SOME ASPECTS OF IODINE METABOLISM I. THE EFFECT OF PHYSIOLOGICAL SALINE AMD DESOXYCORTICOSTERONE ACETATE ON THYROID 1-131 UPTAKE IN RATS II. THE METABOLISM OF 1-131-LABELED THYROXINE IN NORMAL AND THYROIDECTOMIZED DOGS By Ellen St. John Monkus AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 19514- Appro v e d ^ ____ .^» ProQuest Number: 10008389 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest, ProQuest 10008389 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 SOTvra ASPECTS OF IODINE METABOLISM I. THE EFFECT OF 'PHYSIOLOGICAL SALINE AND DESOXYCORTICOSTERONE ACETATE OH THYROID 1-131 UPTAKE IN RATS II. THE METABOLISM OF I-131-LABELED THYROXINE IN NORMAL AND THYROIDECTOMIZED DOGS By Ellen St. John Monkus Experiments were conducted to determine the acute effect of desoxycorticosterone acetate (DCA) and physiological saline (0.9# NaCl ) on the thyroid uptake of radioactive iodine. Con­ comitant plasma 1-131 levels were measured to determine whether there was a positive correlation between plasma 1-131 level and thyroid 1-131 uptake. The three hour thyroid uptake of 1-131 was increased in adrenalectomized rats after 1.25 mg* of DCA per 100 gm. body weight was given intraperitoneally immediately after intravenous injection of 20 uc. of 1-131 with 8.5 ugm. of carrier 1-127 as Nal. The plasma level and the urinary output of radioactivity were not changed by the treatment. The one and one-half hour and three hour uptake of 1-131 in adrenalectomized rats were increased after 10 cc. of 0.9^ NaCl per 100 gm. body weight was given under the same conditions. In this case the blood level of radioactivity was decreased and the urinary output of radioactivity was in­ creased. A study was made to measure the absolute thyroid circu­ lation of the rat. The arterio-venous 1-131 difference for the thyroid was found to be ten per cent of the arterial level under the conditions of the experiment. The estimated 2 absolute thyroid circulation of these rats was calculated to be 23 of blood per mg, of gland per minute. A comparison was made of the metabolism of 1-131 labeled L-thyroxine in intact and thyroidectomized dogs in an attempt to determine the mechanism of the tolerance to exogenous thyroactive materials seen in normal human beings and intact dogs as compared to the athyreotic. A comparison of the rate of fall of radioactivity in the blood and the rate of its appear­ ance in urine and feces after an intravenous injection of 60 ugm. of 1-131 labeled L-thyroxine was made in normal and thy­ roidectomized dogs, which had been pretreated for ten days with 6 ugm, of non-radioactive thyroxine per day. No differ­ ence was seen in total fecal output of radioactivity at seven days after injection of the labeled thyroxine. Combined total thyroidal-urinary 1-131 content of the intact dogs equaled urinary 1-131 output of the thyroidectomized dogs at seven days. The plasma levels of radioactivity of both groups were described by the sum of three semi-logarithmic regression lines having half-times of 0.9ip, 8.7 and 69 hours in intact dogs and 0.9l|-, 9•Ip and 63 hours in thyroidectomized dogs. The second half-time of 8.7 hours found in intact dogs was statistically significantly less than the 9*ip hours of the thyroidectomized dogs by a "t" test. No difference between the groups was seen in the final blood levels and in the final rate of disappearance of the radioactivity. SOME ASPECTS OF IODINE METABOLISM I. THE EFFECT OF PHYSIOLOGICAL SALINE AND DESOXYCORTICOSTERONE ACETATE ON THYROID 1-131 UPTAKE IN RATS II. THE METABOLISM OF 1-131-LABELED THYROXINE IN NORMAL AND THYROIDECTOMIZED DOGS By Ellen St. John Monkus A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Physiology 19511 DEDICATION Frank and John Monkus. ACKNOWLEDGEMENTS The author wishes to express her appreciation to Dr. E. Paul Reineke for his stimulating guidance and helpful criticism throughout the course of this investigation. The aid of Dr. William D. Baten in the statistical analysis of these data is gratefully acknowledged. The author wishes to express her gratitude to Mr. Jack Monroe and Mr. Howard Hardy for their help and patience and to Fouad Soliman, Wallace Friedberg, Cheng Chun Lee, and William Baker for their technical assistance. Thanks is due to Dr. B. V. Alfredson for the use of the facilities of the Department of Physiology and Pharmacology and to the Michigan Agricultural Experiment Station for support of the project under which this work was done. TABLE OF CONTENTS Page GENERAL INTRODUCTION 1 Metabolic Circuit of Iodine, 1 Iodide Metabolism: 1 Intrathyroidal Metabolism of Iodine: 2 Extrathyroidal Metabolism of OrganicIodine: 3 Control of Thyroid Secretion: I4. The Effect of Physiological Saline and Desoxycorti­ costerone Acetate on Thyroid 1-131 Uptake in Rats. 5 The Metabolism of Thyroxine in Normal and Thyroid­ ectomized Dogs. 7 1-131 COUNTING METHODS 9 Sample Preparation. 9 Plasmas, Whole Bloods, and Urines: 9 Thyroids: 10 Muscles: 10 Feces: 10 Counting Time. 10 Comparison with an Aliquot of Injected Dose, 11 Counting Correction Factors. 11 Losses of Counts in the Dead Time ofthe Counter: 12 Geometry: 12 Self-Absorption: 12 Physical Decay: 17 THE EFFECT OF PHYSIOLOGICAL SALINE AND DESOXYCORTI­ COSTERONE ACETATE ON THYROID 1-131 UPTAKE IN RATS 19 Introduction. 19 Physiological Saline: 19 Desoxycorticosterone Acetate: Materials and Methods. 20 22 Diet: 22 Body Weights: 23 Injection of 1-131: 2b Injection of 0.9$ NaCl and DCA: 25 Adrenalectomy: 25 Samples for Counting: 25 Special Conditions and Methods of Experiment 5: 2? Statistics: 28 Experimental and Results. 28 Experiments 1 and 2: 30 Experiment 3* 35 Experiment Ip: 38 Experiment S* 3& Discussion of Results. IpO THE METABOLISM OP THYROXINE IN NORMAL AND THYROID­ ECTOMIZED DOGS ip9 Introduction. 1^9 Materials and Methods. 51 Diet: 52 Thyroidectomy: 52 Pretreatment with Thyroxine: 52 Injection of Radioactive Thyroxine: 53 Blood Samples: 53 Urine and Feces Samples: 53 External Thyroid Counts: 5b Statistics: 55 Experimental and Results Experiment 1: Experiment 2: Experiment 3: Discussion of Results. CONCLUSIONS Appendix 1 GENERAL INTRODUCTION More is known about the thyroid than about any other endocrine gland. Since the early clinical discoveries, in­ vestigations of thyroid physiology have passed through the important milestones of the first use of replacement therapy by Murray (1891), the discovery of the high iodine content of the gland by Baumann (1 896), and the observation that thyroid feeding enhances metabolic rate by Magnus-Levy (1895)* Fol­ lowing these studies came the first observation that injected iodine accumulates in the thyroid gland (Marine, 1915) and the discovery (Kendall, 1915) and synthesis (Harington and Barger, 1927) of the thyroid hormone thyroxine. In the past two decades the newer methods of m o d e m re­ search have been focused on the thyroid gland. In this the­ sis two aspects of iodine metabolism have been studied using 1-131; therefore, a summary of the metabolism of iodine is desirable in order to understand how the problems investi­ gated fit into the total scheme of thyroid function* Metabolic Circuit of Iodine. A number of investigators, among them Brownell (1951 )> Albert (1952), Higgs (1952) and Gross and PItt-Rivers (1953) have reviewed the various facets of iodine metabolism and their Interaction with each other. Their conclusions can be outlined as follows: Iodide Metabolism: The thyroid gland is capable of ex­ tracting iodide from the blood and maintaining a gland to 2 blood ratio of approximately twenty-five (in normal rats, Vanderlaan and Vanderlaan, 1914-7)* make its hormone. It uses this iodide to If 1-131 is injected Intravenously, it is distributed into a volume which approximates the extracellu­ lar fluid space (Wallace and Brody, 1937)* Besides this, it is rapidly taken up by the thyroid, and it is also excreted in the urine. The mechanism by which the thyroid gland concentrates iodide is not known. It is believed that the iodide must be oxidized before it can be bound into organic form but free iodine has never been positively identified in the thyroid gland. Intrathyroidal Metabolism of Iodine: After the iodide has been concentrated in the gland In inorganic form, it is oxidized and combined Into a series of iodine containing amino acids. Two of these, dilodotyrosine and thyroxine, were identified chemically many years ago. Recently in­ vestigations using paper chromatography and radioauto­ graphy have resulted In the discovery and identification of two other amino acids, mono io do tyrosine (Fink and Fink, I9I4-8 ) and triiodothyronine (Gross, Leblond, Franklin and Quastel, 1950; Gross and Pitt-Rivers, 1952a). The iodine containing amino acids in the thyroid gland are chiefly found In a protein, thyroglobulin, but recently small amounts of them have also been located in free form (Gross, Leblond, Franklin, and Quastel, 1950). 