Iatrogenic thyrotoxicosis and the role of therapeutic plasma exchange.

Thyroid storm or severe thyrotoxicosis results from extreme thyroid hormone elevation. Therapy includes medical management to prevent hormone production, release, recycling, and peripheral conversion while stabilizing adrenergic tone. Thyroid dysfunction is the usual cause but it can be due to excessive thyroid hormone ingestion. Therapeutic plasma exchange (TPE) has been used to rapidly remove protein-bound thyroid hormone. American Society for Apheresis guidelines make a weak recommendation to perform TPE in selected patients in the treatment of thyrotoxicosis based on low quality evidence. We present a case of excessive thyroid replacement hormone ingestion treated by TPE. The patient presented with the clinical picture of thyroid storm, including cardiovascular compromise and massively elevated total and free T3 (525 ng/dL, nl 80-200 ng/dL and 28 pg/mL, nl 2.0-3.5 11 pg/mL), which failed medical therapy. A single, one plasma volume TPE was performed. Both total and free T3 demonstrated substantial declines immediately after TPE with the patient's mental status returning to near-normal. Thyroid hormone extraction efficiency and collection efficacy were calculated as 37.1% and 40.8%, respectively. Prior to discharge on day 6, the patient's compounding pharmacy indicated that a "bad batch" of bovine thyroid gland derived replacement hormone had been produced. TPE appears to be effective in removing protein bound thyroid hormone in extreme iatrogenic thyrotoxicosis.

Anti-ruthenium antibodies mimic macro-TSH in electrochemiluminescent immunoassay.

BACKGROUND: Macro-hormones are circulating conjugates of hormones with immunoglobulins, which often artefactually elevate biochemical test results. Particularly when causing only moderate elevation no suspicion will be raised. By far the most frequently encountered macro-hormone is macro-prolactin. Here we report a female patient with rheumatoid arthritis who had persistently and grossly elevated thyroid stimulating hormone (TSH) but normal free thyroxine in electrochemiluminescent assays. Although clinically euthyroid, she was put on thyroxine therapy which caused hyperthyroid symptoms. METHODS: An analytic interference by macro-TSH was assumed by dilution experiments, polyethylene-glycol-precipitation, the addition of a heterophilic antibody blocking reagent and size exclusion chromatography. RESULTS: Further workup, however, revealed the presence of anti-ruthenium antibodies. CONCLUSIONS: To our knowledge this is the first report of anti-ruthenium antibodies selectively interfering with a TSH assay and causing erratic gross elevation of TSH mimicking macro-TSH.

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC.

This paper deals with the chiral separation of triiodothyronine (T3) and thyroxine (T4) by HPLC and micro-HPLC. The separation of T3 and T4 is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic) phase was used. In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T3 and T4. l-4-Hydroxyproline was bonded to 3 microm and the glycopeptide antibiotics were bonded to 3.5 microm silica gel and separations were accomplished by microbore HPLC columns (10 cmx1 mm I.D.). With both techniques and all chiral selectors investigated T3 and T4 were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.

A new radioimmunoassay for plasma L-triiodothyronine: measurements in thyroid disease and in patients maintained on hormonal replacement.

A new procedure for the radioimmunoassay of l-triiodothyronine (T(3)) in human plasma is described in which the iodothyronines are separated from the plasma proteins before incubation with a specific antiserum to T(3). The antibody bound and free T(3) are separated with dextran-coated charcoal. In this system, the mean recovery of T(3) added to plasma was 97.9% and both in vitro conversion of l-thyroxine (T(4)) to T(3) and cross-reaction between T(4) and the anti-T(3) antibody were undetectable (less than 0.1%). The assay procedure allowed the measurement of T(3) in up to 0.5 ml of plasma resulting in improved assay sensitivity (6 ng/100 ml). The mean plasma T(3) in normal subjects was 146+/-24 ng/100 ml (sd). Mean T(3) concentration was increased in hyperthyroidism (665+/-289 ng/100 ml) and decreased in hypothyroidism (44+/-26 ng/100 ml). In patients with severe hypothyroidism, plasma T(3) was between 7 and 30 ng/100 ml. Plasma T(3) concentration was relatively constant throughout the day in three euthyroid subjects. In contrast, in hypothyroid subjects on replacement therapy with T(3), a T(4): T(3) combination or desiccated thyroid plasma T(3) was markedly elevated for several hours after ingestion of the medication. Plasma T(3) was unchanged throughout the day in patients treated with T(4). Thus, insofar as plasma T(3) levels are concerned, replacement therapy with T(4) appears to mimic the euthyroid state more closely than other preparations.

