Chondroitin Sulfate Isomers Research Articles

Low urinary excretion of heparan sulfate in three patients with Lowe's syndrome

Urinary glycosaminoglycans were isolated with the cetylpyridinium chloride (CPC) precipitation method and the excretion of individual species of urinary glycosaminoglycans in three patients with Lowe's syndrome was compared with that of age-matched control children by means of electrophoresis on cellulose acetate membranes and by quantification of hexosamine contents. Total daily excretion of urinary glycosaminoglycans in the patients seemed to be normal, but the relative excretion of urinary heparan sulfate was significantly reduced and ranged from 26 to 46% of the age-matched control mean, when calculated on the basis of relative glucosamine content in urinary glycosaminoglycans. Although electrophoretograms of urines from patients with Lowe's syndrome suggested some excess of low sulfated chondroitin sulfate corresponding in mobility to dermatan sulfate, the enzymatic subunit assay employing chondroitinases did not disclose any significant differences in the excretion pattern or in the degree of sulfation of chondroitin sulfate isomers between Lowe's syndrome and control children.

Intermittent hyperinsulinaemia and arterial glycosaminoglycans in dogs

The effect of insulin on the glycosaminoglycan content of the arterial ground substance was compared in age-matched alloxan-diabetic dogs, hypophysectomized dogs and normal controls. Hyaluronic acid and the three sulphated components, heparan, dermatan and isomeric chondroitin sulphates were quantitatively determined in the arch, thoracic and abdominal aorta, and in the carotid, coronary, iliac, renal and mesenteric arteries. Diabetic animals were either well or poorly controlled. Six well controlled dogs with a fasting mean plasma glucose level of 8.5 mmol/l were given around 30 units of porcine insulin/day and showed fluctuations in plasma insulin concentrations between 24 and 175 mU/l. Four poorly controlled dogs with a mean fasting plasma glucose level of 18.5 mmol/l received an average of 14 units of insulin/day, and the fluctuations ranged from 8 to 113 mU/l. Eight normal, untreated controls showed mean fluctuations between 15 and 22 mU/l. Within 14 weeks of insulin treatment, well controlled animals displayed glycosaminoglycan alterations in five arterial segments, usually involving more than one glycosaminoglycan constituent. In poorly controlled animals the number of segments that showed glycosaminoglycan alterations was the same, but abnormalities were limited to one of the sulphated components only. The coronary arteries displayed identical glycosaminoglycan alterations in the two groups of diabetic dogs, namely a significant rise in dermatan sulphate content (p < 0.01, mean±SEM) from the normal value of 1.12±0.03 mg/g dry defatted tissue to 1.31±0.03 mg/g in well controlled animals and to 1.37±0.08 mg/g in poorly controlled animals. In order to ascertain that the abnormalities in the glycosaminoglycan chemistry were related to hyperinsulinaemia rather than hyperglycaemia, six hypophysectomized dogs were treated with 1.5 U/day of porcine protamine zinc insulin, for 3 weeks. Average plasma glucose levels were in the order of 4.3 mmol/l and plasma insulin levels fluctuated between 9 and 27 mU/l; the latter represented twice the peak value seen in five untreated hypophysectomized dogs. The resulting chemical changes in the glycosaminoglycan content were in line with those encountered in diabetic animals, including the rise in dermatan sulphate content of the coronary arteries. These results indicate that: (1) hyperinsulinaemia produced by injections of insulin causes alterations of the arterial glycosaminoglycan content; (2) not all segments of the arterial tree are equally responsive to insulin and (3) the coronary arteries have a particularly insulin-sensitive dermatan sulphate metabolism.