3 It Is believed that the amino acid moiety of these com­ pounds is already in peptide linkage at the time of its iodinatlon. The iodination of tyrosine occurs quite readily; dilodotyrosine can be formed from tyrosine and iodine in vitro at approximately body pH, Then the formation of thyroxine Is believed to occur from two molecules of dliodo tyro sine * This is thought to be an oxidative process, but the enzyme systems involved have not been definitely determined. Extrathyroidal Metabolism of Organic Iodine; hormone is released directly into the blood stream. The thyroid In the adult animal the major effect of the hormone Is the mainte­ nance of the normal basal metabolic rate. It also has a role in growth and development. The major iodine containing amino acid found in the blood is thyroxine. Until recently it was believed to be the active form of the thyroid hormone. Small quantities of other io­ dine containing amino acids including triiodothyrone have been found in the blood by the use of paper chromatography and radioautography. Triiodothyronine has been found to be more potent than thyroxine by several assay techniques (Gross and Pitt-Rivers, 1952b; Gross, Pitt-Rivers and Trotter, 1952). The problem of what is the active form of the thyroid hormone is now being reconsidered, and a final decision In regard to it has not yet been made. The plasma organic iodine is not dialysable and is loosely bound to one of the plasma proteins. Investigation by the method of paper electrophoresis (Gordon, Gross, 0»Conner and h Pitt-Rivers, 1952) showed that organically bound 1-131 travels with one of the o(-globulins. The thyroid hormone is de-iodinated in the process of exerting its hormonal effect. The resulting iodide is either reused by the thyroid gland or excreted. Control of Thyroid Secretion: The levels of these dif­ ferent substances and the rates of their conversion from one to another are gauged to supply an adequate amount of hormone to the animal. The rate at which hormone is released from the thyroid is controlled by the anterior pituitary. The pituitary produces thyroid stimulating hormone (TSH) which controls both the rate of release and the rate of manufacture of thyroid hormone. A reciprocal relationship has been shown to exist between the thyroid and the pituitary. If the blood level of thyroid hormone falls, the pituitary is stimulated to produce more TSH which in turn stimulates the thyroid to produce more hormone. The mechanism by which the level of thyroid hormone in the blood controls the rate of TSH secretion is not understood nor has the precise timing involved been investigated. Thus it can be seen that the basic facts regarding what happens to iodine In the body have been established. major forms it takes are known. The Nevertheless, there are many questions which have not been answered. Continual investi­ gations are being made in search of a deeper understanding of the function of the thyroid gland In the manufacture, storage, and release of the thyroid hormone and the function of the 5 thyroid hormone in growth and development and in the control of metabolic rate. The work reported in this thesis concerns two aspects of iodine metabolism which are discussed in the following sec­ tions . The Effect of Physiological Saline and Desoxycorticosterone Acetate on Thyroid 1-131 Uptake in Rats. Although the thyroid gland has the triple function of manufacture, storage and release of its hormone in response to the demands of the whole animal, its most unique property is its ability to concentrate iodine. Since the first use of radioactive iodine in the study of thyroid physiology by Hertz, Roberts and Evans in 193®, many studies have been made of the thyroid uptake of radio­ activity, chiefly with I-I31, and the effect of different agents and procedures upon it. Thyroid 1-131 uptake is one expression of the gland *s capacity to manufacture its hor­ mone. Thyroid uptake is controlled at least in part by the anterior pituitary since TSH increases thyroid uptake (Keat­ ing, Rawson, Peacock and Evans, 191+5) and hypophysectomy brings about a sharp decrease in it (Leblond, Sue and Chamo­ rro, 19li0). Certain chemical substances are also believed to affect the thyroid gland directly to alter its uptake of radioactivity. Most explanations of the effect of such agents upon thyroid uptake have been made by use of one of the two mechanisms just mentioned. 6 Thera are also, however, extrathyroidal mechanisms which theoretically could influence thyroid uptake. is the level of radioactivity in the blood. thyroid circulation. One of these Another is the Even though the functional status of the thyroid remained the same, a decreased thyroid 1-131 up­ take might occur under these circumstances. Although Albert, Tenney, and Ford (1952) suggested that decreased availability of 1-131 to the thyroid gland due to decrease in blood level of 1-131 might be a factor in the decreased thyroid, uptake seen after cortisone, no study has been made of the effect that changes in blood level of radioactivity might in prac­ tice have on thyroid uptake. Decreased levels of 1-131 in the blood could arise either because of an increase in the urinary 1-131 output or an increase in the volume of dilution of 1-131 in the body without known effect on thyroid gland function. No studies have been made of the direct effect of changes in urinary 1-131 clearance or volume of dilution of I-13I on thyroid up­ take. Iodide Is known to distribute itself in a volume which Is approximately equal to the extracellular fluid volume. For this reason a preparation was sought which would have either an expanded or a contracted extracellular fluid volume. It was decided to use physiological saline (0*9% NaCl) intraperitoneally in fairly large quantity to expand extracellular fluid volume. It was thought that this treat­ ment would also partition an increased amount of 1-131 to 7 the urine. These two factors should result in lowered plasma 1-131 levels which might lead to decreased thyroid 1-131 up­ take . Desoxycorticosterone acetate (DCA) was selected for study because it is a steroid hormone active in the mainte­ nance of salt and water balance. Boatman et al. (1952) ob­ served increased thyroid 1-131 concentration ratios after chronic treatment of rats with DCA. They also found increased circulating levels of 1-131 in their DCA treated animals and suggested that the increases in 1-131 uptake seen were the result of increased availability of the isotope to the gland due to increased blood levels. By determining 1-131 uptake by the thyroids of normal rats and of rats after physiological saline and DCA, it was proposed to study the correlation between 1-131 uptake by the thyroid and plasma levels of radioactivity. The Metabolism of Thyroxine in Normal and Thyroidectomized Dogs. The major circulating thyroactive substance is thyroxine (Taurog and Chaikoff, 19^6; Laidlaw, 19^9)* Investigations in regard to its metabolism under different conditions are fundamental in elucidating the peripheral action of the thy­ roid hormone. Clinicians have suspected for many years that there is a difference in the effect of exogenous thyroid materials in normal and athyreotic individuals (Means, 1937). This ob­ servation was carefully studied by Winkler and his co-workers (l9li-3) who found that myxedematous individuals could be main­ tained in good health and normal metabolic status for many years by a daily dose of one to three grains of U.S.P. thy­ roid. If more was given to them, they would develop exces­ sive metabolic rates, cardiac symptoms, and nervousness. On the other hand, certain normal individuals could tolerate daily doses of from three to six grains without elevation of basal metabolic rate or symptoms of toxicity. Danowski, Man and Winkler (19I4-6) attempted to confirm this observation in dogs and to determine the mechanism of the tolerance In normal animals. They found no significant difference between normal and thyroidectomized dogs. On the other hand, Borgman (l9lj-9) saw distinct differences in the response of normal and thyroidectomized dogs to considerably smaller doses of exogenous thyroprotein or thyroid substance. A study was set up to compare the metabolism of thyroxine in intact and thyroidectomized dogs. This was done by a com­ parison of the rate of fall of radioactivity In the blood and the rate of its appearance in urine and feces after intra­ venous injection of 1-131 labeled L-thyroxine. 