Hydration of polymeric components of cartilage--an infrared spectroscopic study on hyaluronic acid and chondroitin sulfate

Hydrated polysaccharides are major constituents of cartilage and play an important role in its water-binding properties. Infrared (IR) spectroscopy and sorption isotherms have been used to investigate the hydration behavior of the glycosaminoglycans hyaluronic acid and chondroitin sulfate. IR-dichroism of the vibrational modes of the pyranose ring is found at relative humidities (RH) smaller than 84%. The IR-dichroism data for the vibrational modes of the pyranose ring have been analyzed with respect to the helical structure of these polysaccharides. The orientation vanishes at higher relative humidities (>84%), because a strong increase in the water uptake occurs in the observed sorption isotherms. Differences in the IR-absorbance of the O-H stretching mode of sorbed water between hyaluronic acid and chondroitin sulfate are shown to be caused by the additional hydration of the sulfate groups. The corresponding H-bonds are weaker than those of the hydration shell of the pyranose rings.     

Effect of gluco-monosaccharides and different conditions on digestion of hyaluronan by testicular hyaluronidase

The changes of molecular size of hyaluronan during enzymatic reaction of bovine testicular hyaluronidase at different conditions are monitored by size exclusion high performance liquid chromatography. The effect of glucuronate, galacturonate, glucosamines and pyridoxin as potential inhibitors of hydrolysis is evaluated. The most effective of all tested inhibitors was the presence of glucuronate which not only inhibited the hydrolysis, but also initiated enzymatic reconstruction by transglycosylation reaction at pH 7.0 and absence of any buffer or salt. That effect was not found in the presence of a salt or with any other of the compounds tested.     

Analysis by high-performance liquid chromatography of hyaluronic acid and chondroitin sulfates

The use of high-performance liquid chromatography for the quantification of glycosaminoglycan disaccharides has been hampered by the inability to isocratically resolve the chondroitinase digestion products 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (delta Di-HA) and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-galactose (delta Di-OS). To overcome this limitation, we have developed a solvent system capable of resolving delta Di-HA, delta Di-OS, 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose (delta Di-6S), and 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulfo-D-galactose (delta Di-4S). Integrator responses were linear from 1 microgram down to 25 ng for delta Di-HA, delta Di-OS, and delta Di-4S and down to 100 ng for delta Di-6S. This method was used to examine changes in the content of urinary hyaluronic acid and chondroitin sulfates isolated from normal individuals and from patients with Lowe Syndrome, Werner Syndrome, and Hutchinson-Gilford Progeria Syndrome. We confirmed that the HPLC method gave results comparable to colorimetric methods.     

Selective hydrolysis of chondroitin sulfates by hyaluronidase

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Two tyrosine residues (Tyr⁴ and Tyr⁷⁶) of succinyl-CoA:3-oxoacid CoA transferase (SCOT) are sensitive to nitric oxide (NO) stress, as assessed by mass spectrometry and site-direct mutagenesis. However, monitoring the SCOT nitration in tissue or cells is challenging. Herein, we describe the development of an assay to detect nitrated SCOT directly using site-specific antibodies; the monoclonal antibodies were generated and screened against nitrated peptides of SCOT. After stringent filtration, two antibodies, anti-SCOT4N and anti-SCOT76N, which specifically recognise Tyr⁴ or Tyr⁷⁶ of SCOT, respectively, were successfully selected. In a cell model over-expressing iNOS in the mitochondria, nitrated SCOT was significantly increased compared with control cells. In addition, in a mouse model of diabetes, nitrated Tyr⁴ and Tyr⁷⁶ in the heart and kidney were higher compared to the control animals. Our results using monoclonal antibodies against nitrated SCOT peptides are in good agreement with the proteomic data.     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

Production of chondroitin sulfate and chondroitin

The production of microbial polysaccharides has recently gained much interest because of their potential biotechnological applications. Several pathogenic bacteria are known to produce capsular polysaccharides, which provide a protection barrier towards harsh environmental conditions, and towards host defences in case of invasive infections. These capsules are often composed of glycosaminoglycan-like polymers. Glycosaminoglycans are essential structural components of the mammalian extracellular matrix and they have several applications in the medical, veterinary, pharmaceutical and cosmetic field because of their peculiar properties. Most of the commercially available glycosaminoglycans have so far been extracted from animal sources, and therefore the structural similarity of microbial capsular polysaccharides to these biomolecules makes these bacteria ideal candidates as non-animal sources of glycosaminoglycan-derived products. One example is hyaluronic acid which was formerly extracted from hen crests, but is nowadays produced via Streptococci fermentations. On the other hand, no large scale biotechnological production processes for heparin and chondrotin sulfate have been developed. The larger demand of these biopolymers compared to hyaluronic acid (tons vs kilograms), due to the higher titre in the final product (grams vs milligrams/dose), and the scarce scientific effort have hampered the successful development of fermentative processes. In this paper we present an overview of the diverse applications and production methods of chondroitin reported so far in literature with a specific focus on novel microbial biotechnological approaches.     