Acidic glycosaminoglycans in human heart valves

Acidic glycosaminoglycans (AGAG) in normal human heart valves were analyzed with an enzymatic assay method using AGAG-lyases. The AGAG content was high in the mitral and aortic valves and to a lesser extent in the tricuspid valve. The amount in the three valves was high at maturation and then decreased with advancing age. No definite difference in the AGAG composition was found among the three valves. The main AGAG was hyaluronic acid (HA) constituting approximately half of the total AGAG, and dermatan sulfate (DS) and chondroitin sulfate (CS) isomers each accounted for 1 4 to 1 5 of the total AGAG. Heparan sulfate (HS) was detected to constitute several % of the total AGAG. A minor amount of oversulfated DS was also present. With advancing age, the proportions of HA and chondroitin 4-sulfate tended to decrease whereas those of chondrotin 6-sulfate, DS and HS tended to increase. A possible function of the valvular AGAG is discussed with respect to the constitutional change.

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation. The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.

An automated version of the periodate-thiobarbituric acid assay for analysis of Δ-4,5 unsaturated uronic acids and its application to the assay of hyaluronic acid and chondroitin sulfates

An automated periodate-thiobarbituric acid assay of Δ-4,5 unsaturated uronic acids which avoids extraction of chromogen has been developed and applied to the analysis of hyaluronic acid and chondroitin sulfates in standard glycosaminoglycan mixtures and in biological samples following digestion with eliminase enzymes. Assay of hyaluronic acid was linear between 0.1 and 2.5 μg of uronic acid, when digested with hyaluronidase from S. hyalurolyticus and use directly in the automated procedure. The measurement of unsaturated disaccharide standards (25–100 μ m) derived from chondroitin sulfates was also linear although the color yields were different. The proportions of chondroitin sulfate isomers were estimated by assay of the unsaturated chondroitin disaccharides which has been separated by thin-layer chromatography.

Compositional changes of urinary acidic glycosaminoglycans in progressive systemic sclerosis

The compositional changes of acidic glycosaminoglycans (AGAG) in the urine of progressive systemic sclerosis (PSS) patients were studied using chondroitinases and heparitinase in appropriate enzyme assays and by electrophoretic characterization. Daily urinary excretion of AGAG in patients with PSS was increased, when compared to normals. The proportion of urinary AGAG in PSS patients, which was undigested by chondroitinase-ABC (most probably representing heparan sulfates (HS)), increased significantly from the normal value. The substance was found to be mainly HS as determined by the electrophoretic pattern, thin-layer chromatographic analysis and by its susceptibility to heparitinase. After digestion of urinary chondroitin sulfate isomers with chondroitinases, the unsaturated disaccharides were mainly separated into 4-sulfated and 6-sulfated disaccharide units by paper chromatography. In all of the patients with PSS, the ratio of the unsaturated 4-sulfated disaccharide to the unsaturated 6-sulfated disaccharide was higher than that in normal subjects. These observations indicate an abnormal turnover of AGAG in patients with PSS.

Structural differences of acidic glycosaminoglycans of leukocytes in leukemic states

1. 1. Acidic glycosaminoglycans (AGAG) were prepared from leukocytes of patients in myeloid leukemic states. The characterization of the AGAG was performed by enzymic methods based on susceptibility of the AGAG to specific chondroitinases, hyaluronidase, and heparitinase. 2. 2. The enzymic results indicated that the AGAG in myeloid leukemic leukocytes were polydisperse. In addition to chondroitin 4-sulfate, which is the main constituent of leukocytes in normal state, myeloid leukemic leukocytes contained increased amounts of hyaluronic acid, chondroitin, and other chondroitin sulfate isomers. 3. 3. The increased proportion of nonsulfated AGAG such as hyaluronic acid and chondroitin to the total AGAG determined as the unsaturated nonsulfated disaccharides was the characteristic finding in the constituents of the AGAG in the process of leukemic leukocytes. The different constituents of the AGAG in leukemic leukocytes at various stages indicate that the heterogeneity of the AGAG reflects different cell metabolism in leukemic stage.

