Simple spectrophotometric quantification of urinary excretion of glycosaminoglycan sulfates

We describe a simple, rapid, precise, and sensitive spectrophotometric method for measuring urinary glycosaminoglycan (GAG) sulfate excretion. The GAG sulfates are precipitated with cetylpyridinium chloride, resuspended in water, and mixed with the basic dye 1,9-dimethylmethylene blue to produce a complex with the polyanionic molecule of sulfated GAGs. Absorbance is read at 535 nm. The standard curve for reaction was linear up to 12 micrograms of the different GAGs: dermatan sulfate, heparan sulfate, keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate. Within- and between-run precision (CV), measured at three different GAG concentrations (normal and pathological), varied from 1.6% to 2.5% and from 1.8% to 4.5%, respectively. Analytical recovery ranged from 71% to 107%. Urinary GAG excretion, measured by this procedure, correlates (r = 0.837; p less than 0.001) with the values obtained with the borate-carbazole reaction (Anal Biochem 1962;4:330-4).     

Increased excretion of urinary glycosaminoglycans in meningococcal septicemia and their relationship to proteinuria

OBJECTIVES: Meningococcal septic shock is a devastating illness associated with an increase in vascular permeability leading to hypovolemia and accumulation of plasma proteins and fluid in tissues. The capillary leak syndrome is often associated with widespread thrombosis in the skin, limbs, and digits. We postulated that the increase in vascular permeability and the intravascular thrombosis might be caused by an inflammation-induced loss of endothelial and basement membrane glycosaminoglycans (GAGs), which play a role in the permeability and thromboresistant properties of the microvasculature. DESIGN: Prospective, single-center observational study. SETTING: University-affiliated meningococcal research unit and pediatric intensive care unit. PATIENTS: Eighteen children requiring intensive care for meningococcal sepsis, 18 children with steroid-responsive nephrotic syndrome, and 18 healthy control children. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serum concentrations and urine excretion of glycosaminoglycans were measured and related to changes in glomerular permeability to plasma proteins. The size-distribution and nature of glycosaminoglycans were defined by Polyacrylamide Gel Electrophoresis and specific enzyme digestion. Urinary excretion of heparan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate were significantly increased in meningococcal disease (MD) relative to healthy controls and children with steroid-responsive nephrotic syndrome. The urinary GAGs in MD were of similar size to those in controls when analyzed after pronase digestion. However, analysis of proteoglycan size before proteolytic digestion showed the urinary GAGs in MD were of lower molecular weight and unattached to proteins. The fractional excretion of albumin and immunoglobulin G in MD increased with severity of disease. Patients with severe or fatal MD had albumin clearances overlapping those seen in steroid-responsive nephrotic syndrome. There was a significant correlation between proteinuria in MD and urinary excretion of heparan sulfate (r2 = 0.611, p < .0001), chondroitin-4-sulfate (r2 = 0.721, p < .0001), and chondroitin-6-sulfate (r2 = 0.395, p < .0001). CONCLUSIONS: The capillary leak in meningococcal disease is associated with increased plasma and urine concentrations of GAGs, which may be proteolytically cleaved from endothelial and basement membrane sites. The correlation between the severity of protein leakage and the urine excretion of GAGs suggests that loss of GAGs may be causally related to the increase in permeability to proteins.     

Antioxidant activity and structural modifications of serum chondroitin sulfate in Graves' disease

