chondroitin-sulfates and Graves-Disease

Disturbances 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.. GAGs, 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.. The 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.

Topics: Antioxidants; Chondroitin Sulfates; Graves Disease; Humans

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.. 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.. 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.. 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.

Topics: Adult; Case-Control Studies; Chemistry, Clinical; Chondroitin Sulfates; Dermatan Sulfate; Extracellular Matrix; Female; Glycosaminoglycans; Graves Disease; Heparin; Heparitin Sulfate; Humans; Male; Middle Aged; Nitrous Acid

The presence of thyroglobulin (Tg) in orbital tissues of patients with thyroid-associated ophthalmopathy (TAO) supports a role of Tg in TAO pathogenesis. To search for Tg-binding sites in orbital tissues, because Tg is a heparin-binding protein, we investigated its binding to glycosaminoglycans (GAGs) that are abundant in orbital tissues: chondroitin sulfate B (CSB) and C (CSC) and hyaluronic acid (HA). Both in solid phase and solution phase assays purified human Tg bound to GAGs. In solid-phase assays, binding was increased by coincubation with heparin or GAGs in solution, or with an antibody against a Tg heparin-binding sequence (Arg2489-Glu2503), possibly suggesting crosslinking of Tg molecules induced by GAGs or by the presumably bivalent antibody. Orbital tissue extracts from TAO patients that contained Tg were subjected to high-salt treatment, which resulted in separation of Tg from GAGs, as observed by column chromatography. After separation from GAGs, the Tg in orbital tissue extracts acquired the ability to bind to immobilized CSB, and heparin enhanced binding, resembling the findings with purified human Tg. Therefore, we conclude that GAGs provide binding sites for Tg in orbital tissues, which may explain the presence of Tg in orbital tissues of patients with TAO.

Topics: Adipose Tissue; Aged; Binding Sites; Chondroitin Sulfates; Dermatan Sulfate; Female; Glycosaminoglycans; Graves Disease; Heparin; Humans; Hyaluronic Acid; Male; Middle Aged; Orbit; Ovalbumin; Protein Binding; Thyroglobulin

Accumulation of glycosaminoglycans in the orbit may play an important role in the development of Graves ophthalmopathy. Therefore, it might be clinically useful to evaluate the concentration of glycosaminoglycans in the orbit in patients with Graves disease. We investigated the concentration of glycosaminoglycans in retrobulbar tissue using in vivo 1H-magnetic resonance spectroscopy.. A model solution of 1% chondroitin sulfate (one of the components of the glycosaminoglycans complex) was initially examined using 1H-magnetic resonance spectroscopy, and the resonance of chondroitin sulfate was identified. 1H-magnetic resonance-spectroscopy spectra of retrobulbar tissue were obtained in 16 normal volunteers (28 eyes) and 23 patients with Graves ophthalmopathy (36 eyes). The 1H-magnetic resonance spectroscopy spectrum of chondroitin sulphate in the retrobulbar in vivo tissue was identified by assignments through lineshape comparisons of spectra of the model solutions in vitro and the retrobulbar tissue in vivo. Chondroitin sulphate-peak/H2O-peak ratios were calculated. To verify the results of in vivo 1H-magnetic resonance spectroscopy, retrobulbar tissue samples from five patients, who underwent orbital decompression surgery, were tested for reactivity to chondroitin sulfate proteoglycan by enzyme-linked immunosorbent assay (ELISA).. Multiple peaks, with a large peak at 5.24 ppm, were observed in 1H-magnetic resonance spectroscopy spectra of the model solution. A peak at 5.24 ppm was also observed in spectra of the retrobulbar tissue in all of the normal subjects and the patients. The mean value of the 5.24 ppm-peak/H2O-peak ratio was 0.1781 (SD = 0.0498, range, 0.0775 to 0.2282) in the normal subjects and 0.2874 (SD = 0.1357, range, 0.1405 to 0.7377) in the patients. The 5.24 ppm-peak/H2O-peak ratios were significantly increased in the patients with Graves ophthalmopathy (P < .01). The 5.24 ppm-peak/ H2O-peak ratio was correlated with the chondroitin sulphate concentration in retrobulbar tissue samples as evaluated by ELISA (r = .69).. This study suggests that 1H-magnetic resonance spectroscopy of the retrobulbar tissue allows us to estimate the concentration of chondroitin sulphate proteoglycan in the retrobulbar tissue. 1H-magnetic resonance spectroscopy of the retrobulbar tissue may be a new clinical tool for the evaluation of Graves ophthalmopathy.

