-phosphate in the presence of Cu²⁺, in a mechanism similar to that previously reported for the degradation of DNA and inactivation of virus in vitro.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: hyaluronic acid, heparitin sulfate, and keratan sulfate

Circular dichroism spectroscopy has been used to study the interactions of hyaluronic acid, heparitin sulfate, and keratan sulfate with cationic polypeptides. The results indicate that the presence of these mucopolysaccharides has an effect in the conformation of poly(L -lysine) and poly(L -arginine), such that the former adopts the “random” form and the latter takes up the α-helical conformation, rather than the “charged coil” form expected at neutral pH. The relative strengths of the interactions can be judged from the melting temperatures above which they are disrupted. Both the stoichiometry and the strength of the interactions depend on the position, number, and type of anionic groups attached to the polysaccharide backbone. Such considerations place the six common mucopolysaccharides in order of increasing strength of interaction: hyaluronic acid < chondroitin 4-sulfate < heparitin sulfate < chondroitin 6-sulfate < keratan sulfate ? dermatan sulfate. These differences should be paralleled by differences in the interaction of the mucopolysaccharides with collagen and fibrous proteins.     

On the structure of proteoglycan core

Core protein from bovine nasal proteoglycan has been obtained by cyanogen bromide cleavage and by removal of most of the glycosaminoglycan side chains by hydrogen fluoride treatment. Amino acid analysis of the cyanogen bromide fragment shows it to consist mainly of proline, serine, glycine and glutamic acid (glutamine). End-group analyses of the fragment and HF stripped core reveal and N-terminal residue to be valine in each case. The stripped core has been subjected to sequencing and some sequential information is presented. Based upon the amino acid analysis, sequence information and other properties, conformation analysis indicates that the most likely conformation is that of a flexible extended chain containing β-turns. The existence of a common N-terminal residue indicates that it is the C-terminal region which lies in the region of the hyaluronic acid backbone in intact proteoglycan. Furthermore, enzymatic cleavage of core protein which occurs in proteoglycan turnover, aging and degenerative diseases, probably does not occur by a stepwise cleavage from the N terminus of proteoglycan but by a more drastic degradation process.     

Depolymerization of Hyaluronic Acid by Low-molecular-weight Amadori-rearrangement Products and Glycated Polylysine

Depolymerization of hyaluronic acid (HA) by low-molecular-weight Amadori-rearrangement products in the presence of Cu^{2 +} was studied as an in vitro model for the glycated protein-mediated degradation of biopolymers. This oxygen radical-mediated depolymerization was found to be specifically accelerated by Cu^{2 +} , and significantly inhibited by catalase, hydroxyl radical scavengers, and metal ion chelators. Glycated polylysine also depolymerized HA. The difference in depolymerization rate between low- and high-molecular-weight Amadori products is discussed.     

Stimulatory effect of prostaglandins on the production of hexosamine-containing substances by cultured fibroblasts (3) induction of hyaluronic acid synthetase by prostaglandin F2alpha.

The mechanism of the stimulatory effect of prostaglandin(PG)F2alpha on the production of hexosamine-containing substances by cultured fibroblasts was studied. Treatment of the cells with 1 microgram/ml of PGF2alpha resulted in a doubled net synthesis of acidic glycosaminoglycans during 20 hrs measured with uronic acid as index, and also resulted in 300 per cent increase of 3H-glucosamine incorporation into hexosamine-containing substances during the first 6 hrs. Fractionation of the PGF2alpha-stimulated hexosamine-containing substances with double isotope technique revealed that hyaluronic acid was the most stimulated component. Prior to the increase of hyaluronic acid, hyaluronic acid synthetase activity was found to be augmented by PGF2alpha as high as 4 times over the control. The augmentation of hyaluronic acid synthetase activity by PGF2alpha did not take place if actinomycin D was simultaneously present in the culture medium, suggesting that PGF2alpha induced the enzyme.     

The optimization of a scaffold for cartilage regeneration

Natural polymers offer various advantages in cartilage tissue engineering applications, thanks to their intrinsic bioactivity and adaptability, which can be exploited for the optimization of scaffold properties. In particular, silk fibroin has multifunctional features driven by the self-assembly of molecular subunits in appropriate environmental conditions. For these reasons, it was used in combination with hyaluronic acid to produce porous sponges for cartilage regeneration. The added amount of hyaluronic acid and the cross-linking with genipin modulated scaffold properties in a synergistic way, showing a strong inter-correlation among macroscopic and microscopic characteristics. Interestingly, hyaluronic acid affected silk fibroin conformation and induced a physical separation between the two material components in absence of genipin. Instead, this was prevented by the cross-linking reaction, resulting in a more interspersed network of protein and polysaccharide molecules partially resembling the structure of cartilage extracellular matrix. In addition, the systematic evaluation of sponge properties and how they can be modulated will represent a significant starting point for the interpretation of the complex outcomes driven by the scaffold in vitro and in vivo.     

Catalase binds Grb2 in tumor cells when stimulated with serum or ligands for integrin receptors

Recent studies have demonstrated that H₂O₂ acts as a second messenger of mitogenic signaling and that catalase is under the regulation of PKA and PKC signaling. Here we examined whether catalase binds any mitogenic signaling molecules. Our results indicated that serum stimulation of HeLa, Caco-2, and LiSa-2 cells, but not BJ-1 and primary human bronchial epithelial cells, resulted in catalase binding to Grb2. Whereas serum deprivation, butyrate, and herbimycin-A negatively regulated the binding, an extended culture of confluent Caco-2 cells resulted in binding of an additional but as yet unidentified molecule to the Grb2–catalase complex. Expression of active catalase nearly 15-fold over control level in Tet-off HeLa cells substantially increased binding to Grb2, and this was sensitive to 3-aminotriazole, a specific catalase inhibitor. Furthermore, fibrinogen, fibronectin, and laminin, but not collagen types I to V, hyaluronic acid, elastin, insulin, EGF, IGF-I, PDGF, or NGF, resulted in binding similar to that of serum. A mutation of tyrosine to phenylalanine at 447 abolished the binding capability of catalase to Grb2 in vitro. These results support the view that catalase ⁴⁴⁷Tyr-Val-Asn-Val binds Grb2 upon phosphorylation in tumor cells when stimulated with serum or ligands for integrin receptors. This is the first report demonstrating that catalase binds a SH2 domain of the molecule and participates in integrin signaling.     