ObjectivesDisturbances in the metabolism of proteoglycans/glycosaminoglycans related to free radical overproduction can be detected in Graves' ophthalmopathy. However, it is not evident whether these disorders appear before the clinical manifestation of ophthalmopathy and contribute to the development of nonthyroidal symptoms. They might also reflect the body's defence against the effects of an autoimmune attack, or they merely result from these disorders. Our study conducted in Graves' patients, free of ocular changes at the time of recruitment, is an attempt at explaining these ambiguities. We decided to investigate the relation between the changes in extracellular matrix components, namely the total glycosaminoglycans (GAGs) and chondroitin sulfates (CS), as well as parameters characterising oxidative stress, i.e. the total antioxidant status (TAS), and the thiol group (TG) concentration.Design, methods, resultsGAGs, TAS and TG were quantified in serum samples obtained from 30 healthy subjects and 30 Graves' patients before and after treatment. An increased serum concentration of total GAGs and CS in Graves' patients before and after treatment was observed. Additionally, increased sulfation of CS in both Graves' patient groups was recorded. The serum TG and TAS concentrations in untreated patients were significantly decreased. Subsequent euthyreosis led to the normalisation of TG concentration. We have revealed a significant correlation between changes of extracellular matrix components, especially chondroitin-4-sulfate, thyroid status parameters, and parameters characterising oxidative stress.ConclusionsThe structural modifications of serum CS in Graves' patients clinically free of ocular complications are probably the expression of systemic connective tissue remodelling. Simultaneously, these modifications may participate in a defence response to free radical damage underlying this pathology.     

The glycosaminoglycans of the human artery and their changes in atherosclerosis.

The changes in levels of glycosaminoglycans (GAGs) of the intima and media of the human artery in atherosclerosis were determined by a recently introduced two-dimensional electrophoresis technique that permits direct measurments of each of these macromolecules. To identify the arterial GAGs, they were fractionated by chromatography on a DEAE-Sephadex A-25 column, and the resulting three fractions (hyaluronic acid [HA], heparan sulfate [HS], and the partially separated chondroitin sulfates B [CSB] and C [CSC]) were analyzed for their electrophoretic mobilities by this electrophoretic method, for their digestability by highly specific hydrolases (leech hyaluronidase, heparinase, and chondroitinases ABC and AC) and for their iduronic acid content. From these studies we concluded that normal and atherosclerotic human aortas contain CSB, CSC, HA, and HS. Further, we demonstrated that CSB is a hybrid consisting of approximately 40% CSA and 60% CSB and that CSC appears to be a polymer consisting essentially of glucuronic acid and N-acetylgalactosamine-6-sulfate. Classical CSA as well as chondroitin (CH) were not present in detectable amounts. In the relatively normal intima, the mean concentrations of the GAGs were found to be 4.7, 20.9, 1.3, and 5.1 mg/g of dry, defatted, decalcified tissue for CSB, CSC, HA, and HS, respectively. With the progression of atherosclerosis, there was a pronounced decrease in the total GAG content (from 32 to 18 mg) associated with a decrease in the CSC and HS levels but without a change in the HA concentrations. Of particular interest, however, was the increase in the CSB level. In the media whose total GAG content averaged approximately 20 mg, no significant changes in these GAG levels were noted with the progression of the disease except for that of CSC. These findings may be important in explaining the increased lipoprotein and collagen deposition in the diseased aorta.     

Effects of helium-neon laser on levels of stress protein and arthritic histopathology in experimental osteoarthritis

OBJECTIVE: To investigate the effect of low-power laser therapy on levels of stress proteins (SPs) in experimental arthritis and their relation to the bioeffects on arthritic cartilage repair. DESIGN: A total of 42 rats with similar degrees of induced arthritis evaluated by means of bone scan were divided randomly into two groups. In the treated group, 21 rats received helium-neon laser treatment; in the control group, 21 rats received sham laser treatment. The changes in chondrocytes of SPs were measured by electrophoresis of proteins extracted from chondrocytes of arthritic cartilage at various time periods. The histopathologic changes and the presence of SP of arthritic cartilage were identified by hematoxylin and eosin stain and by immunostains of SP72 antibody individually from frozen sections of arthritic cartilage. RESULTS: SP density increased markedly in rats after laser treatment and was closely related to the repair of arthritic cartilage. Furthermore, the pathohistology of arthritic cartilage improved significantly with the decline of SP levels in the follow-up period. CONCLUSION: Helium-neon (632 nm) low-power laser can enhance SP production in arthritic chondrocytes. The extragenic production of SP is well correlated with the therapeutic effect of low-power laser in preserving chondrocytes and the repair of arthritic cartilage in rats.     