Enzymatic studies of urinary isomeric chondroitin sulfates from patients with mucopolysaccharidoses. The application of high performance liquid chromatography

The high-performance liquid Chromatographic (HPLC) method for the determination of unsaturated sulfated disaccharides is a comprehensive and reliable method which expedites enzymatic studies of isomeric chondroitin sulfates. Responses for these unsaturated disaccharides derived from urinary chondroitin sulfates were linear from 100 ng to 10 μg injected and good quantitation was obtained for 25 μl or less of samples placed on the column. This method which is more sensitive and accurate than methods now being used has considerable potential for the chemical diagnosis of patients with mucopolysaccharidoses and for the clarification of glycosaminoglycan structure. The isomeric chondroitin sulfates in urines from patients with mucopolysaccharidoses were studied by enzyme digestion with chondroitinases followed by HPLC determination of the sulfated unsaturated disaccharides produced. Evaluation by HPLC of the unsaturated 4-sulfated disaccharide produced by digestion of the urinary GAG with chondroitinases ABC and AC revealed rapidly and quantitatively the large amounts of dermatan sulfate present in Hurler, Hunter, and Maroteaux-Lamy urines. Chondroitin 4-sulfate predominated in Sanfilippo urinary isomeric chondroitin sulfates whereas chondroitin 6-sulfate and chondroitin 4-sulfate were shown to be present in nearly equal amounts in Morquio urine. An oversulfated chondroitin sulfate was detected in small amounts in some of these urines. This was demonstrated by the detection of an unsaturated disulfated disaccharide after digestion with chondroitinase ABC but not with chondroitinase AC.

Heterogeneity of Human Urinay Glycosaminoglycans

Crude urinary glycosaminoglycans (GAG) obtained from normal adult men were fractionated by ethanol-fractionation, followed by Dowex 1 column chromatography. The resulting acidic subfractions were qualitatively analyzed for monosaccharide composition and examined by electrophoresis on cellulose acetate membrane and paper chromatography before and after mucopolysaccharidase digestions. Some of the major subfractions were also examined by gel filtration and infrared spectral analysis. The result showed that urinary GAG were extremely heterogeneous in the molecular size, negative charge and the chemical composition. Hyaluronic acid, heparan sulfate, chondroitin sulfates A and C, chondroitin, dermatan sulfate and keratan sulfate were found in wide distribution in these acidic subfractions. Of these GAG, chondroitin sulfate isomers were distributed into almost all acidic subfractions, indicating to be the most heterogeneous.

Constitutional variations of acidic glycosaminoglycans in normal and arthritic bovine articular cartilage proteoglycans at different ages.

Proteoglycans extracted from normal and arthritic bovine articular cartilage of various ages were fractionated and purified under associative and dissociative conditions. After proteolytic digestion, the composition of the acidic glycosaminoglycans (AGAG) in the proteoglycans was determined enzymatically by digestion with chondroitinase-AC II, chondroitinase-ABC, Streptomyces, hyaluronidase and keratanase. Under both associative and dissociative conditions, uniform distribution of chondroitin sulfate (CS) isomers from proteoglycans of different ages was observed: With increasing age, the relative proportion of 4-sulfated disaccharide units in total AGAG decreased, whereas that of 6-sulfated disaccharide units increased. The relative proportion of 4-sulfated disaccharide units in total CS and the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units were greater in arthritic cartilages than in normal cartilages of the same ages. At all three ages studied, the relative proportion of 4-sulfated disaccharide units in sequential fractions increased with the decrease of cesium chloride (CsC1) density, as the proportion of 6-sulfated disaccharide units decreased. The relative proportions of hyaluronic acid (HA) and keratan sulfate (KS) increased with age. The AGAG components of cartilage proteoglycans were distributed with a certain regularity in the fractions of CsCl density gradients, but underwent changes with increasing age and in arthritic process.

Reducing terminals and molecular weights of glycosaminoglycans in normal human urine.

