g::2:3:3:,_,_::_::_. WON ? u’. l E DEGRAD .3 i. ‘0". {YER} .0.‘ THESIS an: my}: ~_ M it}: i (rim 5:. '5': ‘2??? ,, x . . 1.. O l 3 c ‘ t .I‘c.‘( . 54-“ :51. L I B R xi R ‘ .___—- ...J ABSTRACT SOME NEW ASPECTS OF THYROXINE DEGRADATION AS OBSERVED FROM STUDIES USING BOBWHITE QUAIL by Kevin M. Etta In the unending search for more reliable indices of thyroid state and function, physiologists have employed many kinds of drugs. Perhaps the most commonly used are the anions like thiocyanate and perchlorate which have been known to block iodide trapping and goitrogens like tapazole and the thiouracils which block hormone synthesis. This study examined the’more controversial subject of the possible extrathyroidal effects of thiocyanate and tap- azol injection on the degradation of 131 I—L—thyroxine in adult male bobwhite quail. Blood samples were taken at regular intervals after drug and labelled thyroxine treat— ment. The longest period of continuing sampling was 45 hours. Some previous studies may have reported as long or even longer periods of sampling. However, none of the earlier studies known to this writer have ever reported a break in the degradation curve. The break in the con- trol curves was exploited to yield a method for taking account of and mathematically correcting for extrathy- roidal recycling of iodine. This is the first time that this correction has been made without the use of anions to block iodide trapping or goitrogens to block hormone synthesis. Kevin M. Etta Thiocyanate, by blocking recycling of metabolized iodine through the thyroid, offered a second method for estimating the best approximation to-date of fractional degradation per hour of L—thyroxine. These two methods served as checks, one for the other. Both thiocyanate and tapazole drastically and significantly reduced thy— roidal retention of iodide. Thiocyanate depressed muscle retention to the same extent as it increased the retention of radioiodide by the gastrointestinal tract. Tapazole significantly increased liver retention as well as total body retention of radioiodine. Tapazole seemed to have the same effects thyroidally and extrathyroidally as have been reported for other goitrogens by many workers. More specifically, tapazole seemed to cause an impairment at some 131I-L-thyroxine.‘ This inter- point in the metabolism of ference showed up as a greatly increased level of blood radioactivity, a reduced excretion of radioactive material via the gastrointestinal tract and a significantly in— creased retention by the liver of the bobwhite quail. Livers have been reported to be a major site of thyroid hormone deiodination. This would place the liver as a primary site of action of the interference of tapazole in peripheral degradation. The observations concerning the extrathyroidal ef- fects of tapazole and other goitrogens would put to seri- ous question those methods for estimation of thyroxine Kevin M. Etta secretion rate which have used goitrogens either in the goiter-prevention technique or in the substitution methods where goitrogens served to give a steeper thyroidal out- put slope. SOME NEW ASPECTS OF THYROXINE DEGRADATION AS OBSERVED FROM STUDIES USING BOBWHITE QUAIL By Kevin M.‘Etta A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1968 /‘ WWI/WV ACKNOWLEDGEMENTS The author wishes to express his deep gratitude and appreciation to Professor E. P. Reineke for his patient guidance in planning and carrying out this study. His wise counsel, foresight and sympathetic understanding have been a great spur in the completion of the author's program at Michigan State University. The writer feels particularly indebted to Professor R. K. Ringer for the supply of experimental birds, the constant personal encouragement which was invaluable and the actual planning and conducting of all the experiments. Special thanks are due to Mrs. Judianne Anderson, _Miss Martha Dewees and Mrs. Sandra Pangborn for their technical help in processing blood samples and in many other ways. Thanks are also due to Mr. Fritz Lorscheider and Dr. w. E. Cooper for statistical suggestions. A sincere appreciation is due to the Agency for In— ternational Development which provided the financial sup- port that made this whole program possible. Professor D. w. Crabb, Director of the Anthropology Program at Princeton University, and Professor J. C. Elliott, Briggs College, Michigan State University have been of immense financial and moral help Just when both were most needed. To them, too, the author is very grateful. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS. LIST OF TABLES LIST OF FIGURES Chapter I. LITERATURE REVIEW. Thyroid Hormone Synthesis Effects of Drugs on Hormone Synthesis and Metabolism . Extrathyroidal Effects of Goitrogens. Extrathyroidal Effects of Goitrogens versus Their Use in Methods for the Estimation of Thyroxine Secretion Rates Effects of Tapazole versus Those of Other Goitrogens, e. g. The Thiouracils . Biological Half— Life (tl/2). II. MATERIALS AND METHODS Chemicals and Drugs Experimental Animals Experiment I Experiment II Tissue Retentions Computations III. RESULTS Experiment I Experiment II Retention by Tissue Systems and Whole Body. . . TDS and Blood Level of Radioiodine IV. DISCUSSION Effects of Thiocyanate Treatment Effects of Tapazole on .Thyroxine Metabolism iii Page ii vi 12 13 16 18 18 19 19 23 2H 25 3o 30 33 42 43 A3 51 Chapter Page V. SUMMARY AND CONCLUSION . . . . . . . 56 BIBLIOGRAPHY . . . . . . . . . . . . . 