Glycosaminoglycans of skin and urine in pseudoxanthoma elasticum: evidence for chondroitin 6-sulfate alteration

Abnormally elevated hyaluronic acid and dermatan sulfate were isolated from lesional skin of a patient with severe pseudoxanthoma elasticum. These glycosaminoglycans (estimated as uronic acid) surpassed the normal controls by 33.6 and 4.8 magnitudes, respectively. Urine chondroitin 6-sulfate of the same patient moved faster by electrophoresis on cellulose acetate than its counterpart isolated from urine of another four patients or from the normal controls. Hyaluronic acid and chondroitin 6-sulfate exceeded their counterparts in normal urine by 2- to 10- and 4- to 18-folds, respectively. These increments correlated with the pseudoxanthoma elasticum severity in three of the five patients studied. The data showed: the first conclusive evidence that dermatan sulfate increased in lesional skin of a patient, with severe pseudoxanthoma elasticum, who also had considerably augmented HA, alteration of the same patient's urine chondroitin 6-sulfate, and diversified urine glycosaminoglycans in pseudoxanthoma elasticum.     

Composition of urinary glycosaminoglycans in a patient with relapsing polychondritis

OBJECTIVES: Several investigators have reported an increase in urinary glycosaminoglycans (GAGs) in patients with relapsing polychondritis (RP). The aim of this investigation is to analyze the composition and structure of urinary GAGs from a Brazilian patient with RP. DESIGN AND METHODS: The identification and structural analyses of the GAGs were made by electrophoresis and degradation with specific enzymes and identification of their disaccharides products by HPLC chromatography. RESULTS: The disaccharide products formed from RP urinary chondroitin sulfate (CS) by action of chondroitin ABC lyase showed a substantial relative increase of nonsulfated disaccharides with a relative decrease of 6-sulfated disaccharides compared to control subjects. In addition, a significant change of the ratio of CS and heparan sulfate was also observed in the RP patient. CONCLUSION: The RP patient analyzed has shown a structural anomaly of the urinary CS and this may contribute to the diagnosis of this disease.     

Galactose 6-sulfate sulfatase activity in Morquio syndrome

We have prepared a new substrate (o-beta-D-sulfo-galactosyl-(1-4)-beta-D-6-sulfo-2-acetamido-2-deoxyglucosyl- (1-4)-D-[1-3H]galactitol), from shark cartilage keratan sulfate, for the assay of galactose 6-sulfate sulfatase activity. Using this substrate, we found there was a striking deficiency of galactose 6-sulfate sulfatase activity, in addition to the known deficiency of N-acetylgalactosamine 6-sulfate sulfatase, in the cultured skin fibroblasts of patients with Morquio syndrome. Our results could be explained by the hypothesis that accumulation of keratan sulfate and chondroitin 6-sulfate in Morquio syndrome is due to a deficiency of galactose 6-sulfate sulfatase and N-acetylgalactosamine 6-sulfate sulfatase activity, which are necessary for the degradation of these two mucopolysaccharides.     

Ultrasound effect on level of stress proteins and arthritic histology in experimental arthritis.

OBJECTIVE: To investigate the effect of ultrasound on the levels of stress protein in experimental arthritis and their relation to the therapeutic effects on arthritic cartilage repair. METHODS: Thirty-six rats with similar degree of early arthritis were divided at random into two groups. In group s, 18 rats received sonication; in group c, 18 control rats received sham sonication. The severity of induced arthritis was evaluated from bone scan with technetium-99m. The presence and changes of stress protein were identified from immunostain of SP72 antibody from frozen sections of arthritic cartilage and from electrophoresis of proteins extracted from chondrocytes of arthritic cartilage. The severity index, density of immunostained chondrocytes, and histopathologic changes at various periods were also studied in each group. RESULTS: The density of stress protein was increased markedly in treated rats after sonication and it was closely related to the repair of arthritic cartilage. Once the pathohistology of arthritic cartilage improved, stress protein declined significantly in the follow-up period. CONCLUSION: Therapeutic ultrasound can enhance stress protein production in arthritic chondrocytes. The extragenic production of stress protein was well correlated with the therapeutic effect of ultrasound to preserve chondrocytes and bring about the repair of arthritic cartilage.     

Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease.