To elucidate the reducing terminals and the molecular weights of urinary glycosaminoglycans (GAG), the reducing power of the GAG in the acidic subfractions obtained in a previous paper (Endo et al. 1980a) was determined by the methods of Park and Johnson and of Milner and Avigard. Number-average molecular weight was calculated from the value obtained by the Park-Johnson method according to the equation of Partridge et al. The results suggested that xylose and hexuronic acid were present at the reducing terminals of the carbohydrate chains of urinary GAG, and also that 10-65% of the reducing terminals were occupied by the hexuronic acid residues. This finding strongly suggested the exertion of endoglucuronidase activity in the catabolism of the tissue GAG, specifically of chondroitin sulfate isomers. On the other hand, number-average molecular weights of urinary GAG in the subfractions ranged from 5,600 to 15,500, although most of them were within the range between 6,000 and 7,500.

Age-dependent constitution of chondroitin sulfate isomers in cartilage proteoglycans under associative conditions.

Associated proteoglycans were prepared with guanidine-HCl from bovine articular cartilage of various ages. They were purified and fractionated by equilibrium centrifugation in cesium chloride (CsCl) density gradients. The compositions of chondroitin sulfate (CS) isomers in associated proteoglycans of articular cartilages of three ages were compared based on the relative amounts of disaccharide units. The results indicated that the proportions of 4-sulfated disaccharide units comprised around 2/3, 1/3, and 1/6 of the total CS in the associated proteoglycans of calf, 18-month-old cow, and 8-year-old cow, respectively. In contrast, the proportions of 6-sulfated disaccharide units in the proteoglycans were in the reverse order; they comprised nearly 1/3, 1/2, and 2/3 of the total CS, respectively, at the three ages. Thus, with increasing age, the ratio of 4-sulfated disaccharide units to 6-sulfated disaccharide units decreased significantly. With the decrease of CsCl density in the gradients, the proportion of 4-sulfated disaccharide units to total CS as well as that of 4-sulfated disaccharide units to 6-sulfated disaccharide units increased in the associated proteoglycans of all ages. The increased ratios of 4-sulfated to 6-sulfated disaccharide units with decreasing CsCl density were significant among the individual proteoglycans: 1.84-2.36 in calf, 0.40-0.89 in 18-month-old cow, and 0.16-0.28 in 8-year-old cow.

High-performance liquid chromatographic determinations of disaccharides resulting from enzymatic degradation of isomeric chondroitin sulfates

The use of Whatman Partisil-10 PAC and Altex LiChrosorb NH 2 bonded phase columns for the high-performance liquid chromatographic separation of the disaccharides obtained from digestion of isomeric chondroitin sulfates with chondroitinases has been investigated. The substituted unsaturated disaccharides in the enzymatic degradation mixtures undergo rapid isocratic separations on both bonded stationary phases. A complete separation can be established within 10 min. The development of these procedures has expedited enzymatic studies of isomeric chondroitin sulfates. The rate of depolymerization of chondroitin sulfates by the action of chondroitinases based on quantitative high-performance liquid chromatographic separations and detection of the disaccharide products was studied.

Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice

In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.

The relationship of hormones to arterial glycosaminoglycans and atherosclerosis

Glycosaminoglycan fractions were measured in representative large and medium sized arteries of normal, hypophysectomized and hormone treated young beagles. Hyaluronate, heparan sulphate, dermatan sulphate and the isomeric chondroitin sulphates were determined in the aortic arch, thoracic and abdominal segments, in the external iliac, superior mesenteric, renal, common carotid and coronary arteries. The hormones used for replacement therapy of hypophysectomized animals were growth hormone, thyroxine, cortisone and the sex hormones testosterone, estrogen and progesterone. The sensitivity to an individual hormone was found to differ in various segments of the arterial tree; the thoracic and abdominal aorta were most responsive but renal and superior mesenteric arteries were relatively inert. The hypothesis is advanced that arteries with a GAG metabolism highly sensitive to hormones are more prone to develop athero-sclerosis than arteries that have a limited sensitivity to alterations in endocrine balance.