Propylthiouracil inhibits the conversion of L-thyroxine to L-triiodothyronine. An explanation of the antithyroxine effect of propylthiouracil and evidence supporting the concept that triiodothyronine is the active thyroid hormone.

6-n-propylthiouracil (PTU) administered to male Sprague-Dawley rats maintained on 2 and 5 mug L-thyroxine (T(4))/100 g body weight resulted in a marked reduction in the rate of conversion of L-thyroxine to L-triiodothyronine (T(3)). These effects could not be ascribed to induced hypothyroidism since the group maintained on 5 mug T(4)/day had normal levels of liver mitochondrial alpha glycerophosphate dehydrogenase. In confirmation of previous studies, PTU also reduced the fractional rate of deiodination of T(3). These observations provide a possible explanation of the many published observations indicating that PTU antagonizes the tissue effects of T(4) but not of T(3). The data suggest that monodeiodination of T(4) but not of T(3) is essential before hormonal effects can be manifested at the cellular level.

Simultaneous measurement of thyroxine and triiodothyronine peripheral turnover kinetics in man.

Serum triiodothyronine (T(3)) kinetics in man have been difficult to define presumably due to the interference of iodoproteins generated during the peripheral metabolism of T(3). The use, in the present study, of an anion-column chromatographic method for separation of serum T(3) as well as thyroxine (T(4)) from these iodoproteins has overcome this technical handicap. Simultaneous measurement of serum (125)I-T(3) and (131)I-T(4) kinetics were performed in 31 subjects from the clinical categories of euthyroid, primary hypothyroid, thyrotoxic and posttreatment hypothyroid Graves' disease, factitial thyrotoxic, and idiopathically high and low thyroxinebinding globulin states. The normal mean T(3) fractional turnover rate (kT(3)) was 0.68 (half-life = 1.0 days), increased in toxic Graves' disease patients to 1.10 (half-life = 0.63 days), and decreased in primary hypothyroid patients to 0.50 (half-life = 1.38 days). The mean T(3) equilibration time averaged 22 hr except in hypothyroid and high thyroxine-binding globulin (TBG) patients where the equilibration period was delayed by 10 hr. The mean T(3) distribution space in normal subjects was 38.4 liters. This was reduced in subjects with high TBG levels (26 liters) and increased in patients with low TBG and in all hyperthyroid states (53-55 liters). The normal serum T(3) concentration was estimated by radioimmunoassay to be 0.106 mug/100 ml. Combined with the mean T(3) clearance value of 26.1 liters/day, the calculated T(3) production rate was 27.6 mug/day. The mean T(3) production rate increased to 201 mug/day in thyrotoxic Graves' disease patients and was reduced to 7.6 mug/day in primary hypothyroid subjects. T(3) production rate was normal in subjects with altered TBG states. The ratio of T(3) to T(4) production rate in normal subjects was 0.31 and was unchanged in patients with altered TBG values. This ratio was increased in all Graves' disease patients with the highest value being 0.81 in the posttreatment hypothyroid Graves' disease group. This apparent preferential production of T(3) may have been responsible for the retention of rapid turnover kinetics for T(3) and T(4) observed in treated Graves' disease patients. The finding that factitial thyrotoxic patients also displayed similar rapid T(3) and T(4) turnover kinetics indicates that these alterations are not a unique feature of Graves' disease per se. When comparing the peripheral turnover values for T(3) and T(4) in man, it is apparent that alterations in metabolic status and serum TBG concentration influence both hormones in a parallel manner; however, changes in metabolic status seem to have a greater influence on T(3) kinetics while alterations in TBG concentrations have a greater effect on T(4). These observations probably relate to the differences in TBG binding affinity and peripheral tissue distribution of these two hormones.

Chiral separation of T3 enantiomers using stereoselective antibodies as a selector in micro-HPLC.