BACKGROUND: The levels and fine structure of complex polysaccharides, glycosaminoglycans (GAGs), were determined in segments of the posterior mitral valve leaflet (MVL) taken from 15 patients affected by mitral regurgitation and degenerative disease and were compared with segments from 15 multiorgan donors. METHODS: MVL GAGs were analyzed by agarose gel electrophoresis, and by HPLC and fluorophore-assisted carbohydrate electrophoresis to evaluate disaccharide patterns after treatment with chondroitinase ABC. RESULTS: GAGs from the control group were composed of approximately 37% hyaluronic acid and 63% chondroitin sulfate/dermatan sulfate with a charge density of approximately 0.61. Chondroitin sulfate/dermatan sulfate polymers contained approximately 23% of the disaccharide sulfated in position 6 on N-acetyl-galactosamine, approximately 38% of the 4-sulfated disaccharide and approximately 2% of the non-sulfated disaccharide (with a 4-sulfated/6-sulfated ratio of 1.7). The total amount of GAGs was 0.66 microg/mg tissue. The total amount of GAGs in patients suffering from mitral regurgitation and degenerative disease was approximately 51.5% higher (although the difference was not significant, probably because of the low number of subjects enrolled in the study). However, significantly higher hyaluronic acid content (approx. +38%, p<0.05) and lower sulfated GAG content (approx. -21%, p<0.005) were demonstrated. As a consequence, the total charge density decreased by approximately 23% (p<0.005). This macro-modification of GAG composition was also followed by a micro-alteration of the structure of the sulfated polysaccharides, in particular with a significant decrease in the 4-sulfated disaccharide (and a parallel increase in hyaluronic acid content) with no modification of the percentage of the 6-sulfated and non-sulfated disaccharides (with a significant decrease in the 4-/6-sulfated ratio). CONCLUSIONS: We assume that changes in the relative amount and distribution of GAGs in posterior MVL in subjects suffering from mitral regurgitation and degenerative disease are consistent with a decrease in the tension to which these tissues are subjected and with an abnormal matrix microstructure capable of influencing the hydration and of conditioning the mechanical weakness of these pathological tissues.     

Turnover of S35-sulfate in epiphyses and diaphyses of suckling rats; nature of the S35-labelled compounds

S(35)-sulfate was injected intraperitoneally- into 7-day-old rats and their long bones were removed after intervals of time. The epiphyses were separated from the diaphyses for analysis. From the diaphyses freed of bone marrow about 82 per cent of the S(35) which they contained was extracted with a 2.5 N solution of sodium hydroxide. More, about 91 per cent of the S(35), was thus extracted from the epiphyses. Dialysis of the extracts against water showed that the fraction of S(35) which was dialyzable decreased rapidly with time. After 1 hour about 80 per cent and 50 per cent of the S(35) in the extracts of diaphyses and epiphyses, respectively were found in the dialysates, after 24 hours about 20 per cent and 4 per cent, and after 120 hours 12 per cent and 1 per cent. Similar values for the S(35) in inorganic sulfate were found when the extracts were chromatographed on an anion exchange resin, dowex-2. The S(35), other than inorganic sulfate, was in the form of bound sulfate, which was released by acid hydrolysis. Uronic acid and hexosamines, primarily galactosamine, were associated with the S(35). Indeed, on paper electrophoretograms and paper chromatograms the major S(35)-labelled component which was seen resembled chondroitin sulfate in its mobility. On the paper chromatograms, also a second S(35)-labelled component with a mobility lower than that of chondroitin sulfate was found. It is unlikely that the latter is a breakdown product of chondroitin sulfate, produced in the course of extraction with the sodium hydroxide solution. In fact, both components were also found in sodium versenate homogenates which had been dialyzed extensively against water. On the basis of these results it is suggested that the greatest part of the S(35)-labelled materials previously demonstrated by autoradiography to be progressively deposited in the metaphyses after 24 hours,-as the concentration of S(35)-sulfate concurrently decreased in the epiphyseal cartilage plates,-are akin to the chondroitin sulfate of the epiphyseal cartilage plates and are derived from the latter.     

