A case of Graves' disease with anti-triiodothyronine antibodies

A case of Graves' disease with high serum thyroxine (T4) and low triiodothyronine (T3) levels which was therefore initially diagnosed as a T4-thyrotoxicosis is reported. Examination of the serum from the patient showed the presence of unusual protein which bound T3. It was later confirmed as IgG class anti-T3 antibodies. In addition to treatment with methylmercaptoimidazole (MMI), the patient was treated with prednisolone for 30 days (total amount 500 mg). Titers of anti-T3 antibodies in the sera were unchanged before and after prednisolone treatment. Our present case indicates that it is clinically important to bear the presence of autoantibodies in mind to account for a possible error in measuring T3 and T4 by radioimmunoassay (RIA). In the case that RIA determination gives an unexpectedly high or low T3 and/or T4 value, the presence of autoantibodies to them should be considered and a test for them is recommended.     

A case of hypothyroidism with simultaneous presence of stimulating type anti-thyrotropin (TSH) receptor antibodies and anti-thyroxine (T4) autoantibodies.

We have examined a hypothyroid patient with stimulating type anti-thyrotropin (TSH) receptor antibodies and without blocking type anti-TSH receptor antibodies. Although she had high serum TSH (240 microU/ml) and low free triiodothyronine (FT3, 0.49 pg/ml) concentrations, which agree with physical findings of hypothyroidism, she had an unusually high free thyroxine (FT4) concentration (3.56 ng/dl). Incubation of her serum with 125I-T4, followed by precipitation with 12.5% polyethylene glycol (PEG) disclosed a higher binding of 125I-T4 (34.4%) than in normal controls, being 5-7%. In addition, binding of 125I-T4 to her serum gamma-globulin was completely displaced by the addition of unlabelled T4. From these results it was concluded that her serum contained anti-T4 autoantibodies. Treatment with synthetic T4 was begun and her thyroid function was monitored by sensitive TSH radioimmunoassay (RIA) and RIA of FT4 after PEG treatment. Since both sensitive TSH RIA and FT4 RIA results after PEG treatment give results concordant with the physical findings, it was concluded that both of the RIA results are useful for the evaluation of thyroid function in patients with thyroid hormone autoantibodies.     

Time-resolved fluorescence immunoassay of thyroxine in serum: immobilized antigen approach

With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.     

An enzymeimmunoassay method for thyroxine determination in serum samples

Here we describe an enzymeimmunoassay (EIA) for thyroxine (T4) in serum, whose performance is comparable to that of a sensitive T4 radioimmunoassay (RIA). In this assay, specific T4 antibody adsorbed on polystyrene beads is used along with T4-horseradishperoxidase as the tracer and omicron-phenylenediamine as chromogen. Several samples were analysed both by this T4 EIA and by using a commercial RIA kit for T4. The results correlate well with a correlation coefficient r = 0.9 and slope = 0.93 (n = 50).     

Characterization of canine triiodothyronine (T3) autoantibodies and their effect on total T3 in canine serum

A study of 3,5,3'-L-triiodothyronine autoantibody (T3 AA) in 18 dogs revealed an average apparent affinity constant for T3 of 2.24 +/- 1.78 X 10(10) M-1, an average T3 binding capacity of 639.3 +/- 666.5 ng/dl and a low thyroxine (T4) cross-reactivity (less than 1%) in all samples tested. A valid radioimmunoassay (RIA) procedure which involved heat treatment of samples for 1 hr at 70 degrees C and assay on Sephadex minicolumns was developed for measuring T3 in the presence of T3 AA. Total T3 was elevated (mean = 374.8 +/- 158.4 ng/dl) in samples in which T4 was in the normal canine range, but T3 was lower (mean = 96.1 +/- 63.3 ng/dl) in samples with T4 values in the hypothyroid range. For each sample the concentration of T3 not bound by T3 AA was calculated from the total T3 concentration, the affinity constant, and the binding capacity. In dogs with normal total T4 concentrations the average calculated T3 not bound by T3 AA was 147.2 +/- 144.4 ng/dl while in dogs with low total T4 the value was 15.7 +/- 26.3 ng/dl (normal canine range is 45-150 ng/dl). Canine samples containing T3 AA were compared to serum from three rabbits actively immunized against T3 to provide anti-T3 for commercial RIA. The rabbit T3-antisera had an average T3 affinity constant similar to those of the canine samples (1.57 X 10(10) M-1), but had average titer, T3 binding capacity, and total T3 values more than 10-fold higher. Our findings indicate that, in dogs with serum containing T3 AA and normal total T4 concentrations, a compensatory mechanism appears to exist to maintain non-T3 AA bound T3 within the range of normal total T3. This compensatory mechanism does not operate in those dogs with insufficient thyroid activity to maintain normal total T4 values.     

