An improved determination of total glycosaminoglycans in body fluids by formation of complexes with quinacrine: changes in amniotic fluid total glycosaminoglycans during normal pregnancies and in pregnancies at risk for mucopolysaccharidoses.

Acidic glycosaminoglycans form insoluble complexes with quinacrine and this has been exploited for their analysis in blood, urine and amniotic fluid. The method is specific for glycosaminoglycans including keratan sulphate and the samples do not have to be deproteinized. Values for normal urine, serum and amniotic fluid are presented. Urinary total glycosaminoglycans excreted by patients with mucopolysaccharidoses were also determined. The normal changes in amniotic fluid total glycosaminoglycans have been measured between 14 weeks' gestation and term, and values are given for amniotic fluid total glycosaminoglycans in several pregnancies at risk for mucopolysaccharidoses. It is suggested that this method is a potentially valuable analytical tool in the pre-natal diagnosis of mucopolysaccharidoses.     

Study of the Hurler syndrome using cell culture: definition of the biochemical phenotype and the effect of ascorbic acid on the mutant cell

Fibroblasts from patients with Hurler syndrome retain a distinctive biochemical phenotype when grown in culture which is characterized by increased synthesis of both nonsulfated and sulfated glycosaminoglycans. Ascorbic acid reinforces the phenotypic expression of the biochemical abnormality, producing not only increased synthesis of sulfated glycosaminoglycans, but selective retention of sulfated glycosaminoglycans within the cell. Although the synthesis of nonsulfated glycosaminoglycans is also increased, these compounds, particularly hyaluronic acid are not retained by the cell but are secreted into the medium.Analyses of urine from patients with Hurler syndrome show increased absolute concentrations of nonsulfated glycosaminoglycans in addition to the expected increase in sulfated glycosaminoglycans. This indicates that the biochemical phenotype as defined in cell culture is not an artifact of the experimental model but reflects the biochemical defect in the patient.Redefinition of the biochemical defect to include nonsulfated as well as sulfated glycosaminoglycans contradicts explanations of this disease which are based on a single structural gene mutation.     

Isolation and characterization of glycosaminoglycans in human plasma.

We have described methodology for the isolation and quantitation of glycosaminoglycans present in human plasma. Plasma glycosaminoglycans can be quantitatively adsorbed on a DEAE-Sephacel ion exchanger and eluted with a salt gradient as two groups: a low-charge fraction and a high-charge fraction. The low-charge fraction consists of chondroitin sulfate with a low sulfate content and the high-charge fraction consists of heparan sulfate, chondroitin sulfate, and keratan sulfate (type I). We have determined the plasma concentration of each of these glycosaminoglycans in six normal human subjects. We have established that none of the glycosaminoglycans in plasma are covalently linked to plasma proteins. All are isolated as complexes with plasma proteins in noncovalent linkages. The glycosaminoglycans in the low-charge fraction are bound with high affinity to a single plasma glycoprotein by a lectin-type bond that can be disrupted by a simple glycoside. The high-charge fraction contains three major proteins and several minor proteins associated with the glycosaminoglycans by both lectin-type and ionic bonding. The plasma proteins associated with glycosaminoglycans represent less than 0.5% of the total plasma proteins. Little is known about the physiologic role of the plasma glycosaminoglycans as components of metabolic processes. Because glycosaminoglycans have been implicated in lipid metabolism and atherosclerosis, we tested all of these compounds, isolated in free form, on the in vitro hydrolysis of triglycerides by lipoprotein lipase. Plasma heparan sulfate stimulated the rate of this reaction severalfold. All other plasma glycosaminoglycans were inactive. Thus, plasma heparan sulfate may play an important role in plasma lipoprotein metabolism.     

Increased glycosaminoglycan accumulation as a genetic characteristic in cell cultures of one variety of dominant dystrophic epidermolysis bullosa.