Protective effect of Withania somnifera root powder in relation to lipid peroxidation, antioxidant status, glycoproteins and bone collagen on adjuvant-induced arthritis in rats

The present investigation was carried out to evaluate the protective effect of Withania somnifera Linn.Dunal (family-Solanaceae), commonly known as Ashwagandha, on adjuvant-induced arthritic rats. Results were compared with those for Indomethacin, a nonsteroidal anti-inflammatory drug. Arthritis was induced by intradermal injection of complete Freund's adjuvant (0.1 mL) into the right hind paw of Wistar albino rats. Withania somnifera root powder (1000 mg/kg/day) and Indomethacin (3 mg/kg/day) were orally administered for 8 days (from 11th to 18th day) after adjuvant injection. The anti-arthritic effect of W. somnifera root powder was assessed by measuring changes in lipid peroxidation, antioxidant status, and glycoprotein levels in plasma and spleen of arthritic animals. In addition, cartilage degradation was also assessed by estimating bone collagen, and urinary constituents in arthritic animals. Results of the present investigation showed significant increase in the level of lipid peroxides, glycoproteins, and urinary constituents with the depletion of antioxidant status and bone collagen in arthritic animals. These biochemical alterations observed were ameliorated significantly by oral administration of W. somnifera root powder (1000 mg/kg body weight) in arthritic animals. The results of this study clearly indicate that W. somnifera root powder is capable of rectifying the above biochemical changes in adjuvant arthritis.     

Content and synthesis of glycosaminoglycans in the developing lung.

The function of lung is fundamentally linked to the connective tissue composition of the alveolar interstitium. The composition and synthesis of one class of interstitial connective tissue components, the glycosaminoglycans (GAG), was determined in lung parenchyma of rabbits at different stages of development. Parenchymal GAG content ranged between 0.2 and 0.4% (wt/wt) of dry weight, with highest concentration in adult lung. There were significant changes in types of GAG present at different ages. Fetal lungs contained a relatively high proportion of chondroitin 4-sulfate while the GAG in lung parenchyma of older animals was predominantly dermatan sulfate, heparan sulfate, and heparin. Methods were developed for the study of rates of synthesis of GAG by incorporation of [1-14C]glucosamine into lung explants. The rate of synthesis of total GAG per cell increased with development to a maximum in lung from weanling rabbits and fell to low rates of synthesis in mature rabbits. Fetal rabbit lung parenchyma synthesized mostly hyaluronic acid and heparan sulfate, while in weanling rabbit parenchyma hyaluronic acid and chondroitin 4/6-sulfate synthesis was greatest. In mature animals, the rates of synthesis of all types of GAG were relatively low but there was a relatively greater emphasis on synthesis of dermatan sulfate and heparin. These results may have significance in changes in lung function during development and in effects on other connective tissue components.     

Enzymatic determination of urinary glycosaminoglycans from orthopedic patients

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis. The GAG compositions of this fraction and the TCA-soluble fraction were determined by digestion with mucopolysaccharidases (chondroitinase AC, chondroitinase B, chondroitinase C, heparitinase and Streptomyces hyaluronidase). When the amount of the crude GAG fraction was small, no significant amount of the TCA-insoluble peptide-bound GAG fraction was obtained. The GAG composition of this case was also determined by the same procedures after direct alkali-treatment of the crude GAG fraction. The data indicated that the proportion of the TCA-insoluble peptide-bound GAG fraction was very small. The alkali-treated TCA-insoluble peptide-bound GAG fraction contained a larger proportion of heparan sulfate than the TCA-soluble GAG fraction. It was clearly demonstrated that the patients with Werner's syndrome and mucopolysaccharidosis I-S (Scheie) excreted large amounts of hyaluronic acid and dermatan sulfate respectively, into urines. It was indicated in most cases that major urinary GAG were chondroitin 4-sulfate, chondroitin 6-sulfate plus chondroitin and heparan sulfate, while minor ones were dermatan sulfate and hyaluronic acid. In addition, the data suggested a wide range of the degree of desulfation or urinary GAG, and the presence of significant amounts of keratan sulfate plus acidic glycopeptides in the urinary GAG fractions. The present data provided more precise information on urinary GAG from orthopedic patients than those reported previously.     

