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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 4 1409-1413
Copyright © 1999 by The Endocrine Society

Original Studies

Increased Sulfatation of Orbital Glycosaminoglycans in Graves’ Ophthalmopathy1

C. Hansen, R. Rouhi, G. Förster and G. J. Kahaly

Department of Endocrinology/Metabolism, Gutenberg-University Hospital, Mainz, Germany

Address all correspondence and requests for reprints to: Prof. George J. Kahaly, University Hospital, Building 303, 55101 Mainz, Germany. E-mail: kahaly{at}endokrinologie.klinik.uni-mainz.de


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Accumulation of interstitial glycosaminoglycans (GAG) in orbital tissue of patients with Graves’ ophthalmopathy (GO) leads to edema, increased orbital pressure, and proptosis. In this study, a new, highly sensitive, high performance liquid chromatography method was developed to determine the altered concentration and biochemical composition of different GAG polymers in orbital connective tissue of 27 GO patients and 18 controls. GAG were isolated by tissue homogenization and digestion, followed by sequential enzymatic GAG hydrolysis and high performance liquid chromatographic analysis of the resulting {alpha},ß-unsaturated disaccharides. High recovery rates of 78 ± 6% (mean ± SE) and a detection limit of 4.0 µg/L (0.01 µmol/L) were obtained. Total tissue GAG amounted to 254 ± 16 µg/g wet tissue wt in patients and 150 ± 13 µg/g (P < 0.0001) in controls. Regarding the GAG polymers, marked differences were detected between patients and controls (chondroitin sulfate, 127 ± 13 vs. 47 ± 5 µg/g; hyaluronic acid, 56 ± 5 vs. 34 ± 4 µg/g; both P < 0.0001; dermatan sulfate, 77 ± 6 vs. 69 ± 6 µg/g; P < 0.05). In patients, chondroitin sulfate was the major GAG component (48 ± 6 vs. 31 ± 5% of total GAG in controls), whereas dermatan sulfate was dominant in controls (46 ± 8% vs. 30 ± 5%). The sulfated disaccharide digestion products were markedly increased (P < 0.0001) in patients, and the ratio of sulfated vs. total disaccharide content was 85 ± 6% vs. 65 ± 5% (P < 0.05) in patients and controls, respectively. As accumulation of negatively charged sulfate residues in GAG disaccharides results in enhanced water-binding capacity, beside inflammation and increased volume of the orbital adipose tissue, the altered structure and nature of sulfated GAG units in the orbit may be responsible for the pathogenic changes in Graves’ ophthalmopathy.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
GLYCOSAMINOGLYCANS (GAG), as major compounds of proteoglycans in the extracellular matrix, are essential for the structure and function of connective tissues. The strong polyanionic charge and hydrophily of GAG components that are fundamental for the water content and electrolyte composition of the extracellular space are due to multiple carboxyl and sulfate residues (1). Furthermore, adhesion and migration of cells as well as matrix organization and signal transduction, e.g. growth factor attachment, and regulation of cytokine production in local inflammatory response are dependent upon the binding to polyanionic GAG carriers (2, 3, 4, 5). These linear polysaccharides are composed of repetitive disaccharides consisting of one hexosamine (D-glucosamine, D-galactosamine) and one uronic acid (D-glucuronic acid, L-iduronic acid). Different substitution and structure of the disaccharides allows the formation of various GAG components, e.g. hyaluronic acid (HA), chondroitin sulfate (CS), and dermatan sulfate (DS), with highly variable charge and chain lengths (6).