58 iv Table AA. AB. LIST OF TABLES Effect of thiocyanate on T1/2, TDS and tissue retention in bobwhite quail; Experiment I . . . . . . . . Statistical constants for Tu degradation curves; Experiment II . . . . Slopes, Tl/2, degration rates and thyroxine distribution space; Experiment II Tissue retentions expressed as percent injected dose. . . Tissue retentions as percent of total or extrathyroidal radioiodine Page 31 35 37 39 A0 Figure LIST OF FIGURES Diagram of in-dwelling silastic cannula. Representative 131I-T degradation curves for control and thiocyanate-treated bobwhite quail in Experiment I . Representative 131I-T degradation curves for control, correc ed control, thiocyanate- and tapazole-treated bobwhite quail . . . . . . Effect of thiocyanate and tapazole on percent of total body radioiodine located in the thyroid and total of extrathyhr roidal radioiodine located in the liver, kidney, muscles and gastrointestinal tract . . . . . . . . vi Page 21 32 36 Al CHAPTER I LITERATURE REVIEW Thyroid Hormone Synthesis A tremendous amount of work has been done in thyroid physiology. Starting from Just before the turn of the century when hardly anything was definitely known about the physiology of the thyroid gland and its products, thy- roid physiology has come a long way to this date when an enormous number of thyroidal functions have been more or less accurately mapped out. Avian thyroid glands are located ventrolaterally to the trachea Just outside the thoracic cage. As in all vertebrate animals, the thyroids in birds are specialized for the uptake and retention of iodide. Twenty to forty percent of an inJected tracer dose of 131I is taken up by the thyroids. The bulk of the remainder is excreted in the urine such that 90 percent of the inJected amount is accounted for by the combined thyroid uptake and uri- nary excretion in normal birds. Only small amounts of iodide appear in the gastric Juice and saliva. Normal rats have been reported to be in iodide balance when 131 they excreted 70 percent of their daily I dose in their urine (Jones and Van Middlesworth, 1960). For convenience of discussion, Richards and Ingbar (1959) have divided thyroid hormone synthesis into three maJor steps: 1. Thyroid gland concentration of inorganic iodide from extracellular fluid. 2. Oxidation of the iodide, by a peroxidase, pre— sumably to elemental iodine within the thyroid. Iodine then iodinates tyrosyl residues to form first mono-iodotyrosine (MIT) and then di— iodotyrosine (DIT). 3. Coupling of the iodotyrosines to form iodothy— ronines like triiodothyronine (T3) and tetra- iodothyronine (T4). The probable sequence of reaction is summarized in Chart I. The MIT, DIT, T3 and TA all occur within the thyroid gland combined in a storage colloid glycoprotein named thy- roglobulin. A protease cleaves the thyroglobulin to re~ lease T3 and TA into the bloodstream while MIT and DIT are degraded by a scavenger deiodinase enzyme to release iodide within the thyroids. This iodide is again avail- able for use by the thyroid glands via intrathyroidal recycling of iodine. The circulating hormone is deio— dinated by a deiodinase and conJugated at peripheral tissue centers most important of which is the liver (Flock and Bollman, 1959). The resulting metabolites are se- creted from the liver through the bile. Most of the io— dide is excreted in the urine, only a small amount ap- pearing in the feces in the normal animal. Some of the iodide released by deiodination is reabsorbed by peripheral Thyroid—) Thyroid Hormone Synthesis I" '— Peroxidase \ | r W 7 2 12 + Tyrosine > H0©CHz-éH-COOH T i up I NH2 Monoiodotyroslne (MIT) l N MIT + Iodine .1, 7‘ HO-{Q-CHz-g-I-COOH I T0 9" Dl-iodotyrosine (DIT) I l N MIT + D | T 9 HO-(E—O—{Q-CH2 iii-000:4 TrlLlodothyronlne (T3) fl I my, DIT + DIT 7‘ HO-P-O CHz- H-COOH I Tetra-lodothyronine (Ts) FIN *The drug depresses the process indicated. Chart | tissues and some is recycled back to the thyroid for fur- ther trapping and reuse--extrathyroida1 recycling. Effects of Drugs on Hormone Synthesis andMetabolism Thiogyanate: (Drug Effects on Synthesis Numerous drugs have been reported to modify or com- pletely block some or most of the steps in thyroid hormone synthesis. Several such drugs have been employed as an aid in the study of thyroid physiology. One such group of anions has been known for a long time to affect the trap- ping or concentration of iodide by the thyroid gland. Perchlorate, thiocyanate and nitrates have been shown to block the ability of the thyroid to trap iodide to varying degrees. Barker (1936) first reported the occurrence of goiter in patients receiving thiocyanate therapy for hy- pertension and the correction of such goiter by the admin- istration of desiccated thyroid. Jones and Van Middles- worth (1960) have shown perchlorate to inhibit the uptake of iodide by the thyroid without influencing thyroxine deiodination in rats. This was suggested by the obser- vation that both control and potassium perchlorate-fed rats were in iodine balance when they excreted 70 per cent 1311 dose in urine. Franklin, Chaikoff of their daily and Lerner (194A) demonstrated that thiocyanate, but not thiouracil, interferred with thyroidal accumulation of iodine. Two years later, Vanderlaan and Bissel (19A6a) demonstrated even more conclusively that thiocyanate blocked thyroidal uptake of iodine. vanderlaan and Vanderlaan (1947) also demonstrated that iodide accumulated during treatment of rats with thiouracil could be rapidly and quantitatively flushed out from the thyroid by the adminis- tration of thiocyanate. Wyngaarden, §£_al, (1952) demon- strated that perchlorate was ten times and nitrates one- thirtieth as effective as thiocyanate in flushing out con- centrated iodide from propylthiouracil-treated thyroid glands. In an attempt to localize the effect of anions like thiocyanate, Vanderlaan and Greer (1950) suggested that the histologic effects of perchlorate would favor either the hypothesis of a thyrotropin regulation of the iodide trapping mechanism or a competition between block- ing agents and the iodides for the ability to get trapped. The concept of competition has since been supported by experiments. The greater the iodide supply to rats, for instance, the less effective would the blocking agents be in their interference with the trapping mechanism. Vander- 1aan and Caplan (1954) have supported the concept that io— dide concentrating capacity was a more sensitive indicator of thyrotropin activity than thyroid weight in goiter studies. All the reports are thus unanimous on the concept that anions like perchlorate, thiocyanate and nitrates can block the iodide trapping mechanism to varying degrees. It is established that thiocyanate and similar types of anions have little effect on the organification of iodine. Richards and Ingbar (1959) have reported that increasingly higher doses of such anions resulted in no appreciable al— terations in the relative proportions of the individual io— dinated amino acids. At extreme reductions of both total and organic uptake of iodine, however, a small decrease in the ratio of the di-iodotyrosine and mono—iodotyrosine