Acidic glycosaminoglycans in human platelets and leukocytes: the isolation and enzymatic characterization of chondroitin 4-sulfate

The acidic glycosaminoglycans (AGAG) in human platelets and leukocytes were studied by enzymatic digestion with chondroitinase and streptomyces hyaluronidase combined with electrophoretic separation on cellulose acetate membranes and partly with gel filtration on Sephadex G-100.Electrophoretic characterization and enzymatic susceptibility indicated that the main AGAG in human platelets and leukocytes are chondroitin sulfates. The occurrence of the unsaturated 4-sulfated disaccharide on paper chromatography only after digestion of the AGAG with chondroitinase-AC indicated that intracellular chondroitin sulfate in human platelets and leukocytes is present exclusively as chondroitin 4-sulfate.It is also suggested that hyaluronic acid exists in small amounts both in platelets and leukocytes, whereas heparan sulfates are present only in leukocytes, possibly as a membranous component.     

Cellular differentiation and the aging process in cartilaginous tissues. Mucopolysaccharide synthesis in cell cultures of chondrocytes

Primary cell cultures of differentiated chondrocytes were shown to produce chondroitin-4-sulfate as the predominant mucopolysaccharide, with suggestive evidence for the synthesis of keratan sulfate and possibly chondroitin-6-sulfate. Chicken embryonic cartilage was shown to be composed mainly of chondroitin-4-sulfate, with a small amount of chondroitin-6-sulfate, but essentially no keratan sulfate. These findings were compared to the data of others, and a hypothesis explaining the aging process in cartilage in terms of cellular differentiation was presented.     

Alterations in serum glycosaminoglycan profiles in Graves' patients.

BACKGROUND: Qualitative and quantitative analyses of glycosaminoglycans (GAGs) in serum obtained from Graves' disease (GD) patients without extrathyroidal complications were carried out to provide a clearer understanding of the role of these macromolecules in the disease pathogenesis. METHODS: GAGs were isolated from-SIMEL (Italian Society of Laboratory Medicine) Inter-associative Study Group on Diabetes Mellitus serum of 17 GD patients before treatment and after attainment of the euthyroid state, as well as from 20 healthy individuals. GAGs were quantified using the hexuronic acid assay and subjected to electrophoretic fractionation. RESULTS: Increased amounts of total GAGs were found in GD patients. Attainment of euthyroidism led to a decrease in, but not normalization of, total serum GAGs level. Electrophoretic analyses of GAGs before and after treatment identified the presence of chondroitin sulfate (CS), heparan sulfate/heparin (HS/H) and dermatan sulfate (DS) in serum from healthy subjects and GD patients. CS was the predominant serum GAG constituent in all subjects investigated. Enhanced CS levels in both GD patient groups were accompanied by increased structural heterogeneity of these compounds. Normalization of thyroid function did not change CS levels. DS levels in serum of untreated GD patients were elevated in comparison to healthy subjects. Anti-thyroid treatment led to a significant decrease in DS to levels below those in controls. DS in all serum samples investigated displayed a similar structure. HS/H levels in serum of untreated GD patients was seven-fold higher than in healthy subjects. In addition, HS/H in untreated GD patients were characterized by higher structural heterogeneity than those isolated from control subjects and euthyroid GD patients. Anti-thyroid therapy led to a decrease in HS/H concentrations towards normal values. CONCLUSIONS: Our results indicate that in the course of GD, the metabolism of particular types of GAGs is regulated by different mechanisms, including a hyperthyroid state and immunological abnormalities. Furthermore, qualitative and quantitative changes in serum GAGs seem to reflect GD-associated systemic changes in extracellular matrix properties.     