Fibroblast cultures from patients with dominant dystrophic epidermolysis bullosa of the albopapuloid variety display deranged glycosaminoglycan metabolism. These cells accumulate increased amounts of sulfated glycosaminoglycans. The mechanism for the greater content of glycosaminoglycans appears to be related to increased synthesis. During the first 6-12 h, intracellular labeled glycosaminoglycans accumulated in the dominant dystrophic epidermolysis bullosa cells at about twice the rate as that of control fibroblasts. In addition, secretion of sulfated glycosaminoglycans was two- to threefold greater than in control cultures. In contrast, both pulse-chase and cross-correction experiments failed to show any evidence for defective degradation of the material. The biochemical trait is genetically specific for albopapuloid dominant dystrophic epidermolysis bullosa, since fibroblasts from patients with other varieties of epidermolysis bullosa did not accumulate increased glycosaminoglycans. The data suggest that in vitro abnormality in glycosaminoglycan metabolism could serve as an important marker for this variety of epidermolysis bullosa and be of genetic and prognostic value in the sporadic patient with epidermolysis bullosa. Although the precise relationship of the defect to the disease has not yet been defined, it is possible that excessive tissue accumulation of glycosaminoglycans may alter collagen fibril deposition, thus, impairing the structural integrity of the skin and leading to posttraumatic blisters and erosions that characterize the disease.     

The Synthesis of Glycosaminoglycans by Cultures of Corneal Stromal Cells from Patients with Keratoconus

Keratoconus is a disease that results in thinning and ectasia of the central cornea. Cultures of corneal stromal cells from patients with keratoconus were established and the synthesis of glycosaminoglycans compared with the synthesis of glycosaminoglycans by normal human corneal stromal cells in culture.Keratoconus and normal control cell cultures were incubated with sodium [(35)S]sulfate and [(3)H]glucosamine for 4 h. After incubation, the labeled glycosaminoglycans were isolated from the medium fractions and cells. Keratoconus and normal control cultures synthesized similar amounts of sulfated glycosaminoglycans independent of the age of donors and(or) the number of subcultures. In contrast to normal control cultures, most of the newly synthesized glycosaminoglycans produced by keratoconus cells were found in the growth medium and much less were in the cell layer.Treatment with glycosaminoglycan-degrading enzymes followed by paper chromatography showed that keratoconus cells, as normal control cells, produced hyaluronic acid and various sulfated glycosaminoglycans. The production of cell layer-related heparan sulfate was markedly reduced in keratoconus cultures. Because heparan sulfate has been shown to be associated with cell surfaces, the decreased heparan sulfate content could reflect changes at this location.     

Therapeutic value of glycosaminoglycans in cancer

Glycosaminoglycans are unbranched polysaccharides composed of repeating units of alternating uronic acids and amino sugars. Most glycosaminoglycans are covalently attached to core proteins to form proteoglycans. Posttranslational modifications result in specific motifs that bind to a large variety of ligands, thus regulating growth factor signaling, cellular behavior, inflammation, angiogenesis, and the proteolytic environment. Dysregulated expression of glycosaminoglycans is present in cancer and reported to correlate with clinical prognosis in several malignant neoplasms. Recent knowledge on the biological roles of these molecules in cancer biology, tumor angiogenesis, and metastasis has promoted the development of drugs targeting them. Pharmaceutical approaches include the use of chemically modified heparins and glycosaminoglycans with defined structures, combination of inhibitors of glycosaminoglycan biosynthesis and polyamine depletion, and biologically active glycosaminoglycan-binding peptides. In addition, glycosaminoglycans are used as tumor-specific delivery and targeting vehicles for toxins and chemotherapeutics. Encouraging results in animal studies and clinical trials show the clinical relevance of glycosaminoglycan-based drugs and the use of glycosaminoglycans as therapeutic targets.     

Cellular glycosaminoglycans in lymphocytes from patients with cystic fibrosis.

Cellular glycosaminoglycans were isolated from lymphocytes from patients with cystic fibrosis and controls. The isolated glycosaminoglycans were fractionated by cellulose acetate electrophoresis, analyzed for glucosamine and galactosamine content, and subjected to hydrolysis with bovine testicular hyaluronidase. The total glycosaminoglycan content, the per cent glucosamine and galactosamine, and the distribution of cellular glycosaminoglycans in circulating lymphocytes in cystic fibrosis were no different from controls.     