oc- curred. PThis decrease indicated an impaired synthesis of thyroid hormone. Extreme doses of anions such as would give these extreme reductions of both total and organic uptake are presumably rarely used in studying normal thy- roid physiology. It is quite possible that such impaired hormone synthesis may be secondary to the primary effect of the anions in blocking iodide trapping. This is spec— ulative, however. It is also speculation whether or not perchlorate and thiocyanate have extrathyroidal effects. Extrathyroidal Effects of Perchlorate and Thiocyanate ' Jones and Van Middlesworth (1960) reported that both control and perchlorate-fed rats were in iodine bal- 131I ance when they excreted 70 percent of their daily dose in urine. If the route and quantity of excreted io- dine was any index to the state of peripheral metabolism of iodinated thyronines, the report would tend to suggest that perchlorate had no effect on peripheral metabolism, specifically, the deiodination of thyroid hormones. Raben (1949) and Greer, g£_§l, (1966) have shown anions like thiocyanate and perchlorate to depress organic binding by the thyroid. Yamada (1967) surmised that such anions prob- ably altered the peripheral metabolism of thyroid hormone by releasing some of it from its binding plasma proteins (Robbins and Rall, 1957), (Ingbar and Freinkel, 1960). In accordance with this hypothesis Yamada reported markedly decreased protein-bound iodine values (p < 0.001) in rats as a result of perchlorate and thiocyanate treatment. He also observed an increase in urinary iodine as well as an increased uptake of iodine by skeletal muscles. It is interesting to note that Yamada did not observe any dif— ference in the muscle retention of iodine between control and perchlorate—treated rats until at least a ten-fold dilution of theplasma from the muscle samples was at- tained. The reduced PBI could be due to an increased rate of excretion along with a block in recycling. It is more difficult to consider muscle retention to be consequen— tially altered when the samples needed a ten-fold dilution to show a difference. The odds are, therefore, still strongly in favor of the concept that thiocyanate and perch— lorate do not have extrathyroidal effects. Goitrogens: Effects of Thiouracils and Methimazole on Hormone Synthesis Thiouracil and Methimazole have been reported to block hormone synthesis at one or other of three possible stages: 1. The oxidation of iodide to iodine. 2. The iodination of mono-iodotyrosine to di-iodo- tyrosine. 3. The coupling of tyrosines to form thyronines. Vanderlaan and Vanderlaan (1947) reported that thiour— acil prevented the oxidation of iodide to iodine. This step is presumably catalyzed by a peroxidase enzyme within the gland. Slingerland, g£_§1, (1959) reported that propyl- thiouracil treatment increased the mono-iodotyrosine to di— iodotyrosine as well as the tri—iodothyronine to tetra—iodo- thyronine ratios. The same study also showed that the con— version of mono—iodotyrosine to di—iodotyrosine was more sen— sitive to prOpylthiouracil treatment than the iodination of tyrosine to mono-iodotyrosine. These results suggested that propylthiouracil probably blocked thyroid hormone syn- thesis at the MIT to DIT or the T3 and T4 steps. Richards and Ingbar (1959) reported that propylthiouracil affected not only the initial oxidation of iodine and the resulting mono—iodination of tyrosine but also the di—iodination of tyrosyl residues as well as their coupling to form the hormonally active iodothyronines. Whatever the precise stage at which the blocking effect of goitrogens is exerted, there can be no doubt as to the fact that goitrogens do in fact block thyroid hormone synthesis. Effect of Goitrogens on Thyroidal Output of Iodine Albert and Tenney (1951) reported that thiouracil accelerated "thyroidal secretion" of iodine six-fold in rats. Flamboe and Reineke (1959) reported a more rapid 1311 output rate in goats treated with thiouracil compared to untreated goats. Tanabe, g£_al, (1965) reported that thiouracil, propylthiouracil and methimazole all signifi- 1311 from the thyroid cantly increased the release rate of gland. Grosvener (1963) demonstrated that propylthiouracil rapidly increased the thyroidal release of iodine. Goi— trogens, therefore, have marked effects on the rate at which the thyroid gland releases iodine in addition to their effects on thyroid hormone synthesis. Such effects are apparently shared by all goitrogens to differing de- grees. Thiouracil is apparently the most potent in ac- celerating iodine output followed by propylthiouracil and then methimazole (Tanabe, g£_al,, 1965). Regarding the interference of goitrogens with thyroid hormone synthesis by blocking recycling of iodine, methimazole is said to be the most effective (Grosvener, 1963; Premachandra, et_al,, 1958; Pipes, §£_al,, 1963). Methimazole has also been employed to obtain a steeper output slope in the substitu- tion method of estimating thyroxine secretion rates by Brooks, 3133;. (1962) and Romack, 2311. (1964). But do tapazole and other goitrogens have any extrathyroidal effects? lO Extrathyroidal Effects of Goitrogens Goitrogen Depression of Metabolic Rate Barker, §t_al, (1949); Andik, gt_al, (1949); Barrett and Gassner (1951) and Stasilli, g£_§1, (1960) have all reported that the thiouracils inhibited the rise in oxygen consumption or metabolic rate that would normally follow the administration of exogenous thyroxine. There is some more or less direct relationship between the metabolic effectiveness of the thyroid hormone and its catabolism in the tissues via the dehalogenating pathways. Variations in the rates of peripheral deiodination of the hormone and its metabolic effectiveness may, therefore, be different indices of the disturbance of metabolic parameters (Escobar and Escobar, 1961). Goitrogen Effects on PBI, Routes of Iodine Excretion and Tissue Retention of Iodides Several other observations have led to the sugges— tion that goitrogens may affect the peripheral metabolism of thyroid hormones. Jagiello and McKenzie (1960) re- ported that rats maintained on 2 ug of thyroxine per day and treated with propylthiouracil produced normal concen- trations of thyroid—stimulating hormone but showed protein— bound iodine values that were twice