Hypoxia differentially enhances the effects of transforming growth factor-beta isoforms on the synthesis and secretion of glycosaminoglycans by human lung fibroblasts

Interstitial lung diseases associated with hypoxia, such as lung fibrosis, are characterized by enhanced production of transforming growth factor-beta (TGF-beta) and increased deposition of extracellular matrix (ECM) molecules, including glycosaminoglycans (GAGs). In this study, we investigated the effect of hypoxia (3% O(2)) on TGF-beta-induced GAG synthesis by primary human pulmonary fibroblasts, established from lung biopsies. Total GAG synthesis was assessed by the incorporation of [(3)H]glucosamine into GAGs associated with the cell layer (cells and ECM) or secreted in the medium. GAGs were isolated and purified by gel filtration, fractionated by electrophoresis on cellulose acetate membranes, and characterized using GAG-degrading enzymes. GAG molecules identified in the cell layer and the medium were: hyaluronic acid, and chondroitin, dermatan, and heparan sulfates. All TGF-beta isoforms time dependently induced [(3)H]glucosamine incorporation into GAGs of the cell layer or the medium. Characterization of individual GAG molecules indicated that this was attributed to dermatan and heparan sulfates in the cell layer and to hyaluronic acid and chondroitin and dermatan sulfates in the medium. Hypoxia enhanced the effect of all TGF-beta isoforms, particularly that of TGF-beta3, on the secretion of hyaluronic acid and chondroitin and dermatan sulfates. In the cell layer, hypoxia stimulated only the effect of TGF-beta2-induced [(3)H]glucosamine incorporation into GAGs. Our data indicate that hypoxia differentially enhances the effect of TGF-beta isoforms on the secretion and deposition of GAGs and may hasten ECM remodeling associated with the pathogenesis of lung fibrosis.     

Contribution of beta-glucuronidase to the degradation of chondroitin 4-sulfate by canine liver lysosomal enzymes.

Oligosaccharides derived from chondroitin 4-sulfate (Ch4-S) and chondroitin were digested by canine liver lysosomes under acidic conditions. The degree of digestion of Ch4-S by hyaluronidase and beta-glucuronidase was examined on the basis of types of the digestion products. Tetradeca- and dodecasaccharides derived from Ch4-S and chondroitin were first digested by hyaluronidase, while the octasaccharide was hydrolyzed by beta-glucuronidase. Decasaccharide was degraded by both hyaluronidase and beta-glucuronidase. The results showed that decasaccharide from Ch4-S served as the largest-molecular-weight substrate for beta-glucuronidase in the degradation of Ch4-S by the enzymes of lysosomes in contrast to the results of the digestion studies of hyaluronic acid (HA). The contribution of beta-glucuronidase to the depolymerization of chondroitin and HA by hyaluronidase was examined in the presence of saccharo-1,4-lactone, a specific inhibitor of beta-glucuronidase, in the reaction mixture. The depolymerization of chondroitin by hyaluronidase was significantly reduced by the addition of saccharo-1,4-lactone. From the results, it is suggested that beta-glucuronidase contributes to the degradation of the even-numbered oligosaccharides which inhibit the action of hyaluronidase in the depolymerization of Ch4-S.     

Age-related changes of plasma glycosaminoglycans.

BACKGROUND: This study was undertaken to reveal how the physiological ageing process influences plasma glycosaminoglycans (GAGs) content and composition. METHODS: GAGs isolated from plasma samples obtained from 105 healthy subjects were quantified using a hexuronic acid assay and subjected to electrophoretic fractionation. RESULTS: We found that total GAGs decreased gradually with advancing age. The electrophoretic analysis allowed identifying chondroitin sulfates, dermatan sulfates and heparan sulfates and heparin in plasma of healthy individuals. Chondroitin sulfates, predominant plasma GAGs, showed declining values with age and displayed higher structural diversity in subjects from the first five decades of life compared to older subjects. An age-related increase in plasma dermatan sulfates content during the first four decades of life followed by a decrease in a later period of life was apparent. Structural heterogeneity of these glycans was found in subjects until 10 years of age. Heparan sulfates and heparin plasma levels were inversely correlated with age until 25 years of age, without age-dependent changes thereafter. These macromolecules displayed higher heterogeneity of structure in subjects over 50 years of age than in younger subjects. CONCLUSIONS: The obtained results indicate that age should be taken into account in all studies investigating blood GAGs alterations during pathological conditions.