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Assessing Graves Ophthalmopathy activity by hyaluronan determination in the tear.
Coelho, B.V.1, Paves, L.2, Mendes, A.1, Kobayashi, E.Y.1, Manso, P.G.2, Pereira, F.Z.1, Maciel, R.M.B.3, Furlanetto, R.P.3, Nader, H.B.1, Martins, J.R.M.1,3.
1 Departments of Biochemistry (Molecular Biology Division), 2Ophthalmology, and 3Medicine (Endocrinology and Metabolism Division), Federal University of São Paulo, São Paulo, Brazil.
Introduction: Glycosaminoglycans (GAGs) consist of complex polysaccharides that participate in the pathogenesis of Graves Ophthalmopathy (GO). Once deposited on the extracellular matrix, these compounds, particularly hyaluronan (HA) and chondroitin sulfate, attract water, resulting in swelling of connective tissue and enlargement of the extraocular muscles. Several attempts to correlate the local increase of GAGs to serum HA or urinary GAGs in GO patients have been made but controversies still exist. Since lacrimal gland can be involved in this process, we proposed to quantify HA concentration in tears from normal subjects and patients with GO, in an attempt to correlate with the inflammatory state of the disease. Method: Tear samples were obtained from 15 normal subjects (30 eyes) without ocular or systemic diseases and from 43 patients (86 eyes) with GO. Schirmer strips were placed in the temporal side of each eye under the eyelid, during 5 minutes, without use of topical anesthetics, in order to collect tears. Total volume were quantified at the strip scale (in millimeters). The strips were dried at room temperature and stored at -20o C until analysis. Inflammatory state of GO was assessed according to the Clinical Activity Score (CAS). Strips were placed on distilled water and HA and protein contents determined by fluorometric (Martins et al., Clin. Endocrinol. 37: 726-33, 2004) and colorimetric procedure, respectively. Results: After elution with distilled water, the mean of total protein was 144.9 ± 78.1 µg/mL (ranging from 37.5-342.6 µg/mL). The mean concentration of HA present in the tear was 18.9 ± 12.6 ng/mg protein (ranging from 3.2-45.0 ng/mg protein) in normal subjects and 35.1 ± 34.7 ng/mg protein in GO (p<0.01). Conclusions: It is possible to extract macromolecules such as proteins and HA adsorbed on Schirmer´s strips and quantify their contents by conventional laboratories assays. In this pilot study, we have already observed the HA content between eyes with GO and normal subjects is statistically different (p<0.01). This new approach could be useful to investigative studies involving ocular and systemic diseases and help to decision making on therapeutic approaches. Supported by: FAPESP and CNPq.
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