9 I-131 COUNTING METHODS Radioactive 1-131 was counted with an end-window (1.9 mg. per sq. cm.) Geiger-Muller tube mounted in a two-inch thick lead shield. The output of the tube was connected to the input of a laboratory scaler (Nuclear Instrument and Chemical Corporation, Model 163 or 165) • Chiefly the beta component of the radioactivity was counted in these experi­ ments since the G-M tube Is a highly efficient detector of beta particles but an Inefficient detector of gamma rays. Sample Preparation. Samples were prepared in round pyrex glass dishes cm. inside diameter, 0.6 cm. deep). (2.2 All samples were set up by weight using the gramatic balance. The samples were put into the weighed counting dish; dish plus sample was weighed; and the weight of the sample was obtained by difference. To all samples, with the exception of the thyroids, a few drops of a mixture consisting of 2 casein, \% phenol, 1% NaHSO^, and 1 % KI were added as an empirical precaution against losses of iodide. Plasmas, Whole Bloods, and Urines: Plasma, whole blood, and urine samples varied between 300 and 1000 mg. in wet weight. Weights between 300 and 600 mg. were preferred but the 600 mg. limit was exceeded in some cases In which it was known that the counting rate would probably be low. It was found that, If a few drops of distilled water were added to the whole blood samples, the hemolysed samples 10 resulting had a tendency to dry into a flat layer instead of an uneven crust which cracked and shattered* It was also found that more uniform samples resulted if whole blood was dried at room temperature. Thyroids: Rat thyroids were placed intact in the center of the counting dish* Dog thyroids were placed on the dish, snipped into small pieces with scissors, and dis­ tributed evenly* Muscles; Rat muscle samples were prepared in a manner similar to that used for dog thyroids. Feces: Dog feces were homogenized In a Waring Blendor with a minimum of water before an aliquot was removed for counting* Fecal samples varied from 300 to 1000 mg* In this case also a limit of 600 mg. was preferred but was ex ­ ceeded where extremely low counts might have been antici­ pated. All samples, with the exception noted, were placed In an oven at less than 80 degrees Centigrade until they ap­ peared to be dry. They were never left there for a period of more than two hours. They were then stored, exposed to air in the laboratory, and were weighed and counted at a later time. Counting Time. Counting was done for varying lengths of time. standards cited by Calvin et al. The (1914-9) were used to de­ termine the number of counts which must be recorded to 11 obtain a given statistical accuracy* Whenever it was feasible, counts were made to better than 5 P©r cent accu­ racy* Background counts were made at least daily and for sufficient time to give 5 P©r cent statistical accuracy to the longest count of the day. Regardless of the counting level all counts were con­ tinued for at least 100 seconds. Comparison with an Aliquot of Injected Dose. Aliquots of the Injection solution were set up with added plasma and treated In the same fashion as plasma samples* On the basis of these aliquots all counts were ex­ pressed in terms of per cent of injected dose. Some of the counts were calculated in terms of per cent of Injected dose per mg. wet weight of the sample. Counting Correction Factors. All of the counting In these experiments was relative, i. e., in comparison with an aliquot of the dose prepared and counted under identical conditions. With two exceptions, which are discussed when they occurred, all counting was beta counting. Whitehouse and Putnam (1953) discuss the errors Involved in this type of counting. They list the order in which corrections should be applied as follows: (1) Losses of counts in the dead time of the counter (2) Background rate (3) Geometry factor (Ij.) Self-absorption and back-scattering. 12 Each of these factors had to be evaluated under the con­ ditions of this laboratory. Losses of Counts in the Dead Time of the Counter: Counting was restricted to rates of less than 100 counts per second* It was thought that below these levels counting rates would be linear. Graph 1 shows that this prediction was correct. These data were collected by recounting six different rat thyroids over several half lives. Each count was done with and without an external absorber. The count with an ex­ ternal absorber was approximately one-eighth the count with­ out it. (This was the only occasion on which external ab­ sorbers were used in these experiments.) Graph 2 shows the losses of counts which occur at high counting rates. 1, hcwever, shows that a less than 100 counts Graph restriction of counting to rates of per second permits the assumption of a negligible loss of counts in the dead time of the counter. Geometry: In these data all samples have been counted on the same shelf during any given experiment. This was either the second or the third shelf of the plastic sample rack within the lead shield. The first shelf was avoided because random distribution, either horizontal or vertical, of samples on the dish can introduce an excessive error in counting when the sample Is placed relatively close to an end window G-M tube (Calvin et al., 191^-9). Self-Absorption: Corrections for the self-absorption of beta particles were made according to the data given In / O o 12 i- o o 8 o GPS with External Absorber 101- k <25 "O 20 1|0 ' ‘ 60 ' “80 CPS without External Absorber Graph 1: Linearity of counting up to 100 cps* 11+ o 100 80 o / o / 60 O/ dp / CPS with External Absorber o o o 20 TpKT CPS without External Absorber Graph 2: Loss of counts at high counting ratio• table 2 of the appendix. These correction factors were cal­ culated from a series of samples of different weights of rat liver labeled with 1-131. The liver had been dried and powdered; the portion which would pass through an ISO mesh screen was used. Each of the points of the curve on which the self-absorption correction factors are based was estab­ lished by a triplicate count of the sample, which was care­ fully stirred and redistributed between counts. The formula for the best semi-logarithmic straight line through the points between L|.0 and llj.0 mg. was used to set up the cor­ rection factor table. The method used to determine this line is discussed later in this thesis. Then samples for counting were restricted to 20 to 120 mg. dry weight; this was achieved by limiting the wet weight of samples. Some of the dog fecal samples exceeded 120 mg. dry weight so correction factors based on the empirical data were added to include higher weights. All samples except these fecal ones fit the restrictions imposed by the theo­ retical curve. Table 2 in the appendix also gives the empirical correction factors used. An idea of the accuracy of the self-absorption and backscattering corrections used was obtained when the laboratory acquired a scintillation counter. Gamma counts were made for a series of rat plasmas and thyroids which had already been counted with the G-M tube. The counts of these samples were rather low and it was not practical to attain better than 10 per cent counting accuracy in some cases; however, a study 3: I u > CD CO o o vO O o U\ m C^\ o CM tieAjT q.'BH P 9 TJQ J° CM aGC^ SID Tr\ —I I c0 PS rd -P PI CD Pi u 'H O bO 1-131 self-absorption correction curve. Pyrex cm. in diameter. Log y - 40.51^4- -0.00138x. o o —I Graph glass dishes 16 O rH• iH CM 17 mad© of the comparative counts answered the following questions: (1) Was there an error introduced by the use of self-absorption corrections which showed a trend as sample thickness changed? This question arose because the dried plasma samples tended to stick to the walls of the dish and correction factors were based on ideally arranged rat liver powder. (2) Was there an error, which showed a trend with thyroid weight, introduced by not using self-absorption cor­ rections for rat thyroids? Both of these questions can be answered in the negative. Graphs 1 and 2 of the appendix show a plot of ratios of the individual beta counts to their respective gamma counts against dry weight of plasma and wet weights of thyroids (thyroid dry