Proptosis, a prominent feature in patients with Graves’ ophthalmopathy (GO), is mainly caused by enhanced GAG deposition in the orbital space (7, 8, 9). Histological examination of orbital tissue in GO demonstrates mononuclear cell infiltration comprising activated, somatostatin receptor-bearing T cells, a few B cells, and macrophages (10, 11, 12). A current hypothesis is that cytokines released by immunocompetent cells stimulate orbital fibroblasts to proliferate and to secrete GAG in GO patients (13, 14). Beside the increased GAG production, an alteration of the structure and distribution pattern of single GAG components may be the cause of the increased water binding capacity of orbital connective tissue in GO (15). To analyze the absolute amount and the structure of these highly hydrophilic polysaccharides, the biochemical composition has to be determined. Consequently, we focused on the development of a sensitive and highly reproducible method that permits the exact determination of the concentration and molecular structure of the GAG components in the orbit.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Orbital tissue was obtained from 27 patients with Graves’ disease and severe GO according to Ref. 16 (median age, 46 yr; range, 32–72 yr; 22 females and 5 males; median duration of GO, 41 months, 2–65 months) undergoing orbital decompression surgery as well as from 18 controls (median age, 55 yr; range, 37–81 yr; 13 females and 5 males) having enucleation surgery. A complete clinical investigation including an ophthalmic and endocrine check-up was performed. Informed consent has been obtained where appropriate, and the reported investigations have been approved by the responsible ethical committee. All patients have been pretreated with steroids (prednisone; starting dose, 50 mg/day) and irradiation (n = 22; total dose, 16 Gray units), whereas immunosuppressive treatment was terminated at least 2 months before orbital surgery. All patients were euthyroid during methimazole treatment (2.5–20 mg/day; n = 17) or after radioiodine therapy (n = 7) or hemithyroidectomy (n = 3). TSH receptor autoantibodies were positive in 10 of 27 GO subjects (RRA, Brahms, Berlin, Germany). In 3 patients, urinary GAG excretion and serum GAG concentration were measured at orbital surgery.

Isolation of GAG

Orbital GAG were isolated from shock-frozen tissue samples (-196 C) after homogenization by Ultratorax type TP 18-10 (Bochem, Weilburg, Germany; three times, 1 min each time, 100 watts), solubilization in 0.1 N sodium hydroxide (10 µL 10 N NaOH/mL solution, 30 min at room temperature), followed by neutralization with 10 N acetic acid, and pronase digestion (50 mg/g wet tissue wt, 24 h at room temperature). After separation of undigested constituents via centrifugation (3000 x g, 20 min at room temperature), GAG were recovered from the supernatant by precipitation with aqueous cetylpyridinium chloride solution (final concentration, 0.5%; Merck, Darmstadt, Germany; 12 h at 4 C), followed by centrifugation (3200 x g, 40 min at 4 C). The supernatant was decanted, and the precipitate was dissolved in 5 mL potassium acetate in ethanol (0.1 mol/L potassium acetate in 99.5% ethanol) and incubated for 12 h at 4 C. Subsequently, the isolated GAG, obtained as a precipitate after centrifugation (3200 x g, 35 min at 4 C), was dissolved in 1 mL high performance liquid chromatography (HPLC) grade water. Quantification of the GAG raw product was performed by a calorimetric test with carbazole (17, 18, 19). In brief, an aliquot (0.2 mL) of each sample was incubated for 10 min with 0.09 mL of the carbazole reagent (7.5 mmol/L in 95% ethanol; Merck) in a boiling water bath in the presence of 0.9 mL borate-sulfuric acid (25 mmol/L sodium tetraborate decahydrate in concentrated sulfuric acid; Merck), leading to the pinkest stain of uronic acids. After cooling to room temperature for 30 min, the extinction was measured at 530 nm, followed by determination of the GAG concentration, expressed as milligrams per 24 h, by reference to a standard calibration curve of hexuronic acid (0–140 mg/L). Multiple sample assays revealed coefficients of variations ranging from 2.9% (49 mg/L) to 11% (6.25 mg/L). GAG were also isolated from 15 mL of the well mixed 24-h urine collections or 1-mL frozen serum samples (stored frozen at -20 C) of three GO patients undergoing orbital decompression surgery in duplicate after separation of insoluble constituents and denatured proteins by trichloroacetic acid precipitation (0.1%; Merck) as previously described (20).

