Chondroitin Sulfate Content Research Articles

Electron microscopic demonstration of proteoglycans in guinea pig epiphyseal cartilage

Guinea pig epiphyseal cartilage was studied ultrastructurally after staining with ruthenium red or Alcian Blue. Extracellularly, the matrix granules were positively stained with both dyes, whereas no apparent staining of the collagen fibrils occurred. Intracellularly, a positive ruthenium red staining of the Golgi vacuole granules was observed. No Alcian Blue staining of the content of the vacuoles was detected. The fresh, untreated cartilage contained about 1.3% hexosamine on a dry weight basis. About three-fourths thereof represented chondroitin sulfate, the remaining part keratan sulfate and glycoproteins. After digestion of thin cartilage slices with hyaluronidase or chondroitinase ABC the chondroitin sulfate content was less than 50% of that in the buffer-incubated controls. Concomitantly, the matrix granules were reduced in size and number or completely absent. Papain digestion removed more than 90% of the chondroitin sulfate and all matrix granules. From these findings it was concluded that the matrix granules contain proteoglycans. Moreover, it seemed reasonable to assume that the Golgi vacuole granules, at least in part, represent intracellular proteoglycans. Signs of secretion of these granules by exocytosis were occasionally observed.

Control of Chondrogenesis in Limb-Bud Cell Cultures by Bromodeoxyuridine

Initial exposure of cultured limb-bud cells (stage 23-24) to 5-bromo-2'-deoxyuridine (BrdU) irreversibly inhibits differentiation to cartilage under three different culture conditions. The inhibition of chondroitin sulfate synthesis is partially reversed by D-xylose in limb-bud cells after treatment with BrdU. The activities of four enzymes involved in chondroitin sulfate production were reduced in BrdU-treated cultures, but the magnitude of decrease was far less than the decrease in glycosaminoglycan synthesis. The slight increase in the turnover rate of sulfated glycosaminoglycans in BrdU-treated mesenchyme was not sufficient to account for the marked decrease in chondroitin sulfate content. The results suggest that BrdU treatment interferes with normal synthesis of chondroitin sulfate core protein in cultured limb-bud cells, but does not greatly diminish enzyme activities or UDP-sugar levels necessary for production of polysaccharide chains.

Replacement of proteoglycans in embryonic chick cartilage in organ culture after treatment with testicular hyaluronidase.

Explants of cartilage from tibiae of 11-12 days chick embryos were grown in organ culture. To one group hyaluronidase was added to the medium during the first 2 days of culture; the treated tissue was then cultured in medium without enzyme for a further 4 days. Control explants grown in hyaluronidase-free medium for 6 days grew rapidly in size and the total hexosamine content more than doubled during this time. After exposure to hyaluronidase, much of the hexosamine was lost from treated cartilage and appeared in the culture medium, but it was mostly replaced in the tissue during the subsequent recovery period. Analysis of cartilage and medium showed that net synthesis of hexosamine increased greatly in treated cartilage. The proteoglycans were extracted by two procedures from control and treated cartilage after 2, 4 and 6 days in culture. The hydrodynamic sizes of the purified proteoglycans were compared by gel chromatography and the composition of the gel-chromatographic fractions was determined. The proteoglycans from controls did not change during culture, but after exposure to hyaluronidase the proteoglycans from treated cartilage were of much smaller size and lower chondroitin sulphate content. During recovery, even though new proteoglycans were formed, they were nevertheless of smaller size and lower chondroitin sulphate content than control proteoglycans. They gradually became more like control proteoglycans during recovery from treatment, but even after 4 days they were not yet the same. After 2 days of treatment with the enzyme, the chondroitin sulphate in the cartilage was of shorter chain length than in controls but during recovery after 4 and 6 days in culture, the chain lengths in control and treated cartilage were similar. It is concluded that the proteoglycans formed in embryo cartilage in response to their depletion by enzyme treatment contained fewer chondroitin sulphate chains attached to the protein moiety of proteoglycans. This may have resulted from a failure under stress to glycosylate the protein moiety to the usual extent; alternatively the synthesis of normal proteoglycans of low chondroitin sulphate content may have increased, thus changing the proteoglycan population.