Topics: Adolescent; Adult; Aged; Chondroitin Sulfates; Decompression, Surgical; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Graves Disease; Humans; Magnetic Resonance Spectroscopy; Male; Middle Aged; Orbit; Protons

Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAG) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with thyroid eye disease (TED). In order to analyze whether the alteration of distribution pattern and biochemical composition of GAG and proteoglycans play a role in the development of the disease, a highly specific high-pressure liquid chromatography (HPLC) method was developed. The concentration of total GAG, chondroitin sulfate A (CA), dermatan sulfate (DS), and hyaluronic acid (HA) was determined in patients and controls, revealing marked differences in urinary concentration of total GAG and HA, as well as an elevation of CA in patients versus controls. Method sensitivity was 0.86 for patients with active disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation. Furthermore, distribution pattern of orbital extracellular matrix GAG exhibited a significant increase in the tissue fractions of CA and HA in patients with TED in comparison to controls. In conclusion, GAG polysaccharides not only play a major role in the pathogenesis of TED but have been successfully introduced as an activity marker of the disease.

Topics: Chondroitin Sulfates; Chromatography, High Pressure Liquid; Glycosaminoglycans; Graves Disease; Humans; Hyaluronic Acid; Orbit; Reference Values; Sensitivity and Specificity

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycans (GAGs) and subsequent expansion of retrobulbar tissue lead to the clinical manifestation of exophthalmos in patients with Graves' eye disease. 2. A highly specific method to determine the concentration and biochemical composition of different GAGs was developed in order to obtain a sensitive test system for the activity of the disease. By means of this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chloride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyaluronate lyase, with HPLC analysis of the resulting alpha, beta-unsaturated disaccharides by anion-exchange chromatography. 3. Concentrations of GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluronic acid (HA) were determined in patients and controls, with high recovery rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 micrograms/l (0.01 mumol/l)], revealing marked differences in urinary concentrations of total GAG and HA, as well as an elevation of CA in patients compared with controls. 4. Method sensitivity was 0.86 for patients with active Graves' eye disease, and 0.87 for patients with untreated ophthalmopathy, whereas specificity was 1.0 for patients with inactive disease. Patients with increased GAG concentration responded well to steroids and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; CA, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; during therapy: GAG, 54.22 +/- 10.94; CA, 20.52 +/- 4.58; DS, 17.65 +/- 3.46; HA, 16.05 +/- 3.69 mg/24 h), whereas GAG excretion increased markedly 2-3 months after stopping prednisone therapy in patients with still active eye disease (GAG, 109.9 +/- 10.51; CA, 63.8 +/- 7.34; DS, 24.1 +/- 5.07; HA, 22.0 +/- 6.28 mg/24 h). 5. This sensitive method determines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.

Topics: Acute Disease; Adolescent; Adult; Aged; Anti-Inflammatory Agents; Chondroitin Sulfates; Chromatography, High Pressure Liquid; Dermatan Sulfate; Female; Glycosaminoglycans; Graves Disease; Humans; Hyaluronic Acid; Male; Middle Aged; Prednisolone; Sensitivity and Specificity

From three patients with pretibial myxedema (PTM) of Graves' disease, a portion of the skin involved was biopsied, analyzed for proteoglycans and the results were compared with those obtained with euthyroid and hyperthyroid subjects without PTM. The tissue specimen was extracted with 4 M guanidine HCl and subjected to subsequent CsCl density gradient centrifugation. Glycosaminoglycan and protein were recovered in the heaviest density fraction in the three specimens obtained from patients with PTM and not from subjects without PTM. From the analysis by Sepharose CL-6B column, glycosaminoglycan was present as a form of proteoglycan because alkaline borohydride treatment released single chain glycosaminoglycan with a molecular weight of 77,000 or 66,000. The digestion with chondroitin ABC lyase revealed that the majority of proteoglycan in the skin tissue was chondroitin sulfate or dermatan sulfate, and heparan sulfate comprised the minor component (14-34%). The rate of proteoglycan biosynthesis was examined by 35S incorporation into glycosaminoglycan's by cultured fibroblasts from PTM and normal skin. Incorporation of 35S into both proteoglycan and single chain glycosaminoglycan was observed in the fibroblasts of PTM patients as well as of those of subjects without PTM, although the rate of synthesis was more pronounced in the former. The rate of synthesis was influenced neither by normal serum or serum from a pretibial myxedema patient. Since proteoglycan accumulation was detected only in the affected skin of PTM patients, the impairment of local degradation and the proteoglycan clearance mechanism may also be involved.

Topics: Centrifugation, Isopycnic; Chondroitin Sulfates; Graves Disease; Heparitin Sulfate; Humans; Molecular Weight; Myxedema; Proteoglycans; Skin Diseases