Pure Thyroxine Research Articles

Bestimmung von Thyroxin im Serum mit einem heterogenen Enzymimmunoassay: Ergebnisse einer gemeinsamen Erprobung

This paper describes the evaluation of a heterologous enzyme immunoassay for the determination of total thyroxine in serum by a group of seven clinical chemical laboratories. The test follows the principles of the enzyme linked immunosorbent assay (ELISA) and uses peroxidase as a marker. The evaluation of analytical reliability yielded the following results within the analytical range from 39 unto 322 nmol/l: 1. Within-batch precision ranged from 3.1 unto 10.4% (coefficient of variation) with single analyses. 2. Between-batch precision ranged from 3.7 unto 20.4% with single analyses. 3. Between-laboratories precision ranged from 5.4 unto 6.8%. 4. Pure thyroxine, added to serum or thyroxine-free serum, gave recoveries between 93 and 120%. 5. Analysis of control sera gave results essentially comparable to the assigned values based upon radioimmunoassays. 6. Analysis of 288 clinical sera gave slightly higher results by the enzyme immunoassay than by the analogous radioimmunoassay from the same manufacturer. 7. Comparison with other methods of analysis (radioimmunoassays, competitive protein ligand assays, hormonal iodine assay) yielded partly comparable, partly higher results. 8. Comparison with the homogenous enzyme immunoassay (EMIT) led to comparable results. 9. Interference due to hyperlipemia or hemolysis was not observed. 10. There might be an interference in hyperbilirubinaemic sera, due to an as yet unknown factor. With respect to practicability the ELISA-test compares favourably with the analogous solid phase radioimmunoassay. The main differences are the absence of radioactive material and a longer shelf-live of reagents. Following the manual procedure the time taken to perform the enzyme immunoassay is slightly longer than for the analogous radioimmunoassay.

Demonstration of antibodies to thyroxine and monoiodothyronine by mixed haemadsorption.

A TECHNICAL procedure for the application of the mixed haemadsorption technique1 to soluble antigens has recently been described2. This technique, which is essentially a mixed antiglobulin reaction, is being investigated because it is potentially a means of titrating antibodies to thyroglobulin in the sera of patients suffering from thyroiditis and other thyroid diseases. In the course of this work, it became of interest to find out whether the reaction with thyroglobulin may depend to some extent on the thyroxine determinants present on the thyroglobulin molecules. Pure thyroxine coated over a glass slide should have its antigenic determinants exposed much in the same way as when thyroglobulin is similarly exposed. Glass slides were therefore coated with a layer of thyroxine applied either as a suspension of microcrystals in phosphate buffered saline (PBS), pH 7.4, containing 1 per cent of normal rabbit serum (NRS) or as a solution in ethanol. Monoiodothyronine was applied as a solution in PBS, pH 7.4, containing 1 per cent of NRS. Human thyroglobulin was applied as a solution in PBS, pH 7.4. The various antigen films were dried at room temperature and all slides except those coated with thyroxine in ethanol were fixed for 5 min in acetone. The antigen films were covered with layers of agar and ‘Perspex’ disks provided with holes, which served as serum reservoirs. Two-fold serial dilutions of the sera containing antibody were added to the holes and the antibodies were allowed to diffuse through the agar layer. The antibody zones produced when the diffusing antibody attached itself to the antigen film on the slide were marked out by red cells provided with an exterior coating of antiglobulin so that they were able to adsorb specifically on to antibodies from the animal species concerned2,3.

FUNCTIONAL RELATIONSHIPS BETWEEN THE LIVER, THE THYROXINE-BINDING PROTEIN OF SERUM, AND THE THYROID*

Various aspects of the effect of liver damage on thyroid function were studied in 35 cases of liver disease (with 30 normal controls) and in a group of thyroidectomized rats made toxic with carbon tetrachloride. Acute, severe, diffuse liver damage (as in infectious hepatitis) temporarily provokes a statistically significant increase in the level of serum protein-bound iodine (PBI), associated with a higher rate of fixation of thyroxine to the serum thyroxine-binding protein (TBP). These abnormal findings are caused by different mechanisms, such as disturbance of biliary thyroxine excretion, and lack of deiodination and glucuroconjugation of thyroxine in the damaged liver cell. Under these conditions, the thyroxine turnover is slower and thyroxine accumulates in the circulation. The increased serum PBI level is not associated with hyperthyroidism, although the content of the PBI in infectious hepatitis is practically built from chromatographically demonstrable pure thyroxine. The data do not confirm the hypothesis of a pathologic binding of thyroxine to plasma protein, but the high TBP level in hepatitis could contribute to the non-utilization of thyroxine in the tissues. On the other hand, experiments in rats show that the damaged liver cell is also the cause of the lack of metabolic response in this particular type of hyperthyroxinemia.