Lipids and acid mucopolysaccharides in arteries and veins of human umbilical cord

1. 1. Various components of neutral lipids, phospholipids, and acid mucopolysaccharides were determined in the arteries and veins of the human umbilical cord. 2. 2. It was found that umbilical vein had a higher content of phosphatidylethanolamine, phosphatidylserine, and chondroitin sulphate A/C when compared with umbilical artery.

Microchemical analysis of human tibial growth cartilage in various forms of dwarfism.

ABSTRACT Microchemical determinations of glycosaminoglycans and collagen were preformed in isolated histological zones from sections of tibial epiphyseal plate biopsies obtained from children with growth disorders (pituitary dwarfism, congenital myxoedema, Turner's syndrome, Noonan's syndrome, mucopolysaccharidosis type VI, vitamin D resistant rickets and achondroplasia). Alternate sections were used for histochemical localization of glycosaminoglycans and proteins. The values were compared with those found in comparable zones of the growth plate from normal children of the same age. The chondroitin sulphate concentration (% of defatted dry wt.) in the normal epiphyseal plate increased from the resting zone towards the proliferating/hypertrophic zone; collagen exhibited a reverse trend. In some of the pathological biopsies the concentration of chondroitin sulphate was slightly decreased whereas that of collagen was slightly increased. A marked increase in the collagen concentration was found in achondroplasia. The solubility profiles of the cetylpyridinium complexes of the chondroitin sulphate fraction showed three main peaks with slight but characteristic differences in the various zones of the normal cartilage plate. Significant shifts in the proportion of these peaks were observed in several pathological biopsies, indicating possible deviations from the normal molecular characteristics of the chondroitin sulphate. Analysis of the main chondroitin sulphate fraction, obtained from pooled samples of normal tibial growth plate after fractionation on the macroscale, indicated that all three peaks contained both chondroitin-4 sulphate and chondroitin-6 sulphate and that they probably differed in their molecular weight.

Chondroitin sulphate in the normal and rheumatoid synovial fluid

The concentration of chondroitin sulphate and hyaluronic acid in the synovial fluids obtained from 25 patients with rheumatoid arthritis and from 9 healthy donors was determined. There was no correlation between the concentrations of the acidic glycosaminoglycans and the grade of local articular changes, but there was a highly significant positive correlation between the concentrations of chondroitin sulphate and hyaluronic acid. This indicates a common origin for these acidic glycosaminoglycans, the synovial lining cells. Probably chondroitin sulphate is also released from the cartilage into the synovial cavity in rheumatoid joints, where there are also osteoarthrotic changes.

Biosynthesis of proteoglycans in cartilage slices. Fractionation by gel chromatography and equilibrium density-gradient centrifugation.

The kinetics of incorporation of [(35)S]sulphate into slices of pig laryngeal cartilage in vitro was linear with time up to 6h. The specific radioactivities of the extracted proteoglycans (containing about 80% of the uronic acid of the cartilage) and the glycosaminoglycans remaining in the tissue after extraction were measured after various times of continuous and ;pulse-chase' radioactivity incorporation. Radioactivity was present in the isolated chondroitin sulphate after 2 min, but there was a 35min delay in its appearance in the extractable proteoglycan fraction. Fractionation of the proteoglycans by gel chromatography showed that the smallest molecules had the highest specific radioactivity, but ;pulse-chase' experiments over 5h did not demonstrate any precursor-product relationships between fractions of different size. Equilibrium density-gradient centrifugation in 4m-guanidine hydrochloride showed that among the proteoglycan fractions the specific radioactivity increased as the chondroitin sulphate content decreased, but with preparations from ;pulse-chase' experiments there was again no evidence for precursor-product relationships between the different fractions. Differences in radioactive incorporation would seem to reflect metabolic heterogeneity within the proteoglycans extracted from cartilage. This may be due either to a partial separation of different types of proteoglycans or to differences in the rates of degradation of the molecules of different size and composition as a result of the nature and specificity of the normal degrading enzymes. The results suggest that molecules of all sizes were formed at the same time.

