Institut Salah Azaiz
H. Zouaghi
Institut de Nutrition
A. Kammoun
Faculté des Sciences University of Sciences, Technology, and Medicine 1006 Tunis, Tunisia
P. Fragu
Institut Gustave Roussy 94805 Villejuif Cedex, France
Recently, using Secondary Ion Mass Spectrometry (SIMS), we showed the presence of high amounts of bound 127I (iodine) in thyroid stroma of multinodular goiters inhabiting a region of goiter endemicity, especially after absorption of high doses of Lugols solution (2% potassium iodide solution) (1).
We tried to identify the iodinated molecules present in these stroma, using immunohistochemical techniques.
The eight patients previously described (1) were included in this study. One did not receive any treatment, two received L-thyroxine (L-T4) (100 µg/day), three received Lugols solution (3.8 mg iodine per day), and the last two patients received Lugols and L-T4 (3.8 mg iodine and 100 µg per day) during a six-month period of treatment.
All were euthyroid, and had a large multinodular goiter before treatment. Their nodules were cold on scintigraphy. After the six-month treatment period, their serum thyroglobulin concentrations dropped, and all subjects underwent subthyroidectomy.
Fragments of nodules were immersed in a Bouin solution for a period of three days. The fixed tissues were dehydrated in alcohol, cleared in toluen, and embedded in parrafin. Sections with a thickness of 3 µm were processed for immunoperoxidase techniques.
For facilities, we gathered the patient who did not received treatment and the two patients receiving L-T4, in a control group, as we did for the SIMS study.
The following rabbit primary antibodies were used: antiperoxidase (CIS International, Paris, France)(4,000 U/mL), antialbumin (1:50), antithyroxine (Immunotech, Marseille, France)(1:1), antitriiodothyronin (1:1) (Netria, London, England), and antithyroglobulin (CIS International)(3,100 U/mL).
The following monoclonal primary antibodies were used: antithyroglobulin (CIS International)(1:1), antiIgG (Dako, Copenhagen, Denmark)(1:50).
The indirect method was performed on rehydrated sections. They were incubated overnight at 4 C with primary antisera diluted in Tris-HCl-NaCl buffer. After washing, the sections were incubated for 1 h at room tempature with a peroxidase labeled antirabbit or antimouse antiserum (Dako; 1:100). Revelation was performed for a few minutes in a freshly prepared diaminobenzidine and hydrogen peroxide (0.03%) solution. The sections were examined in a Leitz microscope. (Ernst Leitz GMBH, Wetzlar, Germany).
Table 1 summarizes the results of 127I concentration measurements
and of antithyroglobulin-like immunoreactivity.
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The fact that no thyroglobulin or thyroxine-like immunoreactivities were observed in stroma of control patients is consistent with the absence of SIMS measured 127I in the same stroma.
Thyroglobulin-like immunoreactivity was less frequently observed than 127I. This may be related to the different sensitivities of the two techniques. SIMS sputters tissues with an energetic primary ion beam, collects the secondary ions emitted, and separates and measures these ions according to their mass (sensitivity = 3.12, 10-4 µg 127I/mg thyroid). Immunohistochemistry detects molecules in situ and needs immunoreactive epitopes accessible to the antibodies used. The sensitivity of this technique depends also on the revelation step used. We performed indirect immunoperoxidase technique, which is less sensitive than other techniques such as peroxidase-antiperoxidase (PAP) or avidin-biotin techniques.
The presence of thyroglobulin-like immunoreactivity in thyroid stroma of these treated patients has to be associated with the diminution of their serum thyroglobulin concentrations after six months of treatment with iodine (Lugols alone: decreased from 118 to 64 mg/L, 1795 to 479 mg/L, and 636 to 379 mg/L; Lugols and L-T4: decreased from 276 to 16 mg/L; 468 to 72 mg/L). This diminution after supplementation with iodine has been pointed out in the past, and it has been suggested that higher iodinated thyroglobulin is less excreted at the basal pole of thyrocytes (2). Our results seem to show that, in case of iodine overload, thyroglobulin-like molecules might be excreted by follicular cells and stored in stroma. They may be also produced by cells of stroma that are not able to form follicles, or they may stay in stroma after the necrosis of producing cells, as it has been shown that iodine overload is toxic for thyrocytes (3).
The presence of thyroglobulin-like immunoreactivity in thyroid stroma of the treated patients does not exclude the presence of other iodinated polypeptides, nor of iodinated lipids, as it has been shown that nonthyroglobulin iodine does exist and is increased in goitrous thyroid tissue, compared with normal tissue (4, 5).
Footnotes
1 Received February 25, 1997. Revision
received June 3, 1997. Address correspondence to: M.V. El May, Institut
Salah Azaiz, Place Bab Saadoun, 1006, Tunis, Tunisia.
References
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