those of normal rats. This suggested that propylthiouracil might have extra- thyroidal effects. Stasilli (1960) reported that thiour— acil probably inhibited peripheral utilization and modified 11 gastrointestinal excretion of iodinated thyronines and io- dides. What doubts that could be entertained about Sta— silli's findings were completely dispelled by Escobar and Escobar (1961) who reported the following: 1. The over-all peripheral deiodination of L-thy- roxine sharply and rapidly decreased following the administration of thiouracil, methylthiour- acil or propylthiouracil. This decrease was shown to be the first effect of the thiouracils clearly detectable, and the delay in the in- crease of fecal excretion was not due only to the time necessary for the formation of feces. It took four days for a sharp decrease in uri- nary iodide and an increase in biliary radio- activity to become apparent. Whereas some de— crease in urinary excretion was observed 5 hours after the onset of PTU administration, a corre- sponding increase in the intestinal and fecal radioactivity was not observeduntil 20 hours after the onset of PTU administration. The decreased deiodination could not be due to a decreased availability of thyroid hormone to per- ipheral tissues, for even where there was an in- creased fecal and a decreased urinary excretion of iodide, the concentration of iodide in the tissues of such rats was not significantly lower in thiouracil-treated rats relative to normal rats. Besides, a decrease in urinary iodide was 12 clearly demonstrable long before biliary secre- tion and/or intestinal plus fecal contents of iodinated compounds were altered. In the light of such eloquent evidences of the extra- thyroidal effects of goitrogens, one is a little surprised to find the rather profuse use of goitrogens in the estima- tion of thyroxine secretion rates. Extrathyroidal Effects of Goitrogens VerEus Their Use in Methods for the Estimation of Thyroxine Secretion Rates Van Middlesworth, gt a1, (1959) reported that propyl- thiouracil-fed rats developed goiter in the presence of normal concentrations of plasma PBI. This finding seemed to bear out the earlier contention of Goldberg, 23.31, (1957) that thyroid—pituitary relationships were not fully explained by a simple feedback model. These findings could mean that a goiter can develop despite low plasma levels of thyroid-stimulating hormone, although this is very unlikely. Alternatively, it might mean that super- normal levels of thyroxine would be required to reduce thyroid weight from a goitrous to a normal state, which is a more acceptable possibility. If this were so, then all the goiter prevention methods that have been used by Tanabe, 33 a1. (1961); Mellen (1961) and Turner, g£_a1. (1959) were in error. In fact, Reineke and Singh (1955) reported that l3 estimates of thyroid secretion rate in rats given thiouracil were about 10 percent higher than those in rats not receiv- ing the drug. It would then appear that goitrogens could not be expected to give accurate estimates of thyroxine se- cretion rates if such goitrogens did, in fact, have extra— thyroidal effects. But, do all goitrogens share the same extrathyroidal effects as have been reported for the thiour- acils? More specifically, does tapazole have the extra- thyroidal effects that have been reported for the thioura- cils? Effects of Tapazole Versus Those of Other Goitrogens, e.g,, The Thiouracils Hershman and Van Middlesworth (1962) reported among several other findings that the effectiveness of inhibit- ing deiodination of thyroxine did not correlate with the antithyroid activities of goitrogens. Further, tapazole was reported to be ineffective in inhibiting deiodination. The two findings were also reported by Tanabe, §t_§£3 (1965). Tanabe and his group further found tapazole to have the same blocking effect on iodide recycling, thy— roidal output of iodide and the same thyroxine secretion rate in cockerels receiving tapazole compared to that for the birds receiving other goitrogens. And yet, tapazole was still interpreted as being different in its lack of inhibition of deiodination compared to other goitrogens. Ingbar and Freinkel (1955), in presenting the thyroxine turnover method for the estimation of thyroxine secretion 14 rate, reported that the amount of thyroxine degraded by the body per day was equal to the amount of thyroxine synthesized by the thyroid for the same period. Empha— sizing much the same concept, Flamboe and Reineke (1959) and Turner, et a1, (1959) reported that since thyroidal iodine release was a measure of rate rather than quantity, neither this parameter nor thyroid uptake of iodine could be considered reliable indices of thyroidal functioning as would be reflected by thyroxine secretion rate. Pre- machandra, §t_a1, (1958) had observed only a slight rela- tionship between estimated thyroxine secretion rates and thyroidal iodide release rate in fowls. In a survey of thyroid physiology, Turner, gt El: (1959), therefore, concluded that the rate of thyroidal release of iodine would only provide an accurate biological half-life of thy- roidal iodine if recycling were blocked. "Only the esti— mation of thyroxine secretion rate would provide quanti- tative data on thyroid gland function," since the thy— roidal uptake or release of iodine were useful only in qualitative studies of thyroid function. If thyroxine degradation accurately reflected thyroxine synthesis (Ingbar and Freinkel, 1955), and if synthesis matched thy- roxine secretion into the blood since the colloid pools of the hormone remained constant, then the only other reliable index for thyroid function is thyroxine degrada- tion. In the light of this, it would be extremely hard 15 to see how tapazole would have similar effects with the thiouracils with regard to thyroxine secretion rate with- out having analogous effects with regard to peripheral degradation or deiodination. Tapazole may, in fact, have similar effects to the other goitrogens and the difference could be in the extent of their effect or the exact point in the degradation process of such effects. Tanabe, g£_a1. reported the following values of thyroxine secretion rates obtained by their modification of the radioiodine technique and by the goiter prevention assay technique: W Goiter Radioiodine Prevention Goitrogen Dose Assay Assay Thiouracil 0.1% of diet 1.47 1.69 Tapazole 0.1% of diet 1.42 1.71 Tapazole 0.05% of diet 1.52 1.36 Tapazole 0.025% of diet 1.40 1.63 The values of thyroxine secretion rate are given in ug of L-T4 per 100 gm body weight per day. First, it is disput— able that TSR can be accurately estimated by either of these methods. Secondly, Tanabe and his group have them- selves pointed out a difference in potency of these goi- trogens regarding iodine release from the thyroid. Could such a difference in potency not explain the emergence, or lack of emergence of significant differences, in both the thyroxine secretion rates and effects on deiodination fol- lowing the treatment of the cockerels with goitrogens? 