weights are too small to be measured accurately by the methods used). The beta counts were corrected for self-absorption before the ratios were calculated. Tables 1 and 2 show that there is no correlation between ratio of betato-gamma count and either plasma wet weight or thyroid dry weight. Since there is no error due to self-absorption of gamma rays at these thicknesses, this means that there is no trend in the counts of these samples with change in either plasma or thyroid weight. Physical Decay: Corrections for physical decay of 1-131 were made according to factors listed in table 1 in the ap­ pendix. Counts were corrected to a zero time which was arbitrarily established in each experiment. These correction factors have been calculated for an 8.00 day half life. The 18 most recent information found for the half life of 1-131 was that of Lockett and Thomas (1953)* They give 8.06^0.02 days. This means that the use of 8.00 days involves introduction of an error no greater than one per cent per half life. The local counting methods then are such that the ob­ served counts per second were corrected in the following order: (1) The background rate was subtracted. (2) The value obtained in (l) was multiplied by the proper correction for physical decay from table 1 in the appendix. (3) The value obtained in (2) was multiplied by the proper correction for self-absorption from table 2 in the appendix. Rat thyroids were not corrected for self-absorption. {Ij.) The value obtained in (3) was calculated in terms of per cent of dose. 19 THE EFFECT OF PHYSIOLOGICAL SALINE AND DESOXYCQRTICQSTERONE ' , ACETATE ON THYROID 1-131 UPTAKE IN RATS r n ■ ■ Introduction . Plasma levels of radioactivity may be one of the factors which influence the uptake of 1-131 by the thyroid gland. The purpose of this study was to evaluate the effect of plasma levels of radioactivity on thyroid uptake in rats which had been given excess physiological saline intraperitoneally and in rats which had received desoxycorticosterone acetate (DCA). Physiological Saline: No Information was found regard­ ing the effect of a large intraperitoneal dose of physiologi­ cal saline on thyroid 1-131 uptake. Storlaasi, Rosenberg and Friedell (1953) however con­ sidered a related problem when they investigated the effect of food and water on thyroid uptake of radioiodine. Their rats had been maintained on iodine deficient diet and dis­ tilled water for four weeks. They measured six and twenty- four hour uptake and excretion of a tracer dose of 1-131 under four different conditions: (1 ) no food and water for 36 hours before and after administration of 1-131 * (2 ) no food and water after administration of 1-131 , (3 ) only water ad libitum, and (Ij.) food and water ad libitum. small numbers of animals (three per group). They used They found that food and water or even water alone tended to increase urinary output and to decrease thyroid uptake at both six and twentyfour hours. 20 Desoxycortlcosterane Acetate? The first study made r e ­ garding the effect of DCA on thyroid uptake of 1-131 was that of Paschkis et al. (1950)* Their primary aim was the study of thyroid function in the "alarm reaction ,11 but they in­ cluded a group of rats which had been pretreated with I4.O mg* of DCA at forty-eight and twenty-four hours* In their ex­ periment DCA failed to influence significantly the four hour uptake of a tracer dose of 1-131 in adult male rats on a normal diet* Money and his associates studied the effects of several adrenal and gonadal steroids on the twenty-four hour uptake of a carrier-free dose of 1-131 In Z$Q to 300 gm. male rats which had been on an Iodine deficient diet for twenty days. They injected 1*0 mg. per day for ten days* (1950) or 15 mg. (1951) of DCA per rat The rats had increased thyroid gland weights and body weights at both DCA levels* They found that 1.0 mg. had no significant effect on thyroid 1-131 uptake ex­ pressed as per cent of dose per gland or per cent of dose per mg. gland per 100 gm* body weight. They showed however that, although thyroid uptake in terms of per cent of dose per gland was not altered, a significant decrease in thyroid up ­ take expressed as per cent of dose per mg. gland per 100 gm. body weight was seen after the 15 mg. dose of DCA. Boatman et al* ratios (1952) found increased concentration (which they define as per cent of administered activity found in an organ divided by the org a n ’s per cent of the body weight) in DCA treated rats three hours after 21 the injection of carrier-free 1-131, They used approximately > 200 gm, rats, half of which had been hemithyroidectomized. The experimental rats were given one mg, DCA intramuscularly in oil three times a week for a period of four weeks. Intact and hemithyroidectomized rats which had received DCA each showed increased thyroidal concentration ratios when compared with untreated animals of the same type. They found the whole blood, plasma, and erythrocyte content of 1-131 to be about thirty per cent higher in the DCA treated animals than in the controls and hypothesized that the increased concen­ tration ratios found in the thyroids of these animals could be a reflection of the increased circulating levels of radio­ activity. In 1952, Gabrilove, Dorrance and Soffer found that goitrogen-treated rats which had received one mg. of DCA per day for a period of either twelve or twenty-two days showed no difference in thyroid weight as compared to control rats. ZIngg and Perry (1953) studied the effect of DCA on thyroid 1-131 uptake and some other Indicators of iodine metabolism in patients in the psychopathic ward of the Winni­ peg General Hospital. control. Each individual served as his own They found decreased thyroid 1-131 uptake, two, four, twenty-six, and fifty hours after oral carrier-free 1-131, after three days treatment with ten mg. of DCA daily in two five mg. doses twelve hours apart. continued during uptake measurements. Treatment was The uptake of 1-12? by the thyroid gland was also significantly depressed. 22 Plasma iodide-127 and renal 1-131 clearance were not signif­ icantly altered by the DCA. No information was found in regard to the effect of intraperitoneal 0*9$ NaCl on thyroid 1-131 uptake* al. Money et (1951) reported a decreased per cent of dose of 1-131 per mg. thyroid gland per 100 gm. body weight in rats after chronic treatment with DCA. A depressed thyroid uptake of 1-131 after DCA treatment likewise was revealed in human beings by Zingg and Perry (1953)* man et al. On the other hand, Boat­ (1952) found increased thyroid 1-131 concentration ratios, along with increased plasma and blood levels of radioactivity, after chronic treatment of rats with DCA. Materials and Methods. Several groups of male albino rats from Carworth Farms, New City, New York, were used in this study. Experiments 1 and 2 were preliminary experiments; experiments 3 and If. were the main ones. Experiment 5 was exploratory towards a method for explaining the results found in 3 said k* Diet: In experiments 1, 2, and 5# the animals were maintained on a ration consisting of: Yellow Corn Meal 35$ Ground Whole Wheat 25$ Whole Milk Powder 20$ Linseed Oil Meal 10$ Alfalfa leaf Meal 6$ Brewerfs Yeast 3$ Table Salt (Iodized) 1$ 23 In experiments 3 and Ij., the animals were given an iodine de­ ficient diet periment, (Remington, 1937) for ten days prior to the ex­ T!Iodine Deficient Diet,n General Biochemicals Inc., Chagrin Palls, Ohio, consisting of: Yellow Corn Meal (grown in iodine 78$ deficient area) Wheat Gluten Brewer's Yeast 2% Calcium Carbonate 1% Sodium Chloride All animals had tap water to drink. Pood was removed from four to six hours before the in­ jection of 1-131. The rats had free access to tap water up until the time of 1-131 injection. Prom then until the time of sacrifice one and one-half to three hours later, they had neither food nor water available. Although starving the animals overnight before performing the experiments would have been desirable, it was found that a large percentage of the adrenalectomized animals passed into a coma if deprived of food for such a long period of time. Body Weights: The rats of the first four experiments, as can be seen from table 3 * were of approximately the same weight. In each of these experiments the rats were care­ fully matched for weight. The experiments were conducted so that animals were injected in the following order: mal No. 1, group 1; animal No. 1, group 2 ; ani­ animal No. 1, group 3; etc., throughout the total number of