This work deals with the application of stereoselective antibodies against L-T3 as a tailor-made chiral selector in micro-HPLC. The separations were performed in microbore columns using commercially available anti-L-T3 antibodies chemically bonded to 5 microm silica gel. The enantiomers of T3 were baseline separated under mild continuous isocratic elution conditions using 10 mM phosphate buffer, pH 7.4. The D-enantiomer eluted with the void volume, while the L-enantiomer was retained by the antibody phase and eluted second. An indirect competitive and non-competitive enzyme linked immunosorbent assay (ELISA) was used for testing the stereoselectivity of anti-L-T3 antibodies.

Evidence that triiodothyronine and reverse triiodothyronine are sequentially deiodinated in man.

We have demonstrated that in patients given a single iv injection of T3, rT3, or, to a lesser extent, T4, all labeled with 125I in the outer or phenolic ring, chromatography of serum on columns of Sephadex G-25 superfine revealed the presence of a labeled material, separate from the administered hormone and from both iodide and iodoprotein. This peak has been termed pre-T3 because it elutes just before the T3 peak. Identification of the various compounds in pre-T3 was carried out by cation exchange chromatography. Pre-T3 generated from [125I]T3 consistently contained labeled compounds with the chromatographic behavior of 3,3'-diiodothyronine and 3'-monoiodothyronine, while pre-T3 generated from [125I]rT3 contained labeled products with the chromatographic mobility of 3',5'-diiodothyronine, 3,3'-diiodothyronine, and 3'-monoiodothyronine. In addition, pre-T3 also contained the glucuro- and sulfoconjugates of these several labeled products. These studies demonstrate that T3 and rT3 undergo progressive and probably sequential deiodination in the peripheral tissues, resulting in the formation of a variety of diiodothyronines and monoiodothyronine, as well as their glucuro- and sulfoconjugates.

Resin hemoperfusion: a method of removing circulating thyroid hormones.

The ability of an extracorporeal hemoperfusion system employing neutral Amberlite resin to bind thyroid hormone and to decrease circulating levels of triiodothyronine (T3), thyroxine (T4), and free thyroxine (FT4) was evaluated in dogs made thyrotoxic by the intramuscular administration of thyroid hormone. Since the resin column and tubing were charged with saline, the effects of hemodilution from this source on serum T3 and T4 was assessed by control perfusion through a column which did not contain any resin. After correction for hemodilution, the mean serum T3, T4 and FT4 decreased during 2 hours of resin hemoperfusion by 39%, 35%, and 46%, respectively. Hormonal clearance rates were calculated in two experiments and the estimated net hormone removed averaged 60.4 mug of T3 and 1990 mug of T4. Hematologic indices and routine chemistries did not change significantly in these dogs during the procedure except for a decrease in mean serum albumin concentration and an increase in mean serum glucose concentration. Hemoperfusion through this resin system seems to be a safe, effective means of decreasing serum T3, T4, and FT4 in thyrotoxic dogs and warrants evaluation for the treatment of thyroid storm in man.

Coupling of iodotyrosine catalyzed by human thyroid peroxidase in vitro.

The coupling of iodotyrosine (coupling reaction) is one of the least studied in the formation of thyroid hormone, particularly in human thyroid diseases. This paper describes a method of measuring iodotyrosine coupling catalyzed by human thyroid peroxidase (TPO) in vitro. There were two important requirements to demonstrate the coupling reaction: 1) thyroglobulin with a low thyroid hormone content, and 2) partially purified TPO. Thyroglobulin with low thyroid hormone content was obtained from Grave's and follicular adenoma tissues after propylthiouracil (PTU) therapy and L-T4 therapy, respectively. TPO was prepared from Graves' thyroid by solubilizing the 100,000 X g pellet of thyroid homogenate with sodium deoxycholate and trypsin, followed by Sephacryl S-300 gel filtration. Before the coupling reaction, thyroglobulin was iodinated with chloramine-T and potassium iodide, followed by dialysis. The coupling reaction was carried out by incubating newly iodinated thyroglobulin with TPO, diiodotyrosine, a coupling stimulator, and a H2O2-generating system (glucose and glucose oxidase) for 20 min at 37 C. After thyroglobulin was digested with Pronase, the thyroid hormone content of the thyroid digest was measured by RIA. Coupling activity was measured by the amount of newly formed T3 (nanograms of T3 per mg thyroglobulin). The time course of coupling reaction showed a progressive increase in coupling activity up to 30 min, and the reaction was temperature and pH dependent, with a pH optimum of 7.0. Coupling activity in the presence of H2O2 and TPO was 43 +/- 5.0 ng T3/mg thyroglobulin (mean +/- SD of triplicate samples), and addition of diiodotyrosine to the H2O2-TPO system caused a nearly 3-fold increase in coupling activity. This method has potential utilization for measurement of peroxidase coupling activity, since there was a linear relationship between the measured coupling activity and the amount of added TPO when the TPO concentration was over 3 micrograms/300 microliter. Methimazole (MMI) and PTU had similar potencies in inhibiting the TPO-catalyzed coupling reaction, whereas MMI was distinctly more potent than PTU as an inhibitor of TPO-mediated iodination in vitro. The different potencies of MMI in the two reactions suggest that different inhibitory mechanisms may be involved in iodination and coupling. The reducing agent, sodium metabisulfite, was also found to be a more potent inhibitor of the TPO-mediated coupling reaction than of the TPO-mediated iodination reaction. The method of iodotyrosine coupling described here may be useful to investigate the coupling step of thyroid hormone formation in human thyroid diseases.