鯨軟骨コンドロイチン硫酸のエタノールによる沈殿に関する研究-I

Chondroitin sulfate (CS) dissolved in water is difficult to precipitate with ethanol in the absence of salts, therefore suitable salts such as sodium acetate, calcium acetate must be added in order to complete precipitation. However, a detailed study concerning the influence of salts on the precipitation of CS with ethanol has not yet been reported. In this paper, the precipitation of whale cartilage CS in aqueous solution with ethanol under various conditions was studied, and the following results were obtained. 1) The higher the CS or salt concentration, the less ethanol was needed to precipitate CS, and there was a linear relationship between the logarithm of CS or salt concentration and the ethanol concentration needed for the precipitation. 2) The decrease in temperature facilitated the precipitation of CS. Over 0°C, there was a linear relationship between the temperature and the ethanol concentration needed for the precipitation. 3) There were little differences in the precipitating power of various anions. In the case of cations, divalent cations possesed greater precipitating power than monovalent ones, and there were considerable differences in the precipitating power of equivalent cations. 4) The fractions obtained by fractionation of CS with ethanol were differed in molecular weight, and the ethanol concentration needed for the precipitation of CS was inversely proportional to the molecular weight.

Fractionation of the glycosaminoglycans of human articular cartilage on ecteola cellulose in ageing and in osteoarthrosis.

Articular cartilage from the lower femoral epiphysis of human autopsy cases was collected in ten age groups from birth to 95 years and in osteoarthrosis of two grades of severity. By chromatography on Ecteola cellulose columns, the different glycosaminoglycans were separated and keratan sulphate was fractionated. The results are consistent with earlier studies using CPC-cellulose column technique, i.e. a higher content of total hexosamine and chondroitin sulphate was found in early childhood. Furthermore, normal articular cartilage in adults showed a higher content and a higher degree of heterogeneity of keratan sulphate than in childhood. In osteoarthrosis, a decreased content of total hexosamine due to a decrease both of chondroitin sulphate and keratan sulphate was found.

Acid mucopolysaccharides in gingivitis and periodontitis.

Human Gingival Tissues was clinically classified as normal or periodontally involved. Acid mucopolysaccharides were extracted from this tissue. The measurable acid mucopolysaccharides extracted were chondroitin sulfules A and C. Concentration of chondroitin sulfate was lower in the tissue classified as periodontally involved when compared to the normal tissue.

Acid Mucopolysaccharides As Cofactor in Formation of Platelet-clumping Substance

Abstract Incubation at 37°C for 30 min or the addition of CaCl2 (5 mM) generated platelet-clumping activity in citrated plasma and EDTA plasma previously acidified to pH 5.5 and in which heparin was added to give a concentration of 0.5 mg/ml. Similar treatment could not produce platelet-clumping activity in saline or denatured plasma that had been heated to 56°C. Heparin, chondroitin sulfate B, or heparan sulfate produced platelet-clumping activity in BaSO4-adsorbed serum over a given pH range. The effective pH range for each compound was; heparin 0.5 mg/ml, pH 5.16 ± 0.09 to 6.31 ± 0.14; chondroitin sulfate B 0.5 mg/ml, pH 5.62 ± 0.09 to 6.88 ± 0.06; 2 mg/ml, pH 5.17 ± 0.16 to 7.18 ± 0.16. The effect of heparan sulfate in generating platelet-clumping activity was estimated only qualitatively at pH 6.0 and proved to be positive. Chondroitin sulfate A, chondroitin sulfate C, and hyaluronic acid failed to yield platelet-clumping activity in BaSO4-adsorbed plasma. The minimal concentrations of heparin and chondroitin sulfate B necessary to induce platelet-clumping activity in the BaSO4-adsorbed serum were 0.5 mg/ml and 0.1 mg/ml, respectively.