Sequential enzymic degradation

Analysis of GAG compounds derived from orbital tissue, urine, and serum samples was performed in duplicate by sequential enzymic hydrolysis (21) with chondroitin lyase AC (EC 4.2.2.5), chondroitin lyase ABC (EC 4.2.2.4), and hyaluronate lyase (EC 4.2.2.1), followed by HPLC analysis of 0.04-mL aliquots of the resulting {alpha},ß-unsaturated disaccharides by anion exchange chromatography as described previously in detail (20). In summary, aqueous solutions of GAG samples containing 10 µg in a final volume of 200 µL were used. Digestion was conducted in 50 mmol/L sodium dihydrogenphosphate buffer with chondroitinase AC, and chondroitinase ABC (both 0.125 U/10 µg GAG of a 2.5 U/mL stock solution; pH 7.9, 8 h at 40 C; Sigma Chemical Co., Deisenhofen, Germany) as well as hyaluronate lyase (4.8 WHO units/10 µg GAG of a 60 WHO units/mL stock solution in 50 mmol/L sodium dihydrogenphosphate, 0.45% NaCl, and 0.01% BSA, pH 6.0; 60 C for 8 h). External standards of 10 µg CS, DS, and HA (Sigma Chemical Co.) were employed in every digestion step and treated under the same conditions.

HPLC analysis

The chromatographic system comprised the following modules: 625 LC inert system (nonmetallic fluid path; dynamic flow rate range, 0.01–5 mL/min), 717 Autosampler (injection volume, 1–2000 mL; 48-vial carousel; Peltier temperature module, 4–40 C), 996 Photodiode array detector (UV/VIS, 190–800 nm; 512 photodiodes; optical resolution, 1.2 nm), Millennium 2010 chromatography software (all from Waters, Eshborn, Germany), and Bio-Sil amino 5S disaccharide columns (250 x 4 mm; aminopropyl groups bound to bonded silica; particle size, 5 µm; pore size, 8 nm) protected by Bio-Sil amino 5S Micro-guard precolumns (30 x 4.6 mm; Bio-Rad, Munich, Germany). Elution was performed at room temperature with a concave gradient of 100–750 mmol/L sodium dihydrogenphosphate, pH 4.0, filtered through a 0.2-µm pore size cellulose acetate filter (Sartorius, Göttingen, Germany) and degassed with an on-line degas unit (four channels; degassing effect, 0.8 parts/million; DG 2410, VDS Optilab, Eshborn, Germany). Extinction of the elute was detected at 200–300 nm, and chromatograms were calculated at 230 nm. Quantification was performed by calibration with a reference kit of the pure disaccharides (Medac, Hamburg, Germany) and by comparison to the digestion products of the external standards of pure CS, DS, and HA.

Statistical analysis

In HPLC chromatograms, product-specific peaks were analyzed according to UV absorption maximum at 230 nm, retention time, and peak area. Distribution of GAG was presented as the mean ± SE. Differences between the GAG concentrations of patients and controls were evaluated by the Wilcoxon two-sample test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Typical chromatograms of disaccharide digestion products of patients and controls are shown in Fig. 1Go. Absorption signals at 230 nm were analyzed according to UV spectra of the corresponding disaccharide digestion products of CS, DS, and HA, respectively. Tissue samples showed well defined chromatograms with eight disaccharide signals gained by CS digestion and six or seven signals gained by digestion of DS and HA, respectively. The detector response was linear from 0.02–800 µmol/L, with a detection limit for HPLC analysis of 0.01 µmol/L (signal to noise ratio, 3.5). Coefficients of variation for intra- and interassay were less than 2.0% and less than 3.7% (GAG concentration, 0.02–1000 µmol/L; n = 15) for enzymic digestion. Overall recovery rates were 85 ± 7% for CS, 71 ± 7% for DS, and 78 ± 7% for HA.



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  		Figure 1. Chromatograms of disaccharide digestion products of tissue CS (a, patients with GO; b, control subjects) and DS (c, patients; d, controls). Chromatograms were obtained by HPLC analysis with UV detection at 230 nm at room temperature (injection volume, 40 µL). Chromatography was performed with a concave sodium phosphate gradient (100–750 mmol/L; pH 4.0; running time, 40 min) on a Bio-Sil amino 5S disaccharide column (250 x 4 mm; flow rate, 0.6 mL/min; injection volume, 10 µL).