Evaluation of the radioimmunoassay (RIA) for detection of Ciguatoxin (CTX) in fish tissues

The results of the radioimmunoassay (RIA) procedure for the direct determination of ciguatoxin (CTX) in fish tissues are presented. The RIA procedure detected CTX in 93% of fish tissues obtained from clinically documented ciguatera cases. The immunoglobulin nature of the reactive serum factor in sheep immunized with CTX was ascertained by DEAE-cellulose chromatography and by the ability to distinguish toxic from nontoxic fish tissues. Furthermore, a longitudinal study with commercial Seriola dumerili proved the RIA procedure to be effective in protecting the consumer from ciguatera poisoning by eliminating the potentially hazardous fishes from the markets. It is suggested that the RIA procedure has potential use in surveying areas for fishery development and is of value in keeping potentially toxic large fish off the market.     

The role of plasmin on the double antibody radioimmunoassay of carcinoembryonic antigen in human blood samples.

Double antibody radioimmunoassay (RIA) of carcinoembryonic antigen (CEA) on a series of freshly drawn out concurrent plasma and serum samples of normal human blood donors showed that the serum CEA values were invariably higher than the corresponding plasma CEA values. Extraneous addition of fibrinogen brought down the serum-CEA level to a value comparable to or less than the corresponding plasma value. The effect of certain factors associated with blood clotting, particularly Ca++, fibrinogen, and the fibrinolytic enzyme plasmin, was investigated. Ca++ was shown to play no role whereas the effect of fibrinogen was shown to be indirect in that it served as a specific substrate for plasmin, thereby preventing the plasmin degradation of the primary antibody used in the RIA. The finding stresses the role of enzymes like plasmin in double antibody RIA in general and may explain some of the anomalous results obtained when testing biologic material containing plasmin-like substances.     

Effect of thyroid hormone on epidermal growth factor-like immunoreactivity and growth velocity in cynomolgus monkeys

To examine the potential role of epidermal growth factor (EGF) in mediating the effects of thyroid hormone on linear growth, we measured serum EGF levels by RIA in cynomolgus monkeys before and during methimazole-induced hypothyroidism, and after 9 weeks of T4 replacement at different doses. Ten castrated prepubertal monkeys were rendered hypothyroid by methimazole (0.0125% in drinking water for 12 weeks). Methimazole was continued, and T4 was then administered for 9-week intervals. Six weeks elapsed between successive T4 doses. The sequence of different T4 doses for each animal was random. Serum EGF level was measured at baseline and at the end of each treatment period with a newly developed RIA using a polyclonal antiserum against human recombinant EGF. Serum EGF level correlated significantly with the level of serum thyroxine but not with serum triiodothyronine, over the thyroxine dosage range of 1-4 micrograms/kg/day (r = 0.41, p less than 0.005). Lower-leg growth rate correlated significantly with serum EGF level over this same thyroxine dosage range (r = 0.41, p less than 0.005). These data are consistent with the hypothesis that EGF may mediate some of the effects of thyroid hormone on skeletal growth.     