Immunohistochemical localization of thyroid hormone in rat thyroid gland.

Direct and indirect immunofluorescence techniques were used to localize the thyroid hormones triidothyronine (T3) and thyroxine (T4) in adult rat thyroid gland. Optimum dilutions of the antisera were established and four tissue fixatives were investigated for usefulness in this technique. Use of antibodies specific for either T3 or T4 resulted in brilliant fluorescence in the colloid pools and apical cytoplasm of follicular cells. In all cases, the adjacent parathyroid gland was devoid of fluorescence. This report demonstrates that these dipeptide hormones can be localized by using immunofluorescence techniques.

Rapid purification of tri-iodothyronine and thyroxine protein conjugates for antibody production.

Thyroxine (T-4) and tri-iodothyronine (T-3) were coupled to human serum albumin (HSA) with carbodi-imide. By adsorption chromatography on Sephadex G-25, fractions containing purified conjugate, but not reversibly-bound T-3 or T-4, were obtained, and this procedure took 5 h; considerably less than the conventional dialysis technique. Highly specific high-titre antisera were produced in rabbits and guinea-pigs by injection of these fractions in Freund's adjuvant.

Isolation of labeled triiodothyronine from serum using affinity chromatography: application to the extimation of the peripheral T4 to T3 conversion in rats.

A method for the isolation of small quantities of labeled 3,5,3' -triiodothyronine (T3) from serum or thyroid extracts is described. Conjugates of rabbit anti-T3 antibody to Sepharose 4B are incubated with 0.5 to 1 ml of human or rat serum at pH 8.6 for 1 hr. The tubes are centrifuged and washed with buffer followed by 6 M guanidine to remove nonspecifically bound labeled thyroxine (T4). The fraction of T3 and T4 bound to the Sepharose conjugate varies depending on the concentration of serum in the initial incubation tubes, the T3 and T4 content, and the specificity of the antiserum used. In a system that contains 0.5 ml of normal human serum, 1 ml of glycine-acetate buffer (pH 8.6), and 0.25 ml settled Sepharose-anti-T3 conjugate, the T3 to T4 binding ratio was generally 150-200, with up to as much as 50% of T3 bound to the pellet. The coefficient of variation of the method is less than 5%, and it may be performed in a matter of hours. There is no detectable conversion of T4 to T3 during the separation process. Using this technique, conversion of T4 to T3 was evaluated in euthyroid rats after injection of 125l-T4. Over the period of 36-72 hr after injection, a ratio of T3 to T4 of 0.74 +- 0.06 x 10-2 (mean +- SE) was present in the plasma. Using the calculated metabolic clearance rates for T3 and T4 in these animals, fractional conversion of T4 to T3 was estimated to be 27%, in good agreement with results obtained by other techniques. This method would appear to be of value for specific isolation of the small quantities of T3 produced from T4 after in vivo or in vitro T4 to T3 conversion.

Purification of thyroxine and triiodothyronine for radioassay with sephadex.