Glycosaminoglycans in the Developing Chicken Cornea

The development and aging of the chicken cornea is accompanied by the stepwise disappearance of the non-sulphated glucosaminoglycan and by the increase of keratan sulphate and chondroitin sulphate content. After hatching, keratan sulphate becomes a predominant part of corneal glycosaminoglycans. Radioactive sulphate incorporates into both sulphated polysaccharides at a similar rate which appears to diminish with the aging of the cornea

Histochemical localization of acid glycosaminoglycans in human articular cartilage.

Fresh human articular cartilage from patients, 23 to 79 years of age, has been examined histochemically for its distribution of acid glycosaminoglycans. The alcian blue stain with different salt concentration enabled us to localize chondroi-tin sulfate and keratan sulfate in the matrix and in the halos. Young cartilage had a fairly even distribution of chondroitin sulfate, and only the matrix of the deepest zone stained less intensely. With increasing age the intensity of the chondroitin sulfate stain decreased and the tendency for a lack of matrix stain in the deeper layer was more pronounced in old than in young cartilage. In young cartilage, keratan sulfate is exclusively found in the matrix; the halos and their immediate surroundings are unstained. With increasing age and distance from the surface this pattern is less marked and keratan sulfate may be found even inside the halos. In the examined material four of the cartilages, here referred to as slightly arthrotic cartilage, showed an acid glycosaminoglycan distribution which differed from normal as well as from arthrotic cartilage. In these cases the chondroitin sulfate concentration was high in the halos while the matrix itself was unstained. The keratan sulfate distribution was even more deviating in showing an unstained matrix and a ring formation around the secondary halos in the upper half, and intensely stained halos in the lower half of the section.

The glycosaminoglycans of human tracheobronchial cartilage.

1. The glycosaminoglycans of human tracheobronchial cartilages from subjects of various ages were liberated by proteolysis of the tissue and purified by ion-exchange chromatography. Purified glycosaminoglycans were fractionated on Dowex 1 resin and cetylpyridinium chloride was used to separate chondroitin sulphates and keratan sulphates occurring in the same fraction. 2. The total chondroitin sulphate content of the cartilages decreased linearly with increasing age. Age-dependent changes in the chemical heterogeneity of chondroitin sulphate were observed, a low-sulphated compound making up 25% of the total glycosaminoglycan at birth but rapidly diminishing in content during the first 6 months of life. Of the total chondroitin sulphate the 6-isomer became rather more prominent than the 4-isomer with increasing age. 3. The total keratan sulphate content of the cartilages increased from trace amounts only at birth to a plateau value by the beginning of the fifth decade. Of the total keratan sulphate approx. 70% was due to a high-molecular-weight compound with a sulphate/hexosamine ratio of 1.5-1.8: 1.0. The degree of sulphation varied between compounds isolated from different individuals. The remaining 30% of the keratan sulphate appeared to be intimately associated with chondroitin 6-sulphate and could only be separated from it after treatment with 0.45m-potassium hydroxide. The hybrid glycosaminoglycans were of lower molecular weight and had a lower sulphate/hexosamine ratio than the major keratan sulphate compound.

A novel fluorophotometric method for determination of hexosamines using pyridoxal and zinc(II) ion

A novel fluorophotometric method for the detection and determination of amino sugars was established utilizing pyridoxal-HCl and Zn(II) ion. This method was highly sensitive; its limit of determination was 1 × 10 −11 mole of amino sugars. The fluorogen of this fluorescence reaction was Zn(II) chelate of N-pyridoxylidene amino sugar containing a 1:1 ratio of amino sugar to Zn(II) ion. The hexosamine content of chondroitin sulfate A and C, hyaluronic acid, and heparin estimated by this method was higher than that obtained by the Elson-Morgan method. Only 2–3 hr of hydrolysis with 2 N HCl was sufficient for the assay of chondroitin sulfate and heparin.