甲亢患者甲状腺激素水平与血脂代谢相关性分析及临床意义探讨

目的:探讨甲亢患者甲状腺激素水平与血脂代谢指标之间的关系。方法:对160例甲亢患者治疗前后的甲状腺激素(TH)水平、血脂水平进行对照分析。结果:甲亢治疗前后与健康对照组比较,游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)明显增高,促甲状腺激素(TSH)明显降低,差异有统计学意义(P<0.01);总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(LDL-C)、载脂蛋白A(apoAΙ)、载脂蛋白B(apoB)均明显降低,且差异具有统计学意义(P<0.01);低密度脂蛋白胆固醇(HDL-C)无明显变化(P>0.05)。结论:甲状腺激素与脂类代谢密切相关,临床上在诊治甲亢患者时,应当加强血脂水平的监测,以便更好地指导临床诊治,为疾病的发生发展、判断预后提供有价值的实验室检测指标。     

Disparity in the response of sex steroid-binding protein and corticosteroid-binding globulin to thyroxine in the primate

There is uncertainty regarding the differential effects of thyroid hormone on the circulating levels of sex steroid-binding protein (SBP) and corticosteroid-binding globulin (CBG). Therefore, we studied the effects of elevated thyroxine (T4) on SBP and CBG concentrations in serum in 4 baboon (Papio anubis) infants between 6 and 7 mo of age. The infants were given levothyroxine in increasing doses (25 to 600 micrograms) over a 1-mo period until the peripheral T4 levels were 3-4 times higher than baseline values. Each animal served as its own control. Blood samples were obtained at 2- to 3-day intervals prior to and during treatment. Serum T4 was measured by RIA. SBP and CBG were measured by diethylaminoethyl cellulose filter paper assays. Elevations in serum T4 resulted in a dose-response increase in circulating SBP concentrations with a maximal increase at the 600-micrograms dose of T4 (p less than 0.001). In contrast, significant elevations in peripheral CBG levels occurred with 50 and 100 micrograms of T4 (p less than 0.025 and p less than 0.01, respectively), but decreased thereafter with higher doses of T4. At the 600-micrograms dose of T4, CBG concentrations returned to baseline values. We conclude that in the baboon, T4 increases the circulating levels of SBP in a graded fashion, while the effect on CBG is biphasic.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Measurement of human FSH by radioreceptor assay

We established a sensitive RRA system for human FSH, employing PMS-treated immature rat ovary. The dissociation constant of the binding of the receptor preparation to NIAMDD human FSH-2 was 1.15 x 10(-10) M. The standard curve was obtained with 0.2-25.6 ng/tube of NIAMDD hFSH-2. Purified hLH, hTSH, and HCG had no significant effect on the binding. When the anterior pituitary homogenates obtained from humans were assayed by this system, the intra-assay and inter-assay coefficients of variation were 11.9% and 13.4% respectively, and the assay values correlated well with those obtained by RIA. This assay is applicable for the measurement of FSH in serum, when the non-specific inhibitor effects of serum are compensated for by the addition of merthiolate and when FSH-free serum is used instead of the buffer for the standard curve. The intra-assay and inter-assay coefficients of variation were 9.31% and 19.7% respectively. The assay values correlated with those obtained by RIA under the same physiological state. The ratio of the assay values RRA/RIA, was dependent upon the physiological state, e.g. 6.29 in men, 3.84 and 4.18 in women at follicular and luteal phase respectively and 2.40 in menopausal women. During the menstrual cycle, our results showed that the value of RRA/RIA derived from serum did not change significantly.     

Validation of the radioimmunoassays for pituitary gonadotropins II. Human follicle stimulating hormone (author's transl)