The use of [125I] thyroxine in the determination of dialysable thyroxine (free T4) places very high demands on the purity of the reagent. Less than 5% dissociated radioactive iodine (as iodide) is enough to influence the results [1]. For this reason several workers have developed methods for the purification of iodo amino acids used in radioassay for thyroid hormones. Two techniques have been used mainly, namely dialysis of incubated tracer-serum mixture [1,2], and adsorption chromatography on dextran gels in strongly basic buffers [3-5]. In this laboratory we have developed an adsorption chromatography method using bovine serum albumin for the separation of [125I] thyroxine from contaminating compounds.

Rapid and effective separation of thyroid hormones and iodide by thin-layer chromatography.

The use of 1,3-dichlorotetrafluoroacetone allows the conversion of the iodoamino acids into a chemical form convenient for following chromatographic separation on silica as an adsorbent and benzene or chloroform as a solvent. The time required for derivatization and TLC separation is less than one hour. The outstanding resolution of the substance of interest, as well as the sensitivity of the radiochemical analysis, makes the method convenient for the rapid determination of minute quantities of thyroid hormones.

An immunological method for the purification of radiolabelled thyroxine.

A method for the removal of iodothyronine and iodide impurities from radiolabelled thyroxine is described. The principle of the method is to bind iodothyronine contaminants to specific antisera, and then to separate the antiserum-bound contaminants and iodide from the thyroxine by column chromatography. As reported here, the method is optimised for the removal of more than 98% of contaminating 3,5,3'-triiodothyronine and 99.8% of contaminating iodide from between 10 ng and 100 ng of thyroxine, where the molar ratio of thyroxine: 3,5,3'-triiodothyronine before purification is greater than or equal to 10:1. Recovery of purified thyroxine is more than 80%. The performance of the method is superior to that of preparative dialysis.

The relative merits of polyethyleneglycol as a separating agent in the radioimmunoassay of thyroid hormones.

Polyethyleneglycol (PEG) has been recommended as a separating agent in the assay of some peptide hormones (Desbuquois, B. and Aurbach, G.D. (1971) J. Clin. Endocrinol. 33, 732) and several substances of low molecular weight (Ratcliffe, J.G. (1974) Br. Med. Bull. 30, 32). In the present study the PEG-separation technique has been modified and adapted for the assay of thyroid hormones. Separation with PEG has the advantage of being cheap, rapid and relatively non-susceptible to disturbances as compared with the charcoal and double-antibody-solid phase techniques. The influence of different buffer systems, varying pH and ionic strength, on the precipitation process with PEG also has been investigated. Of the different systems tested barbital buffer containing 0.1% human serum albumin proved to be the best, preferably in the presence of bovine gamma-globulin. In the radioimmunoassay of T3 variations in pH and ionic strength are of minor importance whereas in the radioimmunoassay of T4 the adherence to a certain pH is recommended. Barbital buffer containing 0.1% bovine serum albumin was inadequate in the T3 radioimmunoassay, while Tris and phosphate buffers did not give satisfying results for either radioimmunoassay.

Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography.

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of D- and L-thyroxine (D- and L-T(4)) and triiodothyronine (T(3)) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from -6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5-500 microg ml(-1) with limits of detection and quantitation lower than 0.1 and 0.5 microg ml(-1), respectively, for both analytes. Further, the determination of 0.1% impurity, of D-T(4) as well as L- and D-T(3) in levothyroxine sodium tablets proved to be feasible.

The binding of thyroxine and tri-iodothyronine to plasma proteins in the chicken at the physiological pH.

Amounts of 125I-labelled thyroxine (T4) and triiodothyronine (T3), representing approximately 50 per cent of the plasma concentration, were incubated with chicken plasma or serum, the proteins were separated by polyacrylamide gel electrophoresis at pH 7.4 and the distribution of the hormones determined. Seventy to 75 per cent of T4 migrated with albumin, about 10 per cent with the alpha-globulin and most of the remainder with a pre-albumin fraction. The percentage attached to the pre-albumin tended to increase with age. Neither barbiturate nor 8-anilino-1-naphthalene sulphonic acid (ANS) affected the distribution. About 50 per cent of T3 was associated with albumin, 30 to 40 per cent with the main alpha-globulin and most of the remainder with an adjacent globulin band. ANS reduced the percentage attached to albumin and increased that associated with the alpha-globulin. Barbiturate had no effect.