Laser nephelometric determination of glycosaminoglycans--method and application

Light scattering due to the formation of insoluble complexes between the long-chain quaternary ammonium salt N-cetylpyridinium chloride and glycosaminoglycans was utilized for a relative simple, sensitive and precise determination of total and specific types of glycosaminoglycans by laser nephelometry. The addition of the ammonium salt to solutions of various glycosaminoglycans in 0.03 mol/l NaCl produces a time-dependent increase in light scattering, which reaches a maximum between 14 and 18 h of complex formation, irrespective of the type of glycosaminoglycan studied. Only keratan sulphate does not generate light scattering, and is therefore not detectable by the procedure. The scattering of laser light by certain types of sulphated glycosaminoglycans (e.g. heparan sulphate, heparin) depends more on the degree of sulphation (charge density) than on chain length within a certain range. Optimum light scattering was found at 28 mmol/l N-cetylpyridinium chloride and at a ionic strength around 0.03 mol/l NaCl. The detection limits and linear ranges of the individual glycosaminoglycans were evaluated. For the determination of chondroitin sulphate, laser nephelometry is at least 8 times more sensitive and much more simple than the modified carbazole method (glucuronic acid). The intra-assay and inter-assay coefficients of variation are about 4% and 7%, respectively. Laser nephelometry is much more sensitive than turbidimetry. Complex synthetic mixtures of glycosaminoglycans and biological fluids were accurately differentiated by successive chemical and enzymatic degradation of the respective glycosaminoglycans followed by the measurement of the resulting reduction of laser light scattering. In synovial fluids from non-inflammatory joint diseases, light scattering (units/ml) was about 4.5 times higher than in synovial fluids from inflammatory articular lesions. In both pathologic conditions nearly all of the light scattering can be attributed to hyaluronic acid.     

Interaction between endogenous circulating sulfated-glycosaminoglycans and plasma proteins

Interaction between endogenous 35S-labelled plasma glycosaminoglycans and proteins in murine plasma was demonstrated by coelution from gel chromatography of circulating 35S-labelled glycosaminoglycans with a wide range of plasma proteins. Autoradiography of electrophoretic tracing of proteins from 35S-sulfate labelled plasma showed that labelled glycosaminoglycans were associated with alpha 1, alpha 2, beta globulins and albumin, but not with gamma globulins. Analysis by gel chromatography on Sepharose CL-6B of delipidated 35S-labelled plasma after either proteolysis or beta-elimination, suggested that 35S-labelled glycosaminoglycan chains were covalently bound to proteins. Lipids were probably involved in the supramolecular assembly of GAGs with plasma proteins, as shown by hydrophobic interaction chromatography. In addition, strong, non-covalent interaction between glycosaminoglycan chains and proteins was responsible for the difficulty in extracting 'free' glycosaminoglycans from plasma. Consistently, ion-exchange chromatography of 35S-sulfate labelled delipidated plasma after alkali treatment, revealed that the anionic properties of glycosaminoglycans were hampered when plasma proteins were present.     

A heparin-like anticoagulant as part of global abnormalities of plasma glycosaminoglycans in a patient with transitional cell carcinoma

A patient is described in whom a circulating heparin-like anticoagulant developed during the terminal course of metastatic transitional cell carcinoma. The anticoagulant, which was identified as heparan sulfate, was the clinical sign of global abnormalities in the patient's plasma glycosaminoglycans. Subsequent analysis disclosed increased amounts of chondroitin sulfate as well as heparan sulfate. In addition, the charge density and molecular weight of the patient glycosaminoglycans and their organization into proteoglycans differed significantly from glycosaminoglycans isolated from normal plasma samples.     

Increase of dermatan sulfate in a case of pulmonary fibrosis.