Correlations between stiffness and the chemical constituents of cartilage on the human femoral head

A method is described, in outline, for measuring the compressive stiffness of articular cartilage on the human femoral head, in vitro. Indentations were measured normal to the articular surface, and from these measurements creep moduli were calculated at 2 sec after application of the load. Correlations between the stiffness and the following quantities were determined: (1) Total glycosaminoglycan content, (2) chondroitin sulphate content, (3) ‘keratin sulphate’ content, and (4) collagen content. These quantities were determined from chemical analyses of the indented areas of cartilage and expressed as a percentage of the dry weight. The following observations were made: there was a statistically significant correlation between the stiffness, expressed as the creep modulus, and the total glycosaminoglycan content. Approximately equal correlation coefficients were recorded between the stiffness and chondroitin sulphate and ‘keratan sulphate’ content respectively; however, at a given concentration, the influence of ‘keratin sulphate’, on the stiffness, was greater than that of chondroitin sulphate. In contrast, the correlation between stiffness and collagen content was considerably less significant.

Comparative studies of aortic acid mucopolysaccharides in fifteen species

1. 1. Aortic acid mucopolysaccharides (AMPS) were studied in fifteen different species including: dusky shark, dog fish, frog, turtle, chicken, pheasant, mouse, rat, guinea pig, hamster, rabbit, cat, dog, cow and rhesus monkey. 2. 2. Phylogenetically close species (e.g. cat and dog, rat and mouse chicken and pheasant) had similar concentration of AMPS in aortic tissue. 3. 3. Thoracic aortic segment had a higher concentration of total AMPS and chondroitin sulfate B than abdominal segment. 4. 4. Chondroitin sulfater B was not observed in aortic tissue of dusky shark and dog fish, the most primitive species subjected to this study.

Cutaneous acid mucopolysaccharides in ascorbic acid deficiency.

Measurements have been made of the concentrations of hyaluronic acid and chondroitin sulphate, the rate of uptake of <sup>35</sup>SO<sub>4</sub> into chondroitin sulphate, and of the molecular weight of the hyaluronic acid in the skin of the guinea-pig during experimental ascorbic acid deprivation. The results indicated that the acid mucopolysaccharides do not play any significant role in this condition.

Hormonal effects on the acid mucopolysaccharide composition of the rat aorta

Summary Both thyroidectomized and methimazole-treated rats had lower concentrations of the sulfated acid mucopolysaccharides in their aortas. This decrease could be prevented or reversed by the administration of thyroxine. The same observations were made in hypophysectomized rats, and it was concluded that the acid mucopolysaccharide (AMPS) changes were due to a lack of thyroid hormone rather than to any changes in thyroid stimulating hormone (TSH) concentration. On the other hand, growth hormone specifically increased the chondroitin sulfate concentration in hypophysectomized rats, and this proceeded independently from the action of thyroxine. Cortisone decreased the amounts of chondroitin sulfate in rat aorta, but it had no effect upon the other AMPS fractions. Estradiol and testosterone administration as well as adrenalectomy and gonadectomy had no effect upon the aortic AMPS, and it was concluded that the rat aorta is mainly dependent upon thyroid hormone for the maintenance of its AMPS composition. The administration of methimazole prevented the accumulation of chondroitin sulfate in the aortas of lathyritic rats. This suggests that hypothyroidism decreases the biosynthesis of aortic AMPS.

The sulphation of chondroitin sulphate in embryonic chicken cartilage.

1. Whole tissue preparations and subcellular fractions from embryonic chicken cartilage were used to measure the rate of incorporation of inorganic sulphate into chondroitin sulphate in vitro. 2. In cartilage from 14-day-old embryos, [(35)S]sulphate is incorporated to an equal extent into chondroitin 4-sulphate and chondroitin 6-sulphate at a rate of 1.5nmoles of sulphate/hr./mg. dry wt. of cartilage. 3. Microsomal and soluble enzyme preparations from embryonic cartilage catalyse the transfer of sulphate from adenosine 3'-phosphate 5'-sulphatophosphate into both chondroitin 4-sulphate and chondroitin 6-sulphate. 4. The effects of pH, ionic strength, adenosine 3'-phosphate 5'-sulphatophosphate concentration and acceptor chondroitin sulphate concentration on the soluble sulphotransferase activity were examined. These factors all influence the activity of the sulphotransferase, and pH and incubation time also influence the percentage of chondroitin 4-sulphate formed.