16 Yamada (1967) has, for instance, reported that the admin- istration of 1 or 3 ug T4 daily for 3 weeks to tapa- zole-treated rats resulted in a progressive increase in PBI with increasing doses of thyroxine. The point is that there is an increase in the protein—bound iodine level which most probably reflects some extrathyroidal effect. Absolute proof of the precise extrathyroidal effects of tapazole may yet come, but evidence certainly makes it extremely hard, if not impossible, to accept the conten- tion that tapazole has no extrathyroidal effects. Even if we were to doubt the possible extrathyroidal effects of tapazole, we cannot but accept the impressive volume of evidence of the extrathyroidal effects of other goi- trogens. Nor can we doubt the correspondence of the thy- roidal effects of tapazole and those of other goitrogens. Biological Half—Life (t1/2) Biological half-lives in different birds do not ap— pear to be widely different. Different groups of workers have, however, reported widely varying values, sometimes for the same species of birds. Biological half-lives are, of course, much shorter in birds than in.mammals. Heninger and Newcomer (1964) reported mean half-lives of 4.9 hours in the cardiac tissue of chickens. This value is close to the tl/2 of T4 in the chicken plasma observed in the study by Singh, Reineke and Ringer (1968). McFarland, Yousaf and Wilson (1964) reported much longer fractional turnover 17 rates of T4 in Japanese quail which when expressed as t1/2 ranged approximately from 17 to 27 hours at 70-90°F. Hendrich and Turner (1967), sampling at 8-hour intervals, reported t1/2 values of 11.4 hours at normal environmental temperatures for fowl. The degradation curves reported in the literature (where such are shown or discussed) usu- ally indicated that these were obtained without correcting for recycling in non-goitrogen-treated control birds. If the animals were treated with goitrogens, then the observa- tions that such drugs had extrathyroidal, as well as thy- roidal, effects were not considered. Some studies did not mention the methods of estimation of biological half-lives. It seems that recycling and the effects of goitrogens would have to be accounted for before any reasonably accu- rate t1/2 values can be obtained. CHAPTER II MATERIALS AND METHODS Chemicals and Drugs 131I-carrier-free L—thyroxine was obtained from Abbott Laboratories as 50 percent propylene glycol so- lutions. The specific activities of the consignments used in the first and second experiments were 23.5 and 41.4 mc per milligram, respectively. In the first ex- periment, the labelled thyroxine was diluted with 0.5 ml quail plasma, 4.1 m1 of 0.9 percent normal physio- 131 logical saline solution and 2.9 m1 of I—L-thy- roxine solution. In the second experiment, 0.2 m1 of 131I-L-thyroxine solution were quail plasma and 2.4 m1 of diluted with 1.4 ml of 0.9 percent NaCl solution. The quail plasma used in both experrments was to reduce ad- sorption of the radioactive material to glassware. La— belled thyroxine solutions were made up to 66.7 uc/ml and 117 uc/ml, respectively. The standards for the first ex- periment had dilution factors of 200 and 160; for the second experiment, the standard had a dilution factor of 50. A freshly made up 40 mg/ml solution of sodium thio- cyanate was used in both experiments. Only 0.5 m1 of this solution was used in each subcutaneous injection per bird. This way, 20 mg of sodium thiocyanate was delivered to the 18 l9 bird at each inJection. A freshly made up 40 mg/ml solu- tion of tapazole (l—methy1-2-mercaptoimidazole) or methi- mazole was used in the second experiment. By using 0.5 ml of this solution per inJection 20 mg of tapazole was de— livered to each bird per inJection. Experimental Animals A total of 36 adult male bobwhite quail (Colinus virginianus) were used in this study. The 21 birds used in the first experiment had weights ranging from 140-170 grams. The weights of the 15 birds used in the second ex- periment ranged from 175 to 209 grams. The quail were all obtained from the poultry pens of the Michigan State Uni- versity Poultry Science Department. Experiment I Fifteen of the 21 bobwhite quail used in this experi- ment were cannulated by their left external Jugular veins according to the Weeks chronic infusion technique. A piece of silastic rubber tubing* 1.70 cm long and internal diameter 0.012 cm was Just sufficient to run from the mid- dle of the extended adult male bobwhite quail neck to the interior of the left auricle. Provided the tubing was kept flushed with at least a 0.9 percent NaCl solution, blood clotting within the tubing did not occur. The outer end of the tubing was then heat-sealed. Figure I shows a *Dow Corning, Midland, Michigan. 