The quantity and composition of acid glycosaminoglycans (mucopolysaccharides) were compared between a fibrotic lung and normal lungs. The acid glycosaminoglycans were isolated by proteolysis, fractionation with ethanol and precipitation with cetylpyridinium chloride, and were identified by chromatography, electrophoresis and incubation with mucopolysaccharide-lyases. Evidence was obtained for the presence of hyaluronic acid, chondroitin sulfate A(C), dermatan sulfate and heparan sulfate. Quantitation of individual glycosaminoglycans by using specific mucopolysaccharide-lyases revealed that the quantity of dermatan sulfate in the fibrotic lung exceeded that in the normal lungs.     

Urinary glycosaminoglycans in patients with hypothyroidism and in healthy subjects

Although the changes in urinary glycosaminoglycans have been investigated in several endocrinopathies, no information was hitherto available on the content and composition of urinary glycosaminoglycans in hypothyroidism. Urinary glycosaminoglycans were therefore investigated in patients with hypothyroidism and in healthy subjects. The total daily excretion of urinary glycosaminoglycans was found to be significantly increased (by 41%) in hypothyroidism. Two electrophoretic bands were always detected in both examined groups: a major band of chondroitin sulphate and a minor band of heparan sulphate. Heparan sulphate and chondroitin sulphate levels were respectively 114% and 42% higher in patients with hypothyroidism than in controls. The respective increases in chondroitin-4-sulphate and chondroitin-6-sulphate were 31% and 41%. The relative quantities of chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate and non-sulphated chondroitin sulphate were unchanged in the two examined groups. The changes observed in the levels of the excreted glycosaminoglycans may reflect the altered metabolism of connective tissue in hypothyroidism.     

Isolation and preliminary characterization of glycosaminoglycans in human plasma

Plasma glycosaminoglycans were isolated, after pronase digestion by precipitation with cetylpyridinium chloride. The glycosaminoglycans in plasma were found exclusively in the protein fraction precipitated by trichloroacetic acid. The concentration of glycosaminoglycans in normal plasma was 168 to 272 μg per 100 ml.

Electrophoresis of plasma glycosaminoglycans in barium acetate buffer on cellulose acetate revealed three fractions, all resistant to streptomyces hyaluronidase but susceptible to testicular hyaluronidase. Electrophoretic mobility of the major fraction was similar to dermatan sulfate and hyaluronic acid. Enzymatic assay with chondroitinases, however, indicated that the major constituent of plasma glycosaminoglycans was undersulfated chondroitin sulfate composed of equimolar amounts of unsaturated non-sulfated disaccharides and unsaturated 4-sulfated disaccharides at the disaccharide subunit levels.

The other two fractions were identical with chondroitin sulfate A and C. The quantity of chondroitin sulfate C was least. Hyaluronic acid and over-sulfated chondroitin sulfate were not demonstrated in plasma.     

Measurement of plasma glycosaminoglycans: an extension of the method

The measurement of plasma glycosaminoglycans has been modified to measure not only the material which is retained on ECTEOLA cellulose but also that which may be retained on a stronger anion exchanger, Dowex 1× 2 resin. The extension of the method may be useful in the study of conditions in which the degradation of glycosaminoglycans may be impaired.     

Studies on the composition of urinary glycosaminoglycans and oligosaccharides in patients with mucopolysaccharidoses who were receiving fibroblast transplants

This communication reports studies on the composition of the urinary glycosaminoglycans and oligosaccharides in mucopolysaccharidosis patients who were being treated by fibroblast transplantation. The urinary glycosaminoglycans were precipitated with 9-aminoacridine, the oligosaccharides remaining in solution. Both fractions were further subfractionated by gel filtration. The glycosaminoglycan subfractions were examined for their content of iduronic acid, glucuronic acid, galactosamine and glucosamine. We found no changes in these parameters in a patient who had been treated by repeated fibroblast transplantations over the course of 4 1/2 years. The amino sugar composition of the oligosaccharide fraction was examined and shown to be unchanged. We also found no changes in the degree of sulphation of the urinary glycosaminoglycans specifically related to the transplant in four patients with Hurler disease and two with Hunter disease. We conclude that fibroblast transplantation does not produce detectable changes in the carbohydrate content or degree of sulphation of the urinary glycosaminoglycans and oligosaccharides.     