The present paper summarizes the experience of the authors in the setting up of the radioimmunoassay (RIA) for human follicle simulating hormone (H-FSH), with the purified antigen for radioiodination, the F-FSH standard and the specific antibodies kindly donated by the National Pituitary Agency of the National Institutes for Arthritis, Metabolic and Digestive Diseases of the National Institutes of Health, Bethesda, MD. U.S.A. The conditions for the RIA have modified somewhat and simplified with respect to the suggested instructions accompanying the reagents. Thus, the amount of Chloramine T and the time of exposure of the labeled H-FSH (H-FSH) has been studied. It is always purified on Sephadex G-100 immediately before addition to the RIA and in this manner it may be used for up to 2 month after labeling when kept at --20 degrees C. Curves obtained at different dilutions of the H-FSH Standard, carried out with phosphosaline buffer, pH 7.4-7.8 (PBS) containing 1 % human serum albumin, or with horse plasma, of with PBS containing 0,25 % serum from non-immune rabbits (RIA Buffer) have been compared iwth those abtained by serial dilutions of sera from post-menopausal with these diluents. The most consistent results were obtained with the RIA buffer as diluent. The redisual error was smaller, and serial of dilution curves of the H-FSH standard were parallel to those of plasma and acetone precipitates of urine from post-menopausal women. Parallelism was not god using those serum. Results using PBS contianing human seum albumin were poor. PBS containing bovine serum albumin was avoided as some batches were found to interfere with the binding of the F-FSH to the antibody. The stability of the different dilutions of the H-FSH standard prepared in RIA buffer was tested. It was found that the standard curves could be prepared, pipetted into the RIA tubes and kept ready, frozen at --20 degrees C for one to two months. This shortens the actual setting up of a given RIA and decreases interassay variation of results. Parallelism of the H-FSH standard curve with serial dilutions (in RIA-buffer) of sera from women on the day of the preovulatory was confirmed. The data obtained in men and women, during stimulation with LH-RH are also given. No cross reactivity was found the HCG or sera from women, in agreement with the fact that the antiserum had been absorbed with HCG. There is, however, a considerable degree of cross reactivity with H-TSH; Thus, sera containing 15 muU/ml H-TSH or more, would give false H-FSH results. Such H-TSH values are not only found in hypothyroid patients, but might be reached during TRH responses to TRH.     

Relationships between sex hormones assessed in amniotic fluid, and maternal and umbilical cord serum: what is the best source of information to investigate the effects of fetal hormonal exposure?

Levels of testosterone (T) (total and free), androstenedione (A4), dehydroepiandrosterone sulphate (DHEAS), sex hormone-binding globulin (SHBG), and estradiol (E2) were measured by radioimmunoassay (RIA) in 156 normal pregnancies (77 male and 79 female fetuses). Samples were obtained from amniotic fluid, 2nd and 3rd trimester maternal serum, and umbilical cord serum at birth. During the critical period of brain differentiation, at the beginning of the second trimester of pregnancy, sex differences in T and A4 were found in amniotic fluid and not in maternal serum. This finding adds to the fact that mostly low and nonsignificant correlations were found for the different androgenic hormones between levels assessed in amniotic fluid and maternal plasma at this particular and very sensitive period of fetal brain development. On the other hand, high correlations were found for the same hormones between the samples of maternal serum in the 2nd and the 3rd trimester. Our data show that, of all available sources, amniotic fluid seems to be the best candidate to investigate the effects of early fetal androgen exposure.     

Analyses of the serum concentrations and antigenic determinants of thyroglobulin in chickens susceptible to autoimmune thyroiditis

These studies revealed an abnormal elevation in serum thyroglobulin (Tg) concentrations of the thyroiditis-prone OS chicks. Their serum levels, measured by a sensitive and specific RIA, remained high for the first 2 wk of age, a time preceding significant lymphoid infiltration of their thyroid glands. The elevated serum Tg levels probably resulted from, or were related to, hyperactivity of the OS thyroid gland. Reducing the activity of the OS thyroid gland by exogenous administration of T4 caused a temporary but significant reduction in thyroidal infiltration and the synthesis of Tg antibodies. In addition to the analysis of serum concentrations of Tg, a competitive binding RIA was developed to determine whether unique antigenic determinants exist on Tg isolated from OS thyroid glands. This was proved to be unlikely because OS chicks produced autoantibodies that fully cross-reacted with Tg isolated from normal chicken thyroid glands. The relationships between intrinsic thyroid hyperactivity, high serum Tg levels, the sensitization of OS thymocytes, and thyroiditis are discussed.     

Serum T3 level in the patients with hyperthyroidism after therapy.