groups; then, 2k animal No* 2, group 1 ; animal No. 2, group 2; etc* ......... All of the animals numbered 1 were matched for weight and so were all the animals numbered 2, 3» etc* Running totals of the weight sums were kept for each group and animals were assigned to the different groups so as to keep the sums as close to each other as possible. The body weights of rats in experiment 5 are to be found in the appendix, tables 3 an<3. ^I-* injection of 1-131: A dose of approximately twenty uc. of 1-131 per rat was injected intravenously* An incision about two cm. long was made under light ether anesthesia on the medial side of the thigh; the 1-131 was injected into the femoral vein; and the incision was closed with a metal skin clip. Injections were made in a total volume of one- half cc. per rat. In experiments 1 and 2, the 1-131 was carrier-free. In experiments 3 , Lj., and 5 > the 1-131 was prepared so that each rat received 8*5 ugm. of carrier 1-127 in the form of Nal. 1-131 injections were made in the order described under body weights. The conditions of these experiments made it necessary to inject the animals over a period of ten to fif­ teen hours. The order of injections used would have mini­ mized the effect of any diurnal variation In thyroid uptake* Dougherty, Gross and Leblond (1951) found no differences in four hour 1-131 uptake by the thyroids of rats, both on normal and iodine deficient diet, injected at 1 p.m., 5 p.m., 9 p.m., 1 a.m., 5 a.m., and 9 a.m. 25 Injection of 0,9^ NaCl and DCA: Salinas, warmed to ap­ proximate body temperature, wa3 injected intraperitoneally. DCA was administered intraperitoneally as a suspension in corn oil. DCA injections were 0.2 cc. per rat in experiments 1 and 2 and 0.1 cc. per 100 gm. body weight in experiments 3 and Lj.. The injections were given as soon as possible after the 1- 131 . Adrenalectomy: Adrenalectomies were performed by a dor­ sal approach from eight to ten days before the experiment. Animals were maintained on 0.25 mg. DCA given subcutaneously in solution in corn oil in a single daily dose. They had tap water to drink. All animals were examined grossly for the presence of adrenal tissue at the time of autopsy. Thanks is due to Dr. Joseph Meites for assistance with part of these examinations. The few animals in which adrenal tissue was found were dis­ carded. Samples for Counting: Blood was obtained by two methods. In experiments 1, 2, and 5> th© rats were anesthetized with ether and blood was removed from the abdominal aorta with a syringe. The animals were sacrificed immediately by opening the chest cavity and cutting the heart. In experiments 3 and l\.9 in which larger numbers of animals were used, the rats were sacrificed by a guillotine method and freely flowing blood was collected in a small beaker. an anticoagulant. Heparin was used as 26 Thyroid glands were dissected f r e e of visible fat and connective tissue and weighed on a 125 rag* capacity Rolleromith balance, tollman and Scow (1953) found a progressive loss of radioactivity from the thyroid glands of thiouracil treated mice between the time of death and the time of re ­ moval of the thyroid glands five to eighty minutes later. If such effects occur in these normal animals in which or­ ganic binding of the iodine occurs, they should have been equalized among the groups by the method of handling used. As soon as possible after sacrifice, the animal3 were placed in the deep freeze. They were removed at a later time and were autopsied as soon as possible in the same order in which they had been injected. Muscle samples were taken from the thigh opposite to the injection site, cleaned of visible fat, connective tissue, blood vessels and nerves and weighed on a 100 mg. capacity Roller-Smith balance. Samples of l+OO through 600 mg. were used. It was not thought to be possible to collect accurate urine samples for only one and one-half hours without special arrangements. In experiment 3, which lasted three hours, the animals were placed in metabolism cages by groups and the total urinary radioisotope output of the group measured. experiment In an attempt was made to collect urine from the rats Individually at one and one-half hours by placing a purse string ligature on the penis (Friedman and Livingstone, 19l|.2) and removing the bladder intact while the rat was still 27 frozen* A few trial rats done by this procedure were suc­ cessful. However, when this method was used in experiment Ij., some of the rats, even those which had received saline, were found to have empty bladders at the time of autopsy. There­ fore, these data have been used only as an indication of the maximum amount of urinary radioactivity to be found at one and one-half hours. Special Conditions and Methods of Experiment 5 ? The last experiment of this group was conducted in rats which were considerably larger, and therefore older, than those of the other four experiments. The rats were anesthetized with nembutal in contrast to the other experiments in which rats were conscious during the uptake period aside from the short period of light ether anesthesia necessary for intravenous injection. These were intact, not adrenalectomized, rats. Just prior to sacrifice, blood was taken from one of the thyroid veins. The rat has a vein leading from the posterior end of the thyroid gland along the side of the trachea. The sharp angle at which the chest rises and the small size of the vein make it impossible to draw blood from it with a hypodermic needle and syringe. It Is possible to expose this vein at a distance of perhaps one cm. posterior to the gland, cut the vein with a small pair of scissors, and collect around 0.2 ml. of blood as it flows from the vein In a syringe. This procedure results in sufficient blood for analysis in approximately two-thirds of the rats. About one cm. length of the vein was exposed and cleaned 26 ft*©© of fascia; the vein was then tied off at the posterior end of the exposed portion and cut anterior to the 3uture. A detailed report of the gross anatomy of the circulation of the rat thyroid gland has not been found. tracheal area of several The thyroidal- rat necks has been examined with a hand lens by Dr. E. P. Reineke and the author. All gross ap­ pearances lead to the conclusion that the vein from which blood was taken drains the thyroid gland. If it drains any other sites, it is probably only the small amount of fat attached posterior to the gland. In these rats arterial blood was removed from the ab­ dominal aorta after the thyroid blood. In some of them gene­ ral systemic venous blood was also removed from the vena cava. The arterial and thyroid venous bloods same conditions in order were counted under the to obtain the thyroid 1-131 arterio­ venous difference. Statistics: Comparisons between the groups in these ex­ periments were made by Analyses of Variances. A probability of less than 0 .05> was accepted as being statistically sig­ nificant . Experimental and Results. The aim in this study was to perform the experiments under such conditions that the amount of 1-131 found in the thyroid gland would represent as nearly as possible the amount of radioactivity that the gland had extracted from the blood up to the time of sacrifice. The per cent of 1-131 found In the thyroid gland, called "uptake” here, is actually 29 the difference between the per cent of 1-131 extracted from •/ ■ the blood and the per cent of I-I 3I returned to the blood* Since the release of I-131-labeled hormone is a slow process relative to the rate of extraction of 1-131 from the blood, the shorter the time period chosen, the more nearly the measured "uptake” represents amount extracted from the blood* Albert (1951) found a mean exponential rate of dis­ appearance of thyroidal 1-131 in rats, between 50 and 165 hours after injection of 1- 131, of 28 per cent per day. This would represent a loss of only 5 par cent for three hours* In these experiments there is no evidence whether or not the rate of labeled hormone release had been changed by the treatments used; in any case a rate double that found by Albert would increase the output to only 8*5 par cent for three hours. Thus it was felt that any reasonable change of output would not quantitatively effect the per cent of dose in the gland if uptake was measured at three hours or less. It was also desired to have the same amount of 1-12? labeled in the different groups. Without attendant measure­ ments of 1-127 , a decrease in uptake of 1-131 does not nec­ essarily mean that a decreased