Hyaluronic acid and chondroitin-4-sulphate treatment reduces damage in carbon tetrachloride-induced acute rat liver injury

Oxidative stress is involved in the pathogenesis of chemically mediated liver injury. Since glycosaminoglycans possess antioxidant activity, the aim of this work was to assess the protective effects of hyaluronic acid and chondroitin-4-sulphate treatment in a model of carbon tetrachloride-induced liver injury. Liver damage was induced in male rats by an intraperitoneal injection of carbon tetrachloride (1 ml/kg in vegetal oil). Serum alanine aminotransferase and aspartate aminotransferase, hepatic malondialdehyde, plasma TNF-alpha, hepatic reduced glutathione and catalase, and myeloperoxidase, an index of polymorphonuclear infiltration in the jeopardised hepatic tissue, were evaluated 24 h after carbon tetrachloride administration. Carbon tetrachloride produced a marked increase in serum alanine aminotransferase and aspartate aminotransferase activities, primed lipid peroxidation, enhanced plasma TNF-alpha levels, induced a severe depletion of reduced glutathione and catalase, and promoted neutrophil accumulation. Intraperitoneal treatment of rats with hyaluronic acid (25 mg/kg) or chondroitin-4-sulphate (25 mg/kg) failed to exert any effect in the considered parameter, while the combination treatment with both glycosaminoglycans (12,5 + 12,5 mg/kg) decreased the serum levels of alanine aminotransferase and aspartate aminotransferase, inhibited lipid peroxidation by reducing hepatic malondialdehyde, reduced plasma TNF-alpha, restored the endogenous antioxidants, and finally decreased myeloperoxidase activity. These results suggest that hyaluronic acid and chondroitin-4-sulphate possess a different antioxidant mechanism and consequently the combined administration of both glycosaminoglycans exerts a synergistic effect with respect to the single treatment.     

Kinetic studies on activation of peptide bond formation by hyaluronic acid.

1. In this study, a cell-free system derived from Escherichia coli has been used in order to examine in detail the effect of hyaluronic acid on peptide bond formation with the aid of puromycin reaction. 2. This reaction is activated by hyaluronic acid. 3. The degree of activation of peptide bond formation depends on the molecular size of hyaluronic acid. 4. The kinetic analysis revealed that the hyaluronic acid acts as a mixed-type nonessential activator. 5. The presence of hyaluronic acid improves about 9-fold the activity status of ternary complex as it can be calculated by k3/k5 ratio.     

X-ray diffraction studies on the connective tissue polysaccharides. Two-dimensional packing schemes for threefold hyaluronate chains.

An extended conformation of sodium and calcium hyaluronate with helical parameters n (number of residues per turn) = 3 and h (axial rise per disaccharide) = 0.95 ± 0.01 nm has been found to pack in a number of two-dimensional arrays. Each of the structures exhibits a variant of a motif consisting of six chains surrounding a “vacant” site. The distribution of chains around this site, together with certain aspects of the periodicity, suggests that there might be a special relationship between this conformation of hyaluronic acid and water.     

Conformational transition of hyaluronic acid: carboxylic group participation and thermal effect

The chiroptical, viscosity and titration studies of hyaluronic acid in mixed organic/water solvent show a reversible conformational transition of the molecule depending upon pH, solvent composition, temperature, and molecular weight. Neither methylhyaluronate nor chondroitin undergoes conformational transition of this type. These results indicate that hydrogen bonding between the protonated carboxylic group and carbonyl oxygen of the acetamido group is directly involved in the conformational change. Results with chondroitin provide further support for the 4-fold helical structure that we have proposed for hyaluronic acid in mixed organic/water solvent. The thermal stability of the conformation has been studied under various pH values and solvent compositions.     

Cartilage proteoglycan aggregates. The link protein and proteoglycan amino-terminal globular domains have similar structures

Cartilage proteoglycan aggregates contain two components (proteoglycan monomer and link protein) which interact with each other and with hyaluronic acid. Data from amino acid sequence analysis are presented that shows that a domain of the proteoglycan, the hyaluronic acid binding region, which interacts with link protein and hyaluronic acid is very similar to link protein in terms of its primary structure. However, the pattern of glycosylation in the hyaluronic acid binding region is different from that found in link protein. After removal of N-linked oligosaccharides, the tryptically prepared hyaluronic acid binding region from rat chondrosarcoma has a mass by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of 43 +/- 2 kDa. The COOH-terminal two-thirds of rat chondrosarcoma link protein, starting at residue 105, has 41.3% identity with a similar region in the hyaluronic acid binding region. We show that, in addition to the hyaluronic acid binding region, proteoglycan contains another region with similarity to the two repeating loop structures in the COOH-terminal two-thirds of link protein. This presumably corresponds to the second globular domain reported in rotary shadowing studies of cartilage proteoglycans. We have deduced the positions of all of the disulfide bonds in the hyaluronic acid binding region and find them to be in the same positions as would be expected from comparison of these sequences with link protein.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.