 
Total tissue GAG were markedly increased in GO and amounted to 0.0175 ± 0.0012% in patients (254.3 ± 16.1 µg/g wet tissue wt) and 0.012 ± 0.001% in controls (149.7 ± 13.6 µg/g wet tissue wt; P < 0.0001). In comparison, the protein concentration in orbital connective/adipose tissue was 0.239 ± 0.12% in patients vs. 0.257 ± 0.14% in controls. Marked differences in the tissue fractions of CS, DS, and HA could be detected (Table 1Go). CS was the major GAG compound in orbital connective tissue of patients, whereas DS was dominant in controls. The ratio of HA content in relation to total GAG concentration exhibited no change. An increase in the several disaccharide digestion products of CS, DS (Di-6S, Di-0S, Di-4S, and Di-diS2;2 P < 0.05), and HA (Di-UA2S; P < 0.0001) was noted in patients compared to controls. The orbital tissue contents of the HA disaccharide Di-HA and the Di-diS1 disaccharide of CS and DS showed no alterations. The ratio of sulfated to total disaccharides was 85 ± 6% in patients and 65 ± 5% in controls (P < 0.05). The GAG contents of orbital tissue, urine, and serum samples obtained at the same time were measured in three patients with severe and clinically active GO and showed a similar distribution (Table 2Go). All urinary and serum data from GO patients were markedly increased (P < 0.0001) compared to control values. There was no correlation between orbital or peripheral GAG values and TSH receptor autoantibody titers.


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  		Table 1. Distribution of the orbital tissue GAG, chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA) as well as disaccharide composition pattern of patients with Graves’ ophthalmopathy and controls obtained by HPLC analysis, presented as total tissue amount (micrograms per wet tissue) and percentage of total GAG (mean ± SE)

 

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  		Table 2. Comparison of total GAG, chondroitin sulfate (CS), dermatan sulfate (DS), and hyaluronic acid (HA) of the orbital, urine, and serum samples of three patients with Graves’ opthalmopathy, obtained by HPLC analysis, presented as the absolute amount and percentage of total GAG

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In the present study, for the first time, the distribution pattern and biochemical composition of the major GAG components, CS, DS, and HA, were quantified in orbital tissue of GO patients and controls by HPLC analysis. A marked increase in all three GAG molecules and a shift to higher sulfated, negatively charged compounds, making them especially potent as water binders, were noted in GO patients. Thus, we hypothesize that structural alteration of GAG may elucidate the massive increase in the orbital volume, leading to an elevated pressure in the bony orbit and displacement of the globe anteriorly.

The different GAG compounds have been analyzed by a combination of specific enzymic digestion and HPLC-anion exchange chromatography of the resulting degradation products. By means of the present method, the total GAG concentration and structural information about disaccharide composition, degree of sulfatation, and proportion of sulfated isomers can be determined. This is achieved by sequential application of the substrate-specific enzymes chondroitin AC lyase for the digestion of CS, chondroitin ABC lyase for the degradation of DS, and hyaluronate lyase for the reaction with HA. Separation of the digestion products from undigested compounds after each digestion step permits the determination of GAG distribution patterns. The method represents a reliable test system for the detection and quantification of GAG components, with high overall recovery rates down to GAG concentrations of 0.01 µmol/L and sensitivities superior to those of other methods used in the past, such as calorimetric tests as well as paper, thin layer, liquid, or gas chromatography; gel electrophoresis; and radiometric assays (22, 23, 24, 25, 26). An additional advantage of the HPLC method is the possibility for automation and its high reproducibility. Furthermore, structural modifications of all GAG components, like altered disaccharide composition, which may reflect pathological alterations, can be directly examined by means of the disaccharide HPLC elution pattern. Regarding the sequence of application of the GAG lyases, recovery rates using the order employed proved to be superior to other methods that used hyaluronate lyase digestion as the first degradation step (27), as CS and DS can also be digested by hyaluronate lyase.