amount of iodine has been concentrated - it might mean only that the 1-131 was diluted by more 1-127. Perry and Hughes (1952) found such a mecha­ nism occurring in renal disease in humans. In a study in which uptake of a carrier-free dose of 1-131 is measured in animals which have been treated chronically with a hormone which is known to have effects on salt and water balance, 30 there is no guarantee that this situation, or its reverse, has not arisen* It was therefor© decided to give the saline and DCA after 1-131 was administered* Another condition of these experiments was the use of adrenalectomized animals* As has been mentioned, intact animals show decreased thyroid 1-131 uptake after various stresses, e. g. formalin injections Kemp, 19l|9), anoxia (Williams, Jaffe and and trauma (Van Middlesworth and Berry, 1951 )9 and tourniquet shock (Hamolsky, Gierlach and Jensen, 19^1)# and after ACTH and cortisone Gabrilov©, and Dorrance, 1951)* (Perry, 1951; Soffer, It was not known how much of a stress the etherization and intravenous injections con­ stituted. Neither was it known how great a stress the intra- peritoneal physiological saline would be. It was also not known what effect on the adrenal-pituitary axis would result from the DCA injection. In order to eliminate the need to evaluate the importance of these parameters In the 1-131 u p ­ take picture, it was decided to perform the experiments in adrenalectomized animals. Table 1 presents the thyroid 1-131 uptakes and plasma 1-131 levels of the rats in experiments 1, 2, 3» and i|. The observations of experiment 5 are reported in tables 3, and 5 of the appendix. Experiments 1 and 2 ? In these two experiments the one and one-half hour thyroid 1-131 uptake after a carrier-free intravenous dose of radioactive Iodine was measured in ad­ renalectomized rats which had been fed the standard laboratory Table Is Thyroid and plasma 1-131 levels in rats treated with physiological saline and desoxycorticorticosterone acetate. Experiments 1 , 2 , and at one and one-half hours and experiment 3 three hours. All rats except groups i|., 5 an<3- 6 of experiment I4. have been adrenalectomized. Group Treatment No. of Animals Mean Body Weight gm. (range) Experiment 1 1 2 Control 10 cc. 0 * 9 $ NaCl 7 7 1511(132 - 168 ) ll+9 (1314.-161+) Experiment 2 1 2 3 Ij- Control 0.1 mg. DCA 0.5 mg* eca 2.5 EC A 9 9 8 10 ll+9 (136-169) 152{132-171) llj.9 (132-162) I53(13li-167) 10 10 10 10 16K 138 -17I4.) 1614.(128 - 182 ) 163 (132- 202 ) 21 20 20 21 20 19 123(1114.-114.0) 120(106-11+2) 120(106-138) 133(112-152) 129(116-111.6) 131(116-158) Experiment 3 1 2 3 ij. Control 10 cc. 0 . 9 $ NaCl/lOO gm. 5 cc. 0.9% NaCl/lOO gm. 1.25 *ng. DCA/lOO gm. l62(ll;l-193) Experiment I4. 1 2 3 ij. 5 6 Control 1.5 nig. DCA/lOO 10 cc. NaCl/lOO Control 1*5 nig. DCA/lOO 10 cc. NaCl/lOO gm. gm. gm. gm. vindicates significantly different from mean of controls P ^£$ 1. Muscle Iodide space in % = s ■ on «> (Hastings & Eichelberger, i ^ s e / m g . plasma * 2. A value of £ 6 * 1 $ has been omitted* No statistics were done for muscle space in these rats. 32 Mean Thyroid Weight mg. Muscle 1-133Space-1% Urine 1-131 $ of Dose Per Rat Plasma . 1-131 % of Dose/mg. x 10-3 Thyro id ^ 1-131 % of Dose Experiment 1 13.7 21 .52 23.9 1.09 0.851*.* 25.0 28 .14. 1.10 1.08 1.10 1.03 1.88 1.66 Experiment 2 34.1 11*..1 12.0 12.2 29.7 31.1+-”- 1*37 142 1.21 1.13 Experiment 3 19 .u 18.3 19.7 17.14- 19.3 24 .2* 2I4..I4.* 19.1). 10.0 19.1| 22.0 9.5 0 •7i|7 0 .148* 0 .515* 0.685 5.61 9.60* 7.61*. 8 .78* 1.20 1.16 0.907* 1.11 0.986 0.8l|9* 0.66 0.65 0.95* O.I4.6 045 0.55 Experiment b 12.3 11.5 12.1 12.9 11.7 12.1 33 ration prior to the experiment. Plasma 1-131, muscle 1-131, and the 1-131 level in the part of the saline infusion which was still present intraperitoneally were measured* In experiment 1, the rats received 10 cc. total of physiological saline intraperitoneally just after intravenous 1-131* Plasma 1-131 was significantly decreased in these animals as compared to the controls. The saline remaining intraperitoneally appeared to be equilibrated with the plasma; 1-131 levels in it averaged 95 P^n cent of the plasma level, with a range of 68.5 to 120.2 per cent. The thyroid uptake of 1-131 was not significantly altered in the saline-treated animals• In experiment 2, there were four groups of rats, a con­ trol group and groups receiving 0.1, 0.5# and 2.5 nig. total of DCA. None of the groups receiving DCA showed any signifi­ cant difference in plasma 1-131 level when compared to the controls. The thyroid uptake of 1-131 was not significantly altered in any of the DCA-treated animals. There was no in­ dication that the smaller doses of DCA showed any effect which was qualitatively different than the largest dose. Considerable thought regarding the situation in these rats lead to the conclusion that if rats were put on an iodine deficient diet for a short period of time in order to decrease their body stores of iodine and then if the 1-131 was given with some 1-127 as carrier, the desired condition of labeling the same amount of 1-127 in each animal would more nearly be attained. An amount of carrier was used which was less than the 10 ugm. which Wolff and Chaikoff (I9I4.8 ) found to show no signs of depressing thyroidal organic bind­ ing of iodine. Halmi Since these experiments have been performed, (195^) found that ’’Low Iodine T©st Diet," made by Nutritional Biochemicals Corporation by the same formula as the iodine deficient diet used here, had no stimulating ef­ fect on thyroid morphology in rats which had been eating It for a period of 19 to 20 days. The rats used in experiments 3 and I4. were placed on the iodine deficient diet for a period of 9 to 10 days; they received 8.5 ugm. of carrier iodide. It was concluded that perhaps more striking differences might be seen at three hours instead of one and one-half without a great sacrifice of the desired experimental con­ ditions. It was anticipated that the data from this experi­ ment would permit determination of whether or not a cor­ relation between thyroid uptake and blood 1-131 levels occurred in saline-treated and normal rats. Two doses of saline were chosen so that, between saline-treated and nor­ mal rats, a variety of blood levels would be attained. It was decided that It would be more acceptable to dose by weight rather than to give each animal a predetermined amount. Five and 10 cc. of saline per hundred grams body weight were decided upon; one of these is larger and the other smaller in amount than the dose of experiment 1 of 10 cc. per rat, equivalent to an average of 6.7 cc. per 100 gm. body weight. A dose of 1.25 mg. of DCA per 100 gm. body 35 weight was chosen; this was slightly less than the largest dose of experiment 3 which averaged 1.6 mg. per 100 gm* body weight. Experiment 3 ; The three hour thyroid uptake of 1-131, injected with 8*5 ugm. of 1-127 as Nal, was measured in ad­ renalectomized rats which had been on an iodine deficient diet for nine days. Plasma 1-131 and muscle 1-131 were also measured. Both 10 cc. of 0.9$ NaCl and 1.25 nig. of DCA per 100 gm. body weight produced a significant increase in thyroid u p ­ take at three hours under the conditions of this experiment. In the case of the saline, the plasma 1-131 level was de­ creased; in the case of DCA, no change in plasma level was seen. Five cc. 0.9$ NaCl per 100 gm. produced no significant alteration in either uptake or plasma level. The pooled urinary output data suggest that there was an increased u r i ­ nary output of radioactivity in the saline treated animals as compared to the controls. DCA. This effect was not seen with 36 Experiment I4.: Both adrenalectomized and intact rats were used in this experiment. As in experiment 3 , the ani­ mals were placed on an iodine deficient diet for ten days and were given 1-131 with 8.5 ugm. of 1-127 carrier. Thyroid uptake, plasma level and urinary output of radioactivity were measured at one and one-half hours. The maximum urinary output of radioactivity in these rats was 2.5 p©i* cent of dose. This was determined by averaging the two highest values found in each group. Adrenalectomized animals given 10 cc. of saline per 100 gm. body weight showed a statistically significantly increased thyroid 1-131 uptake which was not demonstrable in intact ani­ mals. Both adrenalectomized and intact animals had signifi­ cantly lower plasma 1-131 levels than their respective con­ trols . Adrenalectomized animals given 1.5 mg* BCA per 100 gm. body weight showed no differences