Thyroglobulin (Tg) present in the serum of normal individuals and patients with thyroid disorders could be partly newly synthesized non-iodinated Tg and partly Tg containing iodine and hormone residues originating from the lumen of thyroid follicles. With the aim of examining the contribution of the latter source of Tg to the elevation of serum Tg concentration in thyroid pathophysiological situations, we devised a procedure to identify thyroxine (T4) and tri-iodothyronine (T3) residues on Tg from unfractionated serum. A two-step method, basedon (i)adsorption of Tg on an immobilized anti-human Tg (hTg) monoclonal antibody (mAb) and (ii)recognition of hormone residues on adsorbed Tg by binding of radioiodinated anti-T4 mAb and anti-T3 mAb, was used to analyze serum Tg from patients with either Graves' disease (GD), subacute thyroiditis (ST) or metastatic differentiated thyroid cancer (DTC). Purified hTg preparations with different iodine and hormone contents were used as reference. Adsorption of purified Tg and serum Tg on immobilized anti-hTg mAb ranged between 85 and 90% over a wide concentration range. Labeled anti-T4 and anti-T3 mAbs bound to adsorbed purified Tg in amounts related to its iodine content. Tg adsorbed from six out of six sera from ST exhibited anti-T4 and anti-T3 mAb binding activities. In contrast, significant mAb binding was only observed in one out of eight sera from untreated GD patients and in 1 out of 13 sera from patients with DTC. The patient with DTC, whose serum Tg contained T4 and T3, represented a case of hyperthyroidism caused by a metastatic follicular carcinoma. In conclusion, we have identified, for the first time, T4 and T3 residues on circulating Tg. The presence of Tg with hormone residues in serum is occasional in GD and DTC but is a common and probably distinctive feature of ST.
L Druetta, H Bornet, G Sassolas and B Rousset
L Druetta, K Croizet, H Bornet and B Rousset
Serum thyroglobulin (Tg) concentration increases in diverse thyroid pathophysiological situations. We examined whether Tg molecules appearing in the serum of patients with Graves' disease (GD), subacute thyroiditis (ST) or differentiated thyroid cancer (DTC) have distinctive biochemical properties. We used ultracentrifugation on sucrose gradient and Western blot to analyze structural parameters of immunoreactive Tg in complete serum from 40 patients with GD, ST or DTC. Purified human Tg was used as reference. Immunoreactive Tg from ST or DTC sera sedimented in a single, rather symmetrical peak as purified 19S Tg. In GD sera without detectable anti-Tg autoantibodies (TgaAb), about 80% of immunoreactive Tg was recovered in a Tg dimer peak that often split into two components; the remaining Tg immunoreactivity (10-30%) sedimented faster and was polydispersed. In GD sera containing TgaAb, immunoreactive Tg was recovered in a peak sedimenting faster than 19S Tg corresponding to immune complexes identified by protein A adsorption. Using a Western blot procedure, optimized to detect 0.1 ng Tg in serum. a single band of Tg, migrating as the intact Tg subunit, was always found in non-reducing conditions; the intensity of the band correlated with the immunoassayable Tg concentration. In reducing conditions, the Tg band obtained with GD or ST sera was decreased by up to 70% compared with that of purified Tg or serum Tg from patients with DTC. In conclusion, serum Tg from DTC is remarkably homogeneous and in the form of dimers dissociable into uncleaved monomers. In contrast, serum Tg from GD or ST is heterogeneous with respect to its sedimentation properties and/or the structural integrity of its polypeptide chains. These data provide information on the processes whereby Tg is released into the circulation.