HPLC GLYCOSAMINOGLYCAN ANALYSIS IN PATIENTS WITH GRAVES DISEASE

1. Orbital accumulation of hydrophilic, interstitial glycosaminoglycan s (GAGs) and subsequent expansion of retrobulbar tissue lead to the cl inical manifestation of exophthalmos in patients with Graves' eye dise ase. 2. A highly specific method to determine the concentration and bi ochemical composition of different GAGs was developed in order to obta in a sensitive test system for the activity of the disease. By means o f this method, GAG excretion in 24 h urine collections of 56 patients and 21 controls was analysed by precipitation with cetylpyridinium chl oride and potassium acetate in ethanol, followed by sequential enzymic hydrolysis with chondroitin AC lyase, chondroitin ABC lyase and hyalu ronate lyase, with HPLC analysis of the resulting alpha,beta-unsaturat ed disaccharides by anion-exchange chromatography. 3. Concentrations o f GAG, chondroitin sulphate A (CA), dermatan sulphate (DS) and hyaluro nic acid (HA) were determined in patients and controls, with high reco very rates [72.2 +/- 5.3%, mean +/- SEM; detection limit, 4.2 mu g/l ( 0.01 mu mol/l)], revealing marked differences in urinary concentration s of total GAG and HA, as well as an elevation of CA in patients compa red with controls. 4. Method sensitivity was 0.86 for patients with ac tive Graves' eye disease, and 0.87 for patients with untreated ophthal mopathy, whereas specificity was 1.0 for patients with inactive diseas e. Patients with increased GAG concentration responded well to steroid s and/or orbital irradiation (before therapy: GAG, 111.49 +/- 40.32; C A, 59.58 +/- 21.34; DS, 25.05 +/- 8.12; HA, 26.88 +/- 11.63 mg/24 h; d uring 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 ma rkedly 2-3 months after stopping prednisone therapy in patients with s till 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 de termines the nature of renally excreted GAGs, reflecting the aberrant synthesis pattern of fibroblasts in patients with Graves' disease.