THE DURATION OF ACTION OF SINGLE DOSES OF THYROID HORMONE1

REPORTS on the duration of action of thyroid compounds vary considerably. As early as1904, Magnus-Levy reported that the effects on the basal metabolic rate of thyroid feeding in myxedematous patients lasted 80 days. Kendall (1917), probably the first to inject pure thyroxin intoanimals, compared the effects of small daily doses and a single large dose. A goat receiving 9 mg.daily was dead at the end of 11 days; the single injection of 150 mg. in another goat produced changes in the electrocardiographic tracings which returned to normal in 2 or 3 days. In contrast to-these results, Kendall (1923) reported that a single dose of thyroxin in a myxedematous patient would exert its physiologic effect for 5 to 6 weeks. Boothby, Sandiford, Sandiford, and Slosse (1925) showed that the rate of decline of the basal metabolism following withdrawal of thyroid administration in myxedematous patients was a straight line, when plotted on semilogarithmic paper.

The Effect of the Active Thyroid Substance on Metabolism and Other Functions in Guinea Pigs

Summary.Different preparations of dried thyroid from normal mammals and human beings and from cases of simple goitre have the same metabolic effect when given by mouth in equivalent daily doses based on the iodine content to mature male guinea pigs, accustomed to the metabolism determinations under standard conditions, while in exophthalmic goitre the iodine of the gland becomes less active.A partly purified product, elityran, also shows the same effect, while pure thyroxin, even when given subcutaneously is definitely less active. No difference was observed between the activities of thyroxin when given by mouth or subcutaneously.The metabolic effect of α‐dinitrophenol given by mouth reaches a maximum in about an hour and falls again rapidly while that of thyroid preparations takes two days to develop and by administration each day reaches a more or less constant level, maintained throughout the period of administration.Administration of iodine or di‐iodotyrosine reduces the metabolism raised by every day injections of thyreo‐stimulating hormone (artificial “Basedow”) and causes a regression of the histological and cytological “Basedow” symptoms in the thyroid gland, but has no effect on the hyperthyroidism brought about by feeding thyroid preparations.Refractoriness to the thyreo‐stimulating hormone was not observed in our case.The quantitative relation between dose and metabolic effect of dried thyroid preparations was studied on 44 animals. On 9 of these no well defined metabolic level could be obtained. The 35 animals showed a fairly regular relationship. The average resting metabolism of the guinea pigs was 100.1 ± 3.4 ml O2/m2/min. The increases were for 0.15 mg J/m2/day 17 ml O2/m2/min., for 0.25–29.0, 0.50–35.7 and 10.00–50.4. This corresponds to an approximately straight line relation between log dose and the absolute rise in metabolism.

Effect of Hormones Upon Cells Grown in vitro: II. The Effect of the Hormones from the Thyroid, Pancreas, and Adrenal Gland

In continuation of our hormone studies we have investigated the effects of thyroxin, adrenalin, cortin, and insulin in varying concentrations upon the growth of fibroblasts isolated from the mouse embryo heart. The method of obtaining material, preparing cell cultures, and determining the progress of cellular growth was identical with the one used in our study of sex hormones (1). The hormones studied were obtained in pure form from Dr. F. A. Hartman9s laboratory, the Parke, Davis, the La Roche, and Lilly Companies. The choice of concentrations was purely empirical and the attempt was made to establish for each hormone the optimal effective concentration. Dilutions ranging from 10 −1 to 10 −9 were examined with regard to their effect on tissue growth, and single as well as repeated doses of the various dilutions were administered. The dilutions were prepared with Tyrode solution and the pH was readjusted for each dilution. Thyroxin: The influence of pure thyroxin on the growth of fibroblasts has been investigated in this country by Vogelaar and Erlichman (12), who attempted to repeat work done in Japan by Semura (9) and Sato (8). Using fibroblasts from adult human thyroids, they failed to find any remarkable differences in growth in media containing thyroxin in a wide range of concentrations. Apparent dissimilarities disappeared in trypsin subcultures made after fourteen-day intervals. Semura had previously found that thyroxin (Squibb) in a concentration of 1: 10,000,000 had no influence on the growth of fibroblasts while good growth was observed in a concentration of 1: 10,000. Thyroxin was also found to postpone by several days the degeneration of cells.