from their controls. Thy­ roid 1-131 uptake and plasma 1-131 levels in the two groups were the same. Similar results occurred when intact animals were compared with their controls. No differences in either thyroid uptake or plasma level of radioactivity were seen. When all three groups of adrenalectomized animals were compared with the three intact groups, the adrenalectomized rats had a statistically significantly greater uptake of 1-131 than the intact rats. Experiment 5 : experiment. The last of this series was a preliminary It points a way toward an attempt in a different 37 direction to determine the mechanism behind the results found in experiments 3 and L}.. The observations of experiment 5 have been recorded in tables 3 , 1±, and 5 of the appendix. The differences between this and the other four rat ex­ periments have already been pointed out. In brief these were very large intact rats which had not been placed on iodine deficient diet but which had received the 8.5 ugm. of carrier 1-127• The rats which received physiological saline were given 10 cc. of 0.9$ NaCl per 100 gm. Blood levels, rather than plasma levels, of radioactivity were measured because insufficient thyroid blood was available conveniently to ob­ tain a plasma sample. These data also can be looked upon as a final control on the influence of circulating 1-131 levels on thyroid up ­ take. Since plasma and blood 1-131 levels would be very high early after an intravenous dose of 1-131 , this experiment was planned to ascertain the behavior of blood levels of radio­ activity at early times. Examination of table 5 of the ap­ pendix shows that 1-131 levels In the blood of normal and saline-treated rats tend to be the same at 6 and 30 minutes but between 60 and 135 minutes after I- 13I, the NaCl-treated rats fall on an average below the controls. Therefore — however the increased thyroid 1-131 uptake after saline in experiments 3 and I4- may be explained -- there is probably no reason to attribute it to increased blood 1-131 levels at any time after 1-131 injection. 38 Experiment 5 also afforded data to calculate a tentative figure for thyroid circulation* All available calculations of thyroid circulation have been based on the assumption of complete plasma clearance of I-I 31. There is also an im­ plicit assumption that there is no return of radioactivity back to the blood during the time used for calculation. Jones (19l|-5) found a minimum perfusion of the thyroid gland of the rat of three to ten volumes of blood per volume of gland per minute. Myant, Pochin and Goldie (1914-9) and Stanley (19li9) concluded respectively that the minimum circulation of the thyroid gland of human subjects was 0.5 and 0.25 volumes of blood per volume of gland per minute. Thyroid 1-131 clearance can be calculated by the classi­ cal method used for kidney clearance. If t * time in minutes U - thyroid uptake of radioactivity, expressed as % of dose, between t^ and B - t2 average % of dose of 1-131 por mg. of blood from t^ to t 2 C » the clearance, the number of mg. of blood per minute from which 1-131 has been com­ pletely extracted. Then C = _____ 5_____ B x (t2 - tl) If it is assumed that the blood is completely cleared of 1-131 in each passage through the gland, the clearance represents the amount of blood per minute which has passed through the gland between ti and t2. If the blood is not completely cleared of 1-131, then it will require more blood to deliver to the gland the amount °f radioactivity which it has collected between tq and t2 . Let P - gland circulation expressed as mg. blood per mg. gland per minute. w - gland weight in mg. where A • % of dose of I -131 in L - systemic arterial blood and TV a % of dose of 1-131 in thyroid venous blood. Then l/D represents the number of mg. of blood which would have to pass through the gland In order for one mg. to be cleared completely of 1-131 , and p m - - - _ __ x -i- x -iBx (t2 - tq) D w In experiment 5, circulation of the thyroid was cal­ culated using the data of 5 and 30 minutes to determine thy­ roid 1-131 accumulation and to calculate the average blood levels of radioactivity. A brief preliminary experiment had shown that arterial blood levels of radioactivity were fall­ ing so rapidly in the period about an hour after Intravenous injection of 1-131 that a variation of a minute or two in the time of collection of the thyroid venous and the systemic arterial blood might have meant that arterial blood levels h a d already fallen below the thyroid venous levels. For this reason thyroidal arteriovenous 1-131 differences were determined between 58 and 133 minutes after 1-131 injection. kO For these rats thyroid circulation can be calculated using the following data: U » 0.693% of dose, graph l\. B s 0.0005lii% of dose/mg. of blood, graph 5 w - 2ij_.l mg. D » 0.10, table 2 (t^Q“ t£>) 3 2l| minutes P x 1 o .io ' x 1 2Ij7T f ■ 23 mg. of blood/mg. of gland/minute As far as the author knows, this is the first time that arterio-venous differences in 1-131 blood level have been measured and hence is also the first time that an absolute figure for the blood circulation of the thyroid gland has been calculated. Discussion of Results. Under the conditions of experiments 3 and I}., intraperitoneal physiological saline produced an increased one and one-half and three hour thyroid 1-131 uptake although at the same time there was a decreased circulating level of radio­ activity in the blood plasma. There was probably also an increased partition of 1-131 to the urine by three hours. In the same experiments, DCA produced an increased uptake of 1-131 by the thyroid at three hours. No change in plasma 1-131 level was found after DCA and there was no indication of a change in urinary 1-131 output. It is necessary 3• Oj— Average uptake 30 min. = 0.669^ Average uptake 6 min, = 0,1?)1| Difference = 0-525 X 2 0 * — □ in X □ □ X □ Uptake 1-131 X Average uptake 6 to 30 minutes 5= 0.693^ Per Gent of Dose Correction for Beta self-absorption s 1-32 □ □ □ X □ 1. 0 - x X Thyroid X x X X Controls > Saline-treated a _J 30 _ _ _ J _ _ _ _ _ _ _ _ _ _ I_ _ _ _ _ _ _ _ _ _ 1— 60 90 120 Time in Minutes Graph ]+: Thyroid 1-131 uptake in control and salinetreated rats. Experiment 5* 1+2 50- per mg. Blood x 10“^- -Bstimated Blood Level at t s 0 At Blood Min, Level Total 1 1 1 1 2 3 3 0.00070 65 62 0.00070 65 62 59 110 59 55 52 50 156 150 3 1+7 b 1 192 ll+l 181+ 1+5 b 1-131 of No, . ......... h$. .001231+ 0.000511+ Total 21+ Average 10 of Dose - Per Cent \. -L- Estimated Pall in RadToadt-ivity | Between 50 a n d l 3 5 minutes 0 12 18 21+ 30 Time in Minutes Graph 5: Average blood level of radioactivity between 6 and 30 minutes. Experiment 5* Table 2? Ratios of* systemic venous to systemic arterial blood and of thyroid venous to systemic arterial blood in control and saline-treated rats, Experiment 5. Rat No. 7 8 Ik Systemic venous Systemic arterial 0.91 1.07 - - thyroid venous Systemic arterial 0.88 1.03 0.78 15 16 1.00 0.95 7 8 13 1.05 0.98 16 0.99 0.89 0.91 0.71 0.85 0.97 Mean 0*993 0.897 — — 1.03 0.92 kb therefore to look elsewhere than blood 1-131 levels for an explanation of what has happened in the treated rats. It cannot be inferred from these data that the mechan­ ism of the changed uptake with saline is the same as that with DCA. There is not enough evidence to support or refute such a contention although it is extremely Interesting that these agents seem to behave in the same way throughout this investigation. By what mechanism then could these changes in uptake have come about? The factors which are thought to influence thyroid 1-131 uptake have been discussed by several authors, notably Oddie (19lj-9), Riggs (1952) and Wo liman (1951i)* Thy­ roid uptake as measured in these experiments is actually the difference between the amount of 1-131 which has been col­ lected from the blood and the amount which has been returned to it. A very early uptake was measured in these experiments so that it can probably be stated that changes in radioactive hormone output have made a negligible change in measured 1-131 uptake. The amount of 1-131 collected from the blood as sum­ marized by the foregoing authors is a function of the follow­ ing factors: (1 ) the blood level of 1- 131 * (2 ) the rate of blood flow through the gland, (3 ) the fraction of the blood iodide transferred to thyroid tissue as blood perfuses the gland, which In turn is the function of (a) the glandrblood ratio of 1-131 in inorganic form and (b) the rate of organic binding of 1-131* h5 The