2O diagrammatic representation of the silastic tubing and its connections. The part of the tubing outside the vein was folded back behind the dorsal part of the neck and firmly sutured between the two raised collars (Fig. 1) down over the back of the quail. This technique allowed the bird to feed without hindrance. At least three days after cannulation, five of the quail were subcutaneously inJected with 20 mg sodium thio- cyanate solution, all of the cannulated birds received a 10 no dose of carrier-free 131I-L—thyroxine through the cannulae. A three-way valve with two attached syringes was used for inJecting the labelled hormone. One syringe 131I-L-T4. The other sy- contained the precise dose of ringe contained 0.9 percent NaCl solution. First, some of the saline was flushed through the cannula to make sure that it was open and that fluid could be easily flushed through it. The labelled hormone was then in- Jected slowly and carefully. Blood was drawn back, Just up to the tip of the 27-gauge needle and flushed back in. This drawing back of blood and flushing in procedure was repeated three times to ensure that the blood would pick up as much of the labelled hormone clinging to the walls of the cannula as possible. Just to make sure that all of the labelled material was flushed into the heart, the 0.9 percent NaCl solution was again flushed through the 21 279uooe-nypoderrnlc needle Seed bumps I 1 (iii Theei- crushed silicone glue ~ poiye’lhylene flblTIg v . sealed end ' silastic tubing sutured down at polyethylene co ior Iefl external Jugular vein Fig. i. Diagram or in-dweiiinq silastic cannula. 22 cannula. It was believed that this technique would ensure almost a 100 percent inJection of the radioactive materi- al. The inJected material would go directly into active circulation from the heart and in drawing blood samples one would be spared the tortuous problem of hitting the tiny bobwhite veins. In order to check what effect, if any, the Weeks chronic infusion technique (1964) had on our l311-L-tny- results, six other birds were given 10 ac of roxine by their right brachial (wing) veins. Blood sam- pling was from the wing vein opposite to that into which the labelled hormone was inJected for the uncannulated birds. In the cannulated birds, sampling was from the can- nula. The cannulae would then be flushed with more saline solution and heat-sealed after each sampling. Sampling commenced three hours after labelled hormone inJection. Subsequently, each bird was sampled at three—hour intervals for nine more hours. This gave four blood samples per bird. Only 0.3 ml of blood was drawn per sample. This meant a total of 1.2 m1 of blood was taken from each bird in 12 hours. Since bobwhite quail probably have a blood volume of 8—10 ml per 100 gm body Weight, the sam- pled volume would not produce an unphysiological condition in the bird by dangerously depleting its blood volume. Each blood sample was spun down by centrifugation at 1000 rpm for two minutes. From each spun sample, 0.1 m1 23 plasma was carefully pipetted using 100 microliter (ul) micropipettes. Background counts were taken on a scintil- lation counter (Nuclear Measurements Corporation) Just prior to plasma counts. Plasma counts were then taken for at least a minute per sample on the same scintillation counter. If the plasma counts were less than ten times the background counts, then counts were taken for at least two minutes. The counts per minute, per 0.1 m1 of plasma, were then multiplied by ten to express them in terms of counts per minute, per ml of plasma. Experiment II Fifteen bobwhite quail, split into three groups of five each, were used. Each of the fifteen birds was intra- venously inJected with 29.25 uc of 131I-carrier-free L—thy— roxine by the left brachial (wing) vein some nine hours before the first blood sampling. Thirty minutes after in— Jecting labelled hormone, five of the birds were given 20 mg of sodium thiocyanate solution subcutaneously. Five more quail got 20 mg of tapazole solution subcutaneously; the remaining five were used as controls and were untreated with drugs. The birds were first sampled nine hours after the inJection of labelled hormone. Sampling continued every three hours for about 15 more hours from the first sampling. After a nine-hour break, sampling was resumed at four-hour intervals for twelve more hours. Hendrich and Turner SE! IEI ‘11.! (wind... NV “NH mile-ll. T .. 24 (1967) had taken eight—hour samples for 40 hours following 131I-L-T4 inJection into chickens. It was felt that such extended periodicity of sampling would gloss over any par— allel processes taking place along with the degradation of thyroxine. The three-or four-hour periodicity of sampling was thus preferred to much longer sampling intervals. This, it was hoped, would help document better than ever before the true shape of the degradation curves for labelled thyroxine. Only 0.1 m1 of blood was drawn for each of ten sam- ples taken over a period of 45 hours. By prior experi- mentation, it was estimated that 0.1 ml of blood, when spun down, yielded at least 20 ul plasma volume. The small volume of blood sampling spread over 45 hours would thus obviate the danger of depleting quail blood volume to an unphysiological state. At the same time, the high dose of radioactivity in the inJected labelled hormone would ensure high enough counts to be obtained even with such small plasma volumes as 20 pl. Such counts would then be computed per milliliter plasma. Tissue Retentions In experiment 1, counts of samples of liver, kidney and spleen as well as whole thyroids were taken. All tissue samples counted were weighed. In experiment II, whole thyroids, livers, kidneys, and washed gastrointes- tinal tracts were counted and weighed. Two chunks of 25 breast muscle, one from either side of the clavicles, were also counted and weighed. Counts for the entire body mus- cle weight were then computed from the counted pieces of muscle. In this experiment, absolute percentages of the in- Jected doses in the thyroids were expressed as percent of total body retention. The absolute percentages of the in- Jected dose in all other tissues were expressed as percent of total extrathyroidal retention. Total body retention was taken to be the summation of the retention by liver, kidney, intestine, muscle and thyroid glands. This presumed that the retention by the exo- and endo-skeleton of the birds would be negligible. In experiment II, TDS for the control birds was cal- culated from the first and uncorrected curve. TDS is de- fined as the space which would be occupied by the labelled hormone if the concentration of the hormone in the tissues were exactly the same as that in the plasma. In controls, this figure does not seem to be affected by the processes involved in degradation and recycling. The corrected curve would, therefore, not give a true reflection of distribu— tion space. TDS for the other groups of birds is calcu- lated from the first curve in tapazole-treated birds or from the only curve in thiocyanate-treated birds. Computations 1. Percent Injected Dose per Milliliter of Plasma The percentage of the inJected dose represented by 1 milliliter plasma count was computed by the following stand- ard procedure: 26 Plasma counts/min. - Background counts/min. x 100 Standard counts/min. - Background counts/min. Background counts/min.: 30 0.1 m1 plasma count per min.: 4881 0.1 m1 plasma count/min. - Background count/min.: 4851 Plasma count/min./mi1: 48510 Mean Standard Count/min.: 19182 Mean Standard Count/min. - Background count/min.:19152 Dilution factor: 200 No. of inJected counts in standard: 19152 x 200 = 3830400 Percentage inJected dose = 3&30130 x 100 = 1.2664 Since tests by thin layer chromatography (by F. L. Lorscheider) haVe shown that only 90% of the total inJected 131I-L-thyroxine, radioactive material was in the form of the calculated percent inJected dose in each milliliter of plasma sample was then raised by 1.1111 to correct for the 10% free radioiodine. 