CALORIGENIC ACTION OF SINGLE LARGE DOSES OF DESICCATED HOG THYROID

Several reports have recently been made from this clinic concerning the effects of administering thyroxine in various forms orally and intravenously in an alkaline solution. 1 These observations have shown that single doses of from 10 to 100 mg. of pure thyroxine administered by mouth or duodenum do not have a demonstrable effect on the basal metabolism of patients with myxedema, whereas the oral administration of single large doses of monosodium thyroxine and thyroxine in alkaline solution causes increases in the basal metabolism of 22 and 63 per cent, respectively, as great on the average as the increase produced by the intravenous injection of an alkaline solution. It seemed important to determine the effect of single large doses of desiccated thyroid of the same iodine content for comparison with these data. We have previously reported observations which suggest that the oral administration of desiccated thyroid and the intravenous injection of

THE EFFECTS OF VARIOUS COMPOUNDS OF THYROXINE ON THE BASAL METABOLISM*†

Many different compounds of thyroxine may be prepared which may have different effects on the basal metabolism. It would be impossible in the time allowed for this paper to discuss in detail even the observations that we have made on the effects of a few compounds. For the purpose of simplicity we shall confine our attention largely to a comparison of the effects of administering thyroxine intravenously in alkaline solution with those of administering thyroxine in various forms by mouth. A Comparison of the Effects of Prolonged Oral Administration of Pure Thyroxine, Monosodium Thyroxine and Desiccated Thyroid with the Effects of Administering Thyroxine Intravenously. In one patient with myxedema we have observed a definite response to the prolonged oral administration of large doses of pure thyroxine suspended in distilled water. The thyroxine was given in daily doses of from 7.5 to 50 in gin. over a period of eight months. Final figures are not yet available, but it would seem that the basal metabolism, ...

EFFECT OF ALKALI ON THE ABSORPTION OF THYROXINE FROM THE GASTROINTESTINAL TRACT

We have just reported observations which show that when thyroxine is administered by mouth in the form of its monosodium salt to patients with myxedema it has only about from one-third to one-fifth as much effect as when it is administered intravenously in alkaline solution (sodium hydroxide). 1 This conclusion was based on the amounts of the two substances which had to be administered every day in order to hold the basal metabolism at the normal level. Pure thyroxine had much less effect than its monosodium salt. Since when thyroxine is dissolved in an excess of sodium hydroxide the disodium salt is presumably formed, it seemed logical to try the effect of administering the hormone by mouth in an alkaline solution. Klein 2 reported that Hoffmann-La Roche thyroxine solution for oral use, Schering thyroxine in alkaline solution with the same p h as the Hoffmann-La Roche solution and Schering's Trinktabletten

The Thyroid and the Rate of Cell Division

Among the many investigations on the thyroid gland, few have dealt directly with the relation of the thyroid secretion to the rate of cell division. These few experiments, with a single exception, have been carried out on cultures of protozoa and since they have been subject to certain difficulties of control, due to the material used, the results have given rise to a rather great divergence of opinion. Nowikoff, Shumway, and others have found that the addition of total thyroid to Paramecium cultures accelerated the rate of cell division and more recently Cori has reported that pure thyroxin exerts the same effect, although the acceleration is less marked. Woodruff and Swingle and Torrey, Riddle and Brodie, on the other hand, have found that the addition of thyroxin depresses the division rate of Paramecia and Torrey reports that thyroxin has the same effect on the eggs of Echinometra and Phallusia. In the present work, of which this paper is a preliminary report, the writer has undertaken to follow critically the effect of thyroxin solutions on the cleavage rate of Arbacia eggs. Previous work has shown that the developing eggs of Arbacia punctulata, properly handled, are very satisfactory for quantitative work. In each experiment, eggs from a single mature female were fertilized with sperm from a single mature male and then placed in experimental and control dishes kept side by side in a large vessel of water, so that errors due to temperature differences were eliminated. Samples of eggs removed at intervals of 1 or 2 minutes from experimental and control dishes were transferred to watch glasses containing formalin which immediately arrested development. Counts of 200 to 300 eggs were made from each sample taken.