Synthesis of hepatic glycosaminoglycans in the early stages of galactosamine hepatitis: a rapid decline of heparan sulfate is followed by elevation of chondroitin sulfate and dermatan sulfate

Administration of a single dose of D-galactosamine to rats causes time-dependent, biphasic changes of total glycosaminoglycan synthesis in liver. A rapidly occurring inhibition is followed by a significantly enhanced (greater than 2 fold) production of 35S-labeled glycosaminoglycans in later stages of injury. Degree and duration of the inhibitory phase are dose-dependent; 50% inhibition is reached at 80 mg/kg and maximum inhibition (nearly 80%) at about 300 mg/kg body weight 2 h after injection of D-galactosamine. The hepatotoxin impairs preferentially the production of heparan sulfate, whereas that of chondroitin sulfate and dermatan sulfate is diminished only slightly and for a rather short period of time. The synthesis of the latter, however, is more stimulated than that of heparan sulfate in later stages of injury. The specific radioactivity of 35S-labeled 3'-phosphoadenosine-5'-phosphosulfate (PAPS) did not change significantly during the course of acute liver damage. Glycosaminoglycan synthesis in regenerating liver was nearly unaffected by D-galactosamine. Uridine at the dose applied partially reversed D-galactosamine-inhibited synthesis of proteoheparan sulfate. In accordance with the labeling studies the content of glucosamine-containing glycosaminoglycans in treated liver decreased, whereas that of galactosamine-containing glycosaminoglycans slightly increased, resulting in a nearly 50% reduction of the glucosamine/galactosamine ratio 5 h after administration of D-galactosamine. Ion exchange chromatographic studies of 35S-labeled specific types of glycosaminoglycans from normal and galactosamine-injured liver revealed only minor structural differences.     

Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans

BACKGROUND: The relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis. METHODS: Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. The results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography. RESULTS: Densitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25x10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. In comparison, DMB assays gave much higher results - up to 200 mg/l and 500x10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB. CONCLUSION: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions.     

Urinary glycosaminoglycans in aspartylglycosaminuria: evidence for disturbed proteoglycan metabolism

An abnormal excretion pattern of urinary glycosaminoglycans was found in patients with aspartylglycosaminuria, a lysosomal storage disorder of glycoprotein metabolism. The mean daily GAG excretion, measured as uronic acids, was within the reference range, though higher than that of matched controls. However, in AGU patients fractionation of isolated urinary glycosaminoglycans revealed markedly increased proportions of heparan sulfate which were nearly 50% of the total glycosaminoglycans. The changes observed in glycosaminoglycan excretion reflect abnormalities of proteoglycan metabolism. They offer further evidence for the presence of a generalized connective tissue disorder in aspartylglycosaminuria. Increase of heparan sulfate may also refer to abnormalities of glycosaminoglycan metabolism in the central nervous system with a possible role in the neurological manifestations of the disorder.     

Excessive Ingestion of Fluoride and the Significance of Sialic Acid: Glycosaminoglycans in the Serum of Rabbit and Human Subjects

Abstract

The levels of sialic acid and glycosaminoglycans were explored in the sera of rabbit and human subjects who ingested fluoride and had clinical manifestation of fluorosis. Changes observed in the level of these chemical constituents in sera possibly reflect changes occurring in calcified and noncalcified tissues due to fluoride intoxication. The ratio of sialic acid content vs glycosaminoglycans revealed there was a 50% reduction in rabbit and human sera. The test is recommended for evaluating the prognosis of fluoride poisoning/fluorosis.     

A new quantitative assay for glycosaminoglycans.

A direct dye-binding technique has been developed to measure total glycosaminoglycans in urine. Fifty or 100 microliters of urine was mixed with a solution of Azure A (10 mg/l) and Azure B (10 mg/l) and the resulting decrease in absorbance at 610 nm was measured. The standard curve with chondroitin sulfate C was linear from 1 to 20 micrograms/assay. The normal value for total urinary glycosaminoglycans in adults was 1.8 +/- 0.6 g/mol creatinine. Other normal values were age-dependent. The assay is inexpensive, simple, precise, sensitive and suitable for screening for the mucopolysaccharidoses in pediatric patients.