1.2664 x 1.1111 = 1.4071 Percent inJected dose retained in the tissues counted was similarly computed. II. Statistical Constants of Curves and TDS Computation In experiment I the mathematical constants describing the Tu degradation curve were determined by graphical analysis. Percent radioiodine dose per m1 of plasma was plotted on the log scale (y) of semi-log paper against 27 time plotted on the arithmetic scale (x), and a line was fitted by inspection. Data of this type fit the general equation, log y = a + bx ‘ (1) where, a = the log y; intercept at inJection time and b = the slope of the regression line. Equation (1) when transformed to natural logarithms becomes, y = e-Xt (2) where, y = % inJected dose/ml plasma at time t e = the base of natural logarithm and x = b x 2.302 where, 2.302 = the factor used to transform loglO to natural logarithms. The quantity e'x was obtained from a table of descending exponentials*. Fractional degradation/hr. = l - e"x (3) _ 0.301 t% - -E¥¥-— . (4) Then, _ 100 TDS, in m1 - anti-log a (5) TDS TDS, in ml/lOO gmflbywt.= gm body wt e 100 In experiment II, the line of best fit for each slope together with the other statistical constants were *Tables of the Exponential Functions ex, 1961, 4th ed., National Bureau of Standards Applied Mathematics Series 14. **the numerical, not the algebraic, value of b. 28 determined by the method of least squares (Li, 1964). Final mathematical treatments were then done as outlined in equations (1) t0 (5)- Significance of differences between the control and drug-treated groups was obtained by either the student t test (Li, 1964) or by the Mann-Whitney U-test (Siegel, 1956). The latter was used where there was no variance homogeneity. III. Correction for Recycling in Control Curves All of the control birds in experiment II showed a break in their T4 degradation curves somewhere between the 15th and 25th hour following the labelled hormone inJec- tion. As the first step in resolving the two slopes, separate regression equations were computed for the 5 plasma radioactivity values obtained during the first 20 hours (Slope I) and the 5 values obtained during the sub- sequent 25 hours (Slope II). The first and steeper part of the curves was consid- l3lI-L- ered to represent predominantly degradation of thyroxine. The second curve could only be due to recycl- ing of metabolized iodine back through the thyroid. Con- sequently, in order to obtain the true degradation rate, it was necessary to separate the exponential of Slope II from that of Slope I. To accomplish this the calculated line for Slope II was extrapolated back to zero time. The values for Slope II were subtracted from those of Slope I at several time intervals. 29 Then, - Log AO Log At ~ t (6) b: where, At = percent inJected dose at time t (hours) and A0 = percent inJected dose at time zero The constant b obtained in this way most nearly 131 approximates the true degradation rate of the I—T4 originally inJected. CHAPTER III RESULTS Descending semi-logarithmic curves of very comparable slopes were obtained by both the Weeks chronic infusion technique (1964) and the veni-puncture method of injections and sampling. Technique did not therefore affect the results. Experiment I The thiocyanate—injected quail showed a significantly shorter G>< 0.001) mean biological half-life than the con— trol birds (Table 1). This suggested that thiocyanate in- 131I-L-thyroxine (Fig.2). creased the rate of degradation of The concept of a heightened rate of degradation was also indicated by the much higher fractional degradation rates per hour shown by the thiocyanate-treated birds relative to the controls. The iodide resulting from the degraded hormone did not, however, appear to be accounted for by an increased retention of radioiodide by any particular organs or tissues. There was no significant increase or decrease in the radioiodide retention by any particular tissue system among those counted. Retention by the spleen was negligibly small with or without thiocyanate treatment- 30 31 TABLE 1.--Effect of thiocyanate on T1/2, TDS and tissue retention in bobwhite quail. Experiment I. A: T1/2 and TDS Probability No. of Group Mean of Signifi- T1/2 Birds and cance by or in Standard Mam-Whitney TDS Group Treatment Error U-Test T1/2 in hours) 16 Control 4.58:0.33 5 SON—injected 2.77:0.16 p < 0.001 Fractional Degradation 10 Control 0.15:0.01 < 0 001 Per Hour 5 SON-injected 0.22:0.001 p ° % Injected 'Dose/ml Plasma 10 Control 2.34:0.15 At Zero Time 5 SCN—inJected 5.70:0.97 p < 0.001 TDS in ml/ 100gm 16 Control , 31.36+2.73 Body Weight 5 SON-injected 114752.05 p < 0.001 B: Tissue Retention Tissues: % . Injected State of Dose Group Whole Thyroid 10 Control 0.36:0.03 5 SCN-injected 0.27:0.10 Liver 11 Control 0.38:0.01 Per gm of Tissue 5 SON-injected 0.34:0.07 Kidney 11 Control 0.56:0.01 Per gm of Tissue 5 SCN-injected 0.48:0.01 Spleen 11 Control 0.29:0.003 Per gm of Tissue 5 SON-injected 0.16:0.001 32 5 I 0 I Control 0 ' SCN- injected 96 injected dose lmi plasma LO .9 .8 .7 .6 .5 l4 .3 1 1 1 1 1 i 1 1 L 1 i 1 i 2 3 4 5 6 7 8 9 IO ii l2 l3 Time (hours) Fig. 2. Representative 13114.4 degradation curves for control and thiocyanate- treated bobwhite quail in experiment l. 33 Thyroxine distribution space was drastically reduced by thiocyanate injection (p < 0.001).* Mean TDS values in the thiocyanate-treated group were a little over one—third of mean control values (p < 0.001).