Serum T3 level in various thyroid diseases was determined in unextracted serum with the Dainabot kit for T3 RIA. The serum T3 level in 33 normal subjects was 0.8-1.6 ng/ml. It was 5.7 +/- 3.5 ng/ml (mean +/- S.D.) in 36 hyperthyroid patients, and undetectable to 0.8 ng/ml in 21 hypothyroid patients. Generally the serum T4 and serum T3 decreased in parallel after radioiodine therapy for hyperthyroidism. However, in some cases the serum T3 level remained high in spite of normalized serum T4 after radioiodine therapy. This state indicated "T3-toxicosis", and hyperthyroidism was apt to recur. When thyroid function was observed for 2 years following radioiodine treatment, the ratio of serum T3 (T3 level before treatment/T3 level after treatment) decreased more significantly as compared with the ratio of serum T4 in euthyroid cases. Serum T3 provides a more sensitive index of thyroid function than serum T4 in euthyroid states after radioiodine or anti-thyroid drug therapy. The present data indicate that the serum T3 level and the T4/T3 ratio are valuable aids in the estimation of prognosis of hyperthyroid patients after various treatments.     

Melatonin and thyroxine response to pollution in white stork nestlings (Ciconia ciconia): aspects of rhythmicity and age

There is growing evidence that ubiquitous environmental contaminants may interfere with vertebrate endocrine systems. The selected endocrine biomarkers are used to indicate the condition of free-ranging populations of wildlife, including avian species. The aim of this study was to determine the impact of environment quality on serum thyroxine (T4) and melatonin (Mel) in white stork nestlings (Ciconia ciconia) living in different locations: small villages in natural areas surrounded by forests and crop fields, near the city and near the copper smelter. We extended our analyses to examine the hormones' day-night changes in conjunction with chicks' age. Total serum T4 and Mel was measured by RIA. T4 level, as a decisive measure of thyroid hormone productivity, was significantly lower in the nestlings exposed to pollutants from the copper smelter. Mel, as a well-known scavenger of free radicals, was elevated in the nestlings in the area near the copper smelter. This study indicates that alteration in T4 and Mel levels could be a useful marker of exposure of nestling wild storks to different toxic substances in field studies. Mel is postulated to be a susceptible defensive molecule as a protective mechanism for organisms.     

Production and characterization of antibodies against nivalenol tetraacetate

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.     

Radioimmunossay for detection of antigen and antibodies to Newcastle disease virus.

When Newcastle disease virus (NDV) is treated with NP-40 and ether a membrane fraction of 150,000 m.w. is obtained. This fraction which is composed of two polypeptides with m.w. of 56,000 and 76,000 was used in a radioimmunoassay (RIA). The assay was developed for both antigen and antibody and was found to be reproducible, specific, and highly sensitive. Titers of 1:51,200 were determined by RIA as compared to 1:4 by agar gel diffusion and 1:200 by hemaglutination inhibition (HI). As little as 5 ng of viral protein were detected by RIA inhibition technique. Labeled antigen could be stored in the presence of serum, KCI and Triton X-100 at -20 degrees C for as long as 6 weeks and retained similar reactivity as fresh reagent.     

Production and characterization of antibodies against nivalenol tetraacetate.

Antibody against nivalenol tetraacetate (tetra-Ac-NIV) was prepared by immunization of rabbits with triacetyl-15-pimelate-NIV conjugated to bovine serum albumin. By using tritiated tetra-Ac-NIV as the test ligand, antibody titers were demonstrated as early as 4 weeks after immunization. Useful antibody for radioimmunoassay (RIA) of tetra-Ac-NIV was obtained 7 weeks after immunization, with one booster injection. Results of competitive RIA revealed that the antibody was most specific to tetra-Ac-NIV. The relative cross-reactivity of this antibody with tetra-Ac-NIV, deoxynivalenol triacetate, and neosolaniol triacetate was found to be 100, 2.2, and less than 1, respectively. Practically no cross-reaction was found with deoxynivalenol, fusarenon X, and NIV. The detection limit for tetra-Ac-NIV by RIA was about 5.0 ng/ml (0.5 ng per assay). The use of this antibody for quantitation of NIV in cereals after acetylation of sample extracts is proposed.