Serum levels of hyaluronic acid and chondroitin sulfate as a non-invasive method to evaluate healing after cartilage repair procedures

Magnetic resonance imaging remains the only non-invasive method to assess the quality of cartilage repair procedures, but ideally would be complemented by other modalities, particularly blood tests. Nganvongpanit and colleagues investigated serum levels of hyaluronic acid (HA) and chondroitin sulfate (CS) for their correlation with tissue quality after cartilage repair with autologous chondrocytes versus subchondral drilling in a dog model. They reported better tissue quality in animals treated with chondrocyte implantation. Serum levels correlated with the histological score of biopsy samples: CS showed a negative (r = -0.69) and HA a positive (r = +0.46) correlation. Many questions remain to be answered before serum markers can provide a reliable, non-invasive tool to assess tissue quality, but these data provide an important foundation for additional research.In the previous issue of Arthritis Research & Therapy, Nganvongpanit and colleagues [1], of Chiang Mai University in Thailand, investigated the potential use of serum biomarkers, such as hyaluronic acid (HA) and chondroitin sulfate (CS), to evaluate healing after cartilage repair procedures. They randomly assigned dogs to treatment with autologous chondrocyte implantation (ACI) versus subchondral drilling (SD) and followed the animals for 24 weeks post-operatively with multiple blood draws and a cartilage biopsy at final follow-up.Cartilage defects are a common diagnosis, encountered in over 60% of knee arthroscopies [2]. While the natural history and pathophysiology of cartilage defects remain controversial, a significant number of patients present with symptoms that warrant surgical intervention. These patients undergo various cartilage repair procedures to repair the damaged articular surfaces, including microfracture, osteochondral autografting, and ACI. Progress in the field of cartilage repair has been impeded in part by the relative lack of adequate instruments to evaluate the quality of the reparative tissue. While histological evaluation is desirable, researchers have found it difficult to recruit patients for a second surgical procedure to harvest a tissue biopsy solely for research purposes. Imaging techniques, especially magnetic resonance imaging (MRI), have made significant progress in recent years. Certain cartilage-specific techniques such as delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and T1-rho and T2-mapping have promise to assess tissue quality by indirectly measuring glycosaminoglycan content [3,4]. However, these techniques are associated with substantial cost and potential risk to the patient from contrast exposure; therefore, the development of alternative non-invasive techniques is desirable. In particular, blood tests, which could be repeated multiple times with minimal discomfort to the patient, would present an ideal method to investigate the maturation of repair tissue after cartilage repair. Beyond the scientific benefit of comparing the relative time courses of healing after different repair techniques, once thresholds are established, biomarkers could provide clinical guidance regarding the point when patients might return to full activities.In their article, Nganvongpanit and colleagues investigated the use of monoclonal antibodies and enzyme-linked immunosorbent assay to quantify serum levels of CS and HA, respectively, in a dog model. They followed two groups treated with either SD or autologous chondrocytes (ACs) for 24 weeks, with blood draws at baseline and every 6 weeks thereafter. Other endpoints included the gross visual evaluation of the reparative tissue as well as histologic grading. Animals treated with ACs demonstrated better visual and histological appearance than those treated with drilling. Three of the five AC biopsies were near normal, and the other two showed at least 50% fill and peripheral integration of the repair site. In the SD group, three of five samples demonstrated complete degeneration, and the other two only inconsistent fill and no peripheral integration with the surrounding articular surface. Histologically, both groups demonstrated some fibrocartilage; however, the AC group also showed hyaline cartilage compared with fibrous tissue in the SD group.Interestingly, serum levels of CS and HA demonstrated different trends at final follow-up after 24 weeks: CS had a strong negative correlation with histological scores (r = -0.69), while HA was positively correlated (r = +0.46). In the AC group, CS levels trended downward over time, a finding the authors interpret as a reflection of the normalizing proteoglycan turnover due to a successful repair with maturing tissue. In the SD group, however, levels remained high, possibly reflecting the progressive damage of the surrounding cartilage seen in these samples. Overall, HA levels also decreased from baseline, with relatively higher values in samples with better histological scores, potentially a sign of normalization of joint homeostasis.This study provided two important findings. First, it added to the mounting evidence of improved histological outcomes with cell-based therapy, such as ACI [5], over marrow-stimulation techniques, such as SD or microfracture. Second, the authors describe two potential candidate factors to follow tissue maturation and healing: HA and CS. Many questions remain to be addressed, such as the correlation of marker levels with defect size, number, and location as well as possible differences between chondral and osteochondral defects and patient gender, age, or weight. However, these preliminary results are promising and provide a foundation for future research.In conclusion, while these findings require larger, confirmatory studies (ideally in human patients), they hold promise for non-invasive monitoring after cartilage repair procedures. Reliable, reproducible, and relatively inexpensive methods to evaluate the quality and maturation of reparative tissue will substantially advance the field of cartilage repair. These tests would potentially enable investigators and industry to develop new technologies aimed at repairing articular cartilage, assist surgeons to select the appropriate procedure for any given patient, and post-operatively, allow an individualized determination of when it is safe for the patient to return to higher levels of activity.