Elevated GAG synthesis of stimulated cultured orbital fibroblasts from GO patients has been reported (28, 29, 30). In this study, analysis of orbital GAG showed that the highly sulfated CS represented the major portion of orbital GAG in patients (48%), whereas DS was dominant in controls (46%). The tissue fraction of HA, which does not contain sulfate residues, remained unchanged (22% vs. 23%). CS was also the most prominent GAG compound in serum and urine samples. A previous publication investigating the biochemical composition of orbital tissue in human cadavers 48 h postmortem by cetylpyridinium chloride-cellulose chromatography reported that HA (51% of the total GAG) and DS (31% of the total GAG) were the two major GAG components (31). The examined control tissue was composed of 73% lipid, 24% water, and 4% dried defatted tissue. Total tissue GAG amounted to 0.18% of the dried defatted tissue, which was comparable to our findings of 0.0175% of wet tissue weight in patients and 0.012% in controls. The different GAG distributions in the two studies may be related to an overestimation of HA caused by coelution of HA with structurally related GAG components at similar salt concentrations, and/or to the fact that the GAG concentration was only measured by quantification of the uronic acid portions of GAG (31). In comparison, by means of the specific enzymic digestion and more sensitive HPLC analysis, all disaccharides present in CS, DS, and HA were completely separated and readily determined.

GAG synthesis of human orbital fibroblasts of GO patients was influenced by several cytokines derived from activated T lymphocytes and macrophages (13, 14, 32, 33, 34, 35, 36). Stimulation of GAG synthesis of orbital fibroblasts by interleukin-1 (IL-1) was significantly inhibited by IL-1 receptor antagonists and soluble IL-1 receptors (32). Furthermore, IL-1ß and transforming growth factor-1ß (TGF1ß) increased the rate of [35S]sulfate incorporation into proteoglycans 2–5 times over the control value in cultured orbital fibroblasts (37). The researchers concluded that IL-1ß and TGF1ß increased [35S]sulfate incorporation into proteoglycan by increasing the net increase in proteoglycan synthesis and by increasing the number of GAG chains attached to core protein in the case of IL-1ß in vitro. The results reported by Imai et al. (37) showed that IL-1ß and TGF1ß did not change the size of GAG chains in vitro. However, stimulation analysis of cultured orbital fibroblasts may not completely correspond to the findings in vivo. In comparison, HPLC disaccharide analysis revealed that GAG contained elongated chains, composed of monosulfated CS, DS disaccharides (Di-6S and Di-4S), and oversulfated CS and DS compounds (Di-diS1 and Di-diS2), showing that significant oversulfation of GAG chains was present in GO patients. Thus, different mechanisms for increased GAG deposition into the orbital tissue are possible, e.g. net increase in proteoglycan synthesis, increase in both the number of GAG chains and the number of GAG-producing cells, elongation, oversulfation, and/or decreased GAG degradation.

Comparable orbital and peripheral GAG distributions in patients with GO were observed. In this sense, multifocal fibroblast activation with resulting overproduction of certain GAG compounds, especially CS, leads to the hypothesis that we are dealing with a systemic autoimmune disease. In conclusion, disaccharide HPLC analysis allowed biochemical determination of connective tissue changes in patients with Graves’ disease for the first time. Whether these alterations were caused by the expression of certain cytokines derived from activated orbital lymphocytes and macrophages warrants further investigations.


   Footnotes
 
1 Presented in part at the 70th Annual Meeting of the American Thyroid Association, October 1997, Colorado Springs, CO. Back

2 Di-0S, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-ene-pyranosyluronic acid)-D-galactose; Di-6S, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-D-galactose; Di-4S, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-D-galactose, Di-diSd, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-b-D-gluco-4-enepyranosyluronic acid)-6-O-sulfo-D-galactose; Di-diSe, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose; Di-triS, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-b-D-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-D-galactose; Di-HA, 2-acetamido-2-deoxy-3-O-(b-D-gluco-4-enepyranosyluronic acid)-4O-sulfo-D-galactose; Di-diSb, 2-acetamido-2-deoxy-3-O-(2-O-sulfo-b-D-gluco-4-enepyranosy-luronic acid)-4-O-sulfo-D-galactose; Di-diS1, Di-diS2, 2-acetamido-2-deoxy-3-O-(x-O-sulfo-b-D-gluco-4-enepyranosyluronic acid)-x-O-sulfo- D-galactose. Back

Received August 25, 1998.

Revised November 2, 1998.

Accepted November 25, 1998.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals