HPLC Glycosaminoglycan Analysis in Patients with Graves' Disease

Abstract

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.

The xylosyltransferase Ι gene polymorphism c.343G>T (p.A115S) is associated with decreased serum glycosaminoglycan levels

“…Serum from healthy blood donors instead of serum from patients was chosen for our study to avoid changes in serum GAGs by pathological mechanisms in patients. Consistent with the literature [16] no association between gender and GAG amount was detected. The XYLT1 SNP c.343GNT in the heterozygote state was associated with a significantly decreased GAG amount compared to serum from blood donors who were non-carriers of all SNPs investigated.…”

“…NM_022167 for XYLT2). [6,7,15,16]. Genetic polymorphisms in the genes relevant for glycosaminoglycan biosynthesis could be a risk factor for development and the severity of diseases that are associated with changes in the GAG metabolism.…”

Elephantiasic Pretibial Myxedema: Insight into and a Hypothesis Regarding the Pathogenesis of the Extrathyroidal Manifestations of Graves' Disease

“…Thus, present investigations focus on sites with obvious clinical disease (orbit and lower extremity) with the intuitive assumption that such localization of disease must involve cellular and/or biochemical differences between different regions of the body. However, previous studies have raised the possibility that Graves' disease may have a systemic, connective tissue systemic component (27)(28)(29)(30)(31)(32)(33). Perhaps the strongest evidence for this notion is increased urinary excretion of mucopolysaccharides or glycosaminoglycans in Graves' patients with, as opposed to without, GO (28,32).…”

“…Perhaps the strongest evidence for this notion is increased urinary excretion of mucopolysaccharides or glycosaminoglycans in Graves' patients with, as opposed to without, GO (28,32). Moreover, Hansen et al found increased urinary GAG excretion in Graves' disease independent of the presence of ophthalmopathy (33). It is difficult to explain increased urinary excretion of GAG with involvement of only a few grams of orbital tissue.…”

Adipose thyrotrophin receptor expression is elevated in Graves' and thyroid eye diseases ex vivo and indicates adipogenesis in progress in vivo

“…The extra-ocular muscles (EOM) can be enlarged in TED, primarily due to oedema, but the myocytes themselves are relatively unaffected (Hufnagel et al 1987). Two further processes increase the contents of the orbit, production of glycosaminoglycans by orbital fibroblasts (Hansen et al 1997) and hypertrophy and hyperplasia of the adipose tissue (Sorisky et al 1996). The increase in fat cell number is the consequence of adipogenesis, in which preadipocytes differentiate into mature adipocytes.…”