Conflicting results have been reported regarding serum lipoprotein(a) (Lp(a)) concentrations in patients with hypothyroidism. We addressed the question whether thyroid autoimmunity could be associated with elevated Lp(a) values independent of the thyroid status. Lp(a) was measured by ELISA in 30 males, 29 premenopausal and 30 postmenopausal females positive for thyroid peroxidase- and/ or thyroglobulin-antibody (T-Abs) and normolipidemic, screened out respectively from 428 male donors, 162 premenopausal donors and 108 postmenopausal females; they were compared with 65 males, 72 premenopausal and 48 postmenopausal females, negative for thyroid antibodies, normolipidemic and matched for age. T-Abs-positive male donors showed serum Lp(a) concentrations significantly increased compared with males without T-Abs (mean 19.7 +/- 15.9 vs 12.7 +/- 17.5 mg/dl; median 17.0 vs 4.0 mg/dl; Mann Whitney U test: P = 0.0000). In premenopausal females no difference could be found between T-Abs-positive and T-Abs-negative subjects (mean 13.2 +/- 16.1 vs 12.3 +/- 13.9 mg/dl; median 5.2 vs 8.7 mg/dl), suggesting an Lp(a) lowering effect of estrogens. The study was, therefore, extended to postmenopausal females. Significantly elevated Lp(a) levels were found in 30 postmenopausal females with T-Abs when compared with 48 postmenopausal females without T-Abs (40.0 +/- 34.2 mg/dl vs 20.7 +/- 19.3 mg/dl; median 32.0 vs 18.0 mg/dl; Mann Whitney U test: P = 0.0002). Finally, 21 postmenopausal, normolipidemic, autoimmune hypothyroid patients on L-thyroxine and euthyroid compared with 48 postmenopausal females without T-Abs also showed increased serum levels of Lp(a) (mean 27.0 +/- 16.8 mg/dl vs 20.7 +/- 19.3 mg/dl, median 25.0 vs 18 mg/dl; Mann Whitney U test: P = 0.0024). Thyrotropin levels in all subjects and patients were within the normal range. In conclusion, our results in males and postmenopausal females with T-Abs and euthyroid show an association between thyroid autoimmunity and increased levels of Lp(a), while the results obtained in premenopausal females suggest that estrogens might interfere with the Lp(a) increase related to thyroid autoimmunity.

Radial immunodiffusion and electroimmunodiffusion were used to measure thyroglobulin, the main component of thyroid colloid, in thyroid fine needle aspiration biopsies. A linear relationship was established between precipitation ring diameter and thyroglobulin concentration by radial immunodiffusion (0.5-3.0 g/1), and between "rocket" height and thyroglobulin concentration by electroimmunodiffusion (0.1-2.0 g/1). A nearly complete correlation was observed between the two methods (r = 0.97). In radial immunodiffusion the ring diameter is dependent on time of diffusion and on the antiserum concentration in the agar gel. In this study, the observation time was standardised at 48 h, and the rabbit anti-thyroglobulin serum concentration at 26 ml/1. The intrathyroidal concentration of thyroglobulin was determined by radial irrummodiffusion and the thyroid fine needle aspiration biopsy of 45 thyroid tumours with different cytological-laboratory-and clinical diagnoses. It was found that in colloid nodules or cysts thyroglobulin is markedly higher than in euthyroid nodular goitre (13.7 ± 11.9 g/1 vs 1.35 ± 0.8 g/l, p = 0.005).In conclusion radial immunodiffusion and electroimmunodiffusion are precise, easy to perform, low cost, non polluting methods, which do not require high sample dilution (in contrast, high sample dilution is necessary for measurement of thyroglobulin in thyroid fine needle aspiration biopsy by radial immunodiffusion). Measurement of thyroglobulin in thyroid fine needle aspiration biopsy provides a quantitative estimate of colloid, an important marker in the differential diagnosis of thyroid nodules.

The high prevalence of subjects in the general population and among patients with thyroid diseases who are positive for serum anti-thyroglobulin antibodies, together with the inconsistency of hybridoma techniques to obtain large amounts of anti-thyroglobulin antibodies, prompted us to prepare anti-thyroglobulin antibodies from human serum. An anti-thyroglobulin antibody positive serum was absorbed with CNBr-activated sepharose 4B conjugated to thyroglobulin and anti-thyroglobulin antibodies eluted by acid pH (3.0), basic pH (10.7) and detergent (SDS 3 g/l or 7 g/l). Preliminary experiments carried out with rabbit anti-human thyroglobulin allowed us to purify an Ig-fraction that retained its anti-thyroglobulin activity by immunoprecipitation and tanned red cell haemagglutination. When subjected to agarose gel electrophoresis and Silver stain, this fraction has the mobility of rabbit lg. Further experiments were carried out using Hashimoto's serum (tanned red cell haemagglutination 1:40620). pH 3.0 was found to give the best yield of stable antibodies. An IgG fraction was eluted at a concentration of 10+/-1.2 mg/l of serum. This fraction has the same electrophoretic mobility as human IgG and is close to pure human anti-thyroglobulin antibody. A dose-response curve was built up at a concentration range between 0.016 mg-2 mg of IgG anti-thyroglobulin antibody per litre. The slope of the curve parallels that of a dilution curve of the whole serum, suggesting that the purified antibodies are representative of the whole antibody population. In conclusion, we provide a method for preparing considerable amounts of highly purified anti-thyroglobulin antibodies from human serum for application in clinical medicine and basic research, and in particular which provides a standard for measuring anti-human thyroglobulin serum antibodies by weight rather than the presently used standard expressed in U/ml. Furthermore, this reagent could act as an ideal immuno-vector for the diagnosis and therapy of differentiated thyroid carcinoma.

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