Rapid and sensitive determination of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography

The separation and quantitative analysis of enzymatic degradation products of isomeric chondroitin sulfates by high-performance liquid chromatography (HPLC) are described. The substituted unsaturated disaccharides which result from digestion of chondroitin sulfates with chondroitinase are quickly separated on polar adsorbents such as silica gel. The UV absorption properties of these unsaturated disaccharides permit UV measurement with detection limits of approximately 100 ng. Their separation by HPLC facilitates the use of enzymatic methods for the determination of chondroitin sulfates A, B and C. The potential of this method in clinical application is demonstrated by quantitative assays of glycosaminoglycans from a normal urine and urine from a patient with Hunter syndrome. The results are consistent with amount of isomeric chondroitin sulfates found in comparable urines by others.

Acid mucopolysaccharides in porcine gingiva.

Acid mucopolysaccharides (AMPS) were extracted from porcine gingiva. The concentration of the uronic acid extracted was 0.23% of the ry‐defatted issue. The AMPS were fractioned with a Dowex 1‐X2 column.The individual AMPS components of the various fractions obtained were determine on the basis of the contents of the hexosamine, unsaturated disaccharides, and chondroitin sulfate isomers. It was suggested from the results that the AMPS of porcine gingiva consisted of large amounts of chondrotin sulfates A and B, moderate amounts of hyaluronic acid and heparan sulfate, and a small amount of chondroitin sulfate C.

Structural subunits of chondroitin sulfate isomers isolated from different aged human kidneys

Acidic glycosaminoglycans (AGAG) were extracted from whole human kidney tissues of different aged subjects and were purified by precipitation with cetylpyridinium chloride and by an ion exchange resin column chromatography. The prepared AGAG were individually digested with chondroitinase-AC and they were further digested with chondroitinase-ABC. The resulting unsaturated disaccharides and the undigested AGAG were identified by paper chromatography, electrophoresis, and other chemical analyses. The AGAG, which were resistant to chondroitinase-AC, were mainly heparan sulfates and their proportion to total AGAG appeared to decrease with the advance of age. Dermatan sulfate plus oversulfated dermatan sulfate were composed of one-fifth to one-third of the AGAG resistant to chondroitinase-AC, and their proportion tended to increase with the advance of age.

Physicochemical Properties of Chondroitin Sulfate

1. The degree of calcium ion binding to chondroitin sulfate was larger than that of sodium ions. The association of calcium ions with the mucopolysaccharide cannot be ascribed solely to electrostatic forces. 2. No differences in the degree of ion binding of chondroitin sulfate isomers A and C were detectable. 3. The spacing between charged groups of chondroitin sulfate was found to be 0.48 nm, which is consistent with the results of X-ray analysis. 4. The characteristic ratio was large. Therefore, rotation about the backbone bonds is highly restricted.

Differences in the Constitution and Distribution of Acidic Glycosaminoglycans in the Intimal and Adventitial Layers of Bovine Aorta

Differences in the constitution of acidic glycosaminoglycans (AGAG) in the intimal layer and in the adventitia of bovine aorta were demonstrated by means of enzymatic assay and electrophoretic characterization after fractionation by ion-exchange chromatography and by gel filtration. The data obtained after ion-exchange chromatography indicated that chondroitin 4-sulfate and chondroitin 6-sulfate were the predominant constituents in the intima, whereas hyaluronic acid, dermatan sulfate, oversulfated dermatan sulfate, and heparan sulfates and a related glucosaminoglycan were the predominant constituents in the adventitia. The molecular weight dependent distribution of the aortic AGAG by gel filtration indicated that hyaluronic acid was prevalent in the high molecular weight fractions from both layers whereas chondroitin, heparan sulfates and the related glucosaminoglycan were present in the low molecular weight fraction, especially in adventitial AGAG. Chondroitin sulfate isomers, dermatan sulfate and oversulfated dermatan sulfate were distributed almost equally in all fractions, but the proportions were different in the two layers.