* Such a drastic reduc- tion had to mean something. It could not, however, be explained on the basis of the retention data for this experiment (Tables 1 and 3). Experiment II A break in the control curves always occurred some- where between the l5th and the 25th hour (Fig. 3). The first and the second curves were significantly different from one another (p < 0.001).* The two curves in the tapazole group of birds were not significantly different. However, "eye- balling" the points clearly showed them to fall into two curves in three out of the five birds. Biological half- 1ives were calculated from the slopes of the first curve, since these were the shortest t1/2 values that could be obtained from this group of birds. Clearly, tl/2 values obtained either from the second curve or from the single curve fitted onto all ten points would be even longer than those obtained from the first curve. Mean tl/2 and frac- tional degradation values were obtained by calculation from corrected slopes of the control birds, slopes of the thio- cyanate—treated and slopes of the first curve of the *By-Mann—Whitney U-test. 34 tapazole—treated birds. Table 2 summarizes the statistical constants used to obtain the results shown here. The tl/2 values so obtained for the control birds were essentially the same as those for the thiocyanate-treated birds. Con- trol mean t1/2 was found to be 8.58 :_l.36 hours and mean tl/2 for thiocyanate-treated birds was found to be 7.50 i 0.76 hours. Mean fractional degradation rates per hour were found to be 8.46 and 8.90 percent for the control and thiocyanate-treated birds, respectively. This finding was rather startling because the data of the first experiment has revealed shorter t1/2 and increased degradation rates for the thiocyanate—injected birds relative to controls. But there, of course, extrathyroidal recycling of iodine had not been accounted for. Biological half-lives and frac- tional degradation rates in percentages per hour calculated from the first curve of the tapazole-treated birds turned out to be 31.74 i_6.69 hours and 2.1 percent per hour, respectively. Tapazole, thus, seems to significantly re- 131 duce the rate of degradation of I-L—thyroxine in bobwhite quail. Retention by Tissue Systems and Whole Body A massive 23.65 percent of the injected dose of radioiodide was retained by control thyroid glands 45 hours 131I-T4. This compares with 0.48 per- after injection of cent in the thiocyanate-treated and 0.15 percent in the tapazole—injected group of birds. These are all group mean 35 .coxm» mm; oaaemm oooan pram on» Hopmm ooao mopfin omm£B* m MOm.H mammm.O mmmOO.O HH ma m OOH.H H:OHO.O1 OaOOO.Ot HH me O ema.H Hamma.O1 OOHOO.O1 HH NH m mOO.H OaHmO.O1 HHOOO.O1 HH HO O HOO.H OOOOm.O ONHOO.O HH me O Hmm.m OOMOO.O1 OHHOO.O1 H as m OmO.H OOHOO.O1 :mHHO.O1 H me O mmm.m HHOmO.Ou HmOOO.O1 H NH m HmO.m OOmHO.O1 OHOHO.O1 H HO O HHH.m NHHOO.OI :HHO0.0- H oOOHm a ”oopoowcHl.de OH mHH.m OHmOO.O1 OOH:O.O1 mm *O Omw.m OOOOO.O1 HmmOO.Ou mm OH mam.m H:NOO.O1 ammm0.0t mm *O mmO.m HOOOO.OI HOHmO.Or Hm OH Oam.a HOOOO.O1 OHHOO.O1 m "OoaooncHuzom m mHO.O HHNNO.O- eooooaaoo m OO:.H OOOOO.O1 mOHOO.O1 HH m aHm.m :HmH0.0u mOMHO.Ou H OQOHm "m .HoapaoO m OOO.H mONmO.O1 eopooaaoo m MOO.H NOOOO.O- mHOOO.On HH m Hmm.m OmmO0.0t OHNNO.O1 H oaon ”a .Hoaaaoo m mOO.O O:OOO.OI ooaooaaoo m mOO.H OmomO.O1 ONOOO.O1 HH m Omm.m ONOH0.0- mOOHO.On H oOOHm "m .HoaocoO m mmO.O OHmmo.O1 Omaooaaoo m mHOO.O OOmHH.O1 mOOOO.O1 HH m OOH.H mHaHO.On mmOHO.Ot H oaOHm "m .HoaocoO m OO:.O HmmmO.Ot Ooaooaaoo m OHO.H maOO0.0- emOHO.O- HH m mom.m mmamm.Oi OOmmO.O1 H oQOHm "H .HoaaeoO mpsfiom mo .02 quohoch w HcoHOHmmmoo momoaw oaoam one QSOHU oHHm coapmaohaoo .HH uQmEHHmQNm .uo>Hzo cowpmommwoo :9 Hon mucmpmcoo HOOHHmemHmII.m mamH> *HO m v nae *Om OIA aaa HHHoaolm coco uazm m one aOHO Hmmo.H + mm.mH OHmm.o + om.m mm.o + om.m wozoo.o + mmo:o.ou oopomncH ImpwcmzooHQB OOOO.H H ma.O em.H H Om.m HmHHO.O H mmOmO.O1 Oooooaaoo H0.0 v nae u HH oaoam .m> H odoam OH.O H mO.mm MHHO0.0 H HHOOO.OI HH oaon OOOO.H H me.mm OO.H H mH.mH mHHOO.O H OOOHO.O- H oaon "Houucoo mHOHHm maso Ammv mHOHHm mopmpm macaw some 90 moHHm vamccmum A m camcsmpm new moaoam use mdsomw m Ham pom .Hano3 new Hzom Hog av moon Em OOH\HE CH mme HcooHom ca machnm COHHwomeoQ pudendum HOOOHHoaeH one m\HH .HH HamEHHomxm .oomam coauznfinpmfim mcfixoamne cam moumm cOHHmomHmoQ .m\HB .moaoam1|.m mqm¢e 38 values. Both thiocyanate and tapazole were, therefore, seen to drastically and significantly reduce thyroidal retention (p = 0.004 in each case relative to control). Thiocyanate depressed the retention of radioiodine in all major systems of the body except the gastrointestinal tract. Retention by the gastrointestinal tract is signifi- cantly increased (p < 0.01) while that by the quail muscle is significantly decreased (p < 0.02) following thiocyanate (treatment. Tapazole significantly increased liver reten- tion. The retention of most other tissues was also in- creased by tapazole administration. Since the first ex— periment had indicated that quail spleens retained a negligibly small amount of the radioiodine injected into the bird, spleen retentions were not taken into account in this experiment. Table 4A and Fig. 4 show body retentions expressed as percent injected dose and including thyroidal retentions. Comparisons were drawn, between the total body retentions (including thyroidal retention) of radioiodine in the con- trol, thiocyanate-treated and tapazole-treated groups of 1311 retention in the birds. Here it was found that the thiocyanate group was only 0.13 times that of the control value (p < 0.01). Tapazole treatment resulted in a radioiodine retention of 0.81 times the control value (p > 0.5). Comparison of body retentions of the groups, .onoaia ecoosam amae .maoppcoo on oommmfioo oosoHoMMHo no cosmOHMHcmHm mo mHHHHQMQOHma 39 . .1 *Om.O A Oaa mm m + new: mm.Om mm.Om mO.O OH.Hm NO.O OO.m OH.O Hmvasoao mm.w: NH.OH mm.m :N.Hm HO.H OH.O OH.O m OO.HH Ha.OH ma.m OH.OH Hm.O HO.H OO.O : HH.mm OO.mm mm.m mm.mm Om.H mm.m O0.0 m OH.mm om.mm Hm.m ma.mH Om.O HO.H ON.O m ON.mm :O.mm HO.O OO.mm :H.H om.m mm.O H copoowsH Imaoumdma mm.o+ *Ho 0 v axe coo: OO.O Oa.a mO.m mO.m mH.O om.O OO.O Hmvasoao Om.m mH.H mm.O O0.0 m0.0 O0.0 aH.H m ma.a. mH.O. 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