third of these parameters has been investigated very extensively. Both the gland:blood ratio of inorganic radio­ active and the total thyroid 1-131 uptake have been studied under many different conditions. As far as the author is aware, no critical study has been made of the effect changes of blood level of I- 13I on thyroid uptake--with the exception of Albert and his co-workers1 investigation of the effect of hypophysectomy on renal clearance of 1-131 (Albert, Tenney and Lorenz, 195>2a)--although either changes in urinary output of 1-131 or changes in volume of dilution of iodide have been suggested as explanation for the differences in thyroid up ­ take seen after cortisone (Albert, Tenney and Ford, 1952). Likewise no study has been made of the effect of the rate of thyroid blood flow on thyroid uptake although it is obvious from gross examination that the hyperplastic, hypertrophied gland must have an increased blood supply. These experiments were planned to examine the effect of blood radioactivity levels on thyroid 1-131 uptake. It was hypothesized that the saline-treated animals would show de­ creased circulating 1-131 levels and this expectation was realized. Along with the saline-treated animals was studied a group which had been treated acutely with DCA, al. Boatman et (1952) had found that chronic treatment with DCA increased thyroid uptake, expressed as per cent of administered activity found in the organ divided by the organ’s per cent of body weight, with an accompanying increase In circulating 1-131 levels. Ij_6 Changes in thyroid, uptake can arise either indirectly through changes in the anterior pituitary secretion of TSH or directly through an effect on the thyroid gland itself* Keating et al. (191-1-5) found in chicks that no elevation in four hour thyroid uptake of 1-131 was seen twenty-four hours after a single dose of TSH although mean acinar cell height and thyroid weight were already increased. wood Stanley and Ast- (191^9) found no increase in accumulative gradient six to eight hours although an increased was noted at twenty-four hours after a single dose of TSH in human subjects. No in­ formation in rats regarding this point had been found; how­ ever, if data from these other species can be extrapolated to the rat, then the effects seen in these experiments oc­ curred too soon after the treatments to have been mediated through the anterior pituitary. Pew studies on thyroid uptake have been made at early times after treatment with any chemical substance. It is be­ lieved that this is the first study in which thyroid 1-131 uptake has been measured when the treatment with a steroid hormone was given after the radioactivity was administered. The increased uptake after either saline or DCA cannot be explained in terms of increased blood levels of radio­ activity. levels. The saline-treated rats showed decreased blood In the rats which were acutely treated with DCA, unlike the chronically treated ones of Boatman et al. (1952) which showed increased circulating I—131 levels, there was no change in blood levels of radioactivity relative to the controls. k7 A return to the factors influencing thyroid uptake leads to the question as to whether it might have been one of the other factors discussed which was changed in these rats. No information has been found regarding whether DCA and saline in doses similar to those used have any effect on the circu­ lation in general or on thyroid circulation in particular. If it was not the gland circulation which has been affected, it may have been a change in the inorganic Iodine uptake or in the rate of organic binding. The experiments conducted here involved the collateral information believed necessary to determine whether plasma levels of radioactivity influence thyroid uptake. The condition of the groups in respect to other factors can only be conjectured. No data are available on which to base speculation in regard to the mechanism of the change which occurred in the NaCl or DCA treated groups; nevertheless, the experiments were planned in such a fashion that it can be stated that those groups which showed an in­ creased uptake of 1-131 had collected a greater amount of iodine in their thyroid glands. Is the situation which has been set up in these rats of sufficient interest physiologically to merit an extended study to determine the mechanism by which it comes about? The 0.9 % NaCl was employed because it was believed that changes of blood I—131 level would be seen with it. It is extremely interesting that such a simple treatment as 10 cc. of physiological saline per 100 gm. body weight would affect thyroid 1-131 uptake in experiments in which adrenalectomized animals were used and stress, acting through the adrenals, was eliminated from consideration^ however, whatever the un­ explained mechanism of this change may have been, there is no reason at present to conclude that it is important in the normal physiology of the thyroid gland. On the other hand, steroid hormones similar to DCA are normally found in the body* Exhaustive studies on the effect of adrenal hormones of the type involved primarily in the regulation of carbohy­ drate metabolism, especially cortisone, on the thyroid gland have been made. Cortisone has been found by several authors to decrease thyroid 1-131 uptake (Money et al. 1950* Perry, 1951) while having no effect on thyroidal secretion rate of labeled hormone (Albert, Tenney and Lorenz, 1952a). Hormones of the DCA type which influence salt and water metabolism have been little studied. Since the thyroid gland is con­ cerned with concentrating an inorganic ion, iodide, the DCA type hormones could conceivably have a physiologic role in thyroid function* The observation of a decreased thyroid uptake after cortisone and the added information that DCA in­ creased thyroid 1-131 uptake when given either chronically or acutely suggest the possibility of a reciprocal effect of DCA and cortisone in the manufacture of thyroid hormone. This is especially true if DCA, like cortisone, affects only thyroid uptake— and there is no information available on this point. It could logically be anticipated that If this were so, DCA might influence the thyroid mechanism for concentrating inor­ ganic iodine and that cortisone could be affecting rate of or­ ganic binding. normal animals. If such a mechanism exists, it could occur in k9 THE METABOLISM OF THYROXINE IN N O R M L AND THYROIDECTOMIZED DOGS Introduction* In 19il3 Winkler and his associates observed a tolerance to chronic oral dosage with thyroid substance in normal human subjects as compared to patients with myxedema. They found certain non-myxedematous individuals who could tolerate as much as six grains of thyroid substance (U.S.P. dried thyroid) daily for long periods of time with no increase in basal metabolic rate Lavietes (BMR) or pulse rate. Winkler, Criscuolo and (19^3) had observed that patients with true myxede­ ma require from 1 to 3 grains daily to maintain normal met a ­ bolic rates. Such patients usually become nervous and 111 if given more. Winkler and his associates suggested three possible ex­ planations for the difference in response to thyroid substance found in the two groups: (1 ) The normal individual may absorb less of the materi­ al from an oral dose than the athyreotic. (2) The normal individual may be less responsive to the level of circulating hormone. He may require a greater plasma protein-bound iodine level (PBI) to produce a given metabolic increase. (3) The normal individual may inactivate, store, or de­ stroy greater amounts of hormone. The first of these explanations is probably eliminated by the observation of Winkler et al. , in the first publication 5o mentioned, that normal individuals, when compared with myxe­ dematous ones, show a quantitatively diminished response in terms of increased BMR to intravenous thyroxine* and Winkler Riggs, Man (191+5) found that normal and athyreotic subjects showed the same relationship between PBI and BMR; however, when they studied the amount of thyroxine necessary to pro­ duce a given increase in PBI, they found that the euthyroid individuals required considerably more thyroxine to produce a given increase in PBI than did the athyreotic. Thus, it appears that the third hypothesis offered by Winkler et al. is the most likely explanation of the difference in response to thyroid substance of normal and myxedematous human beings. Danowski, Man and Winkler (19i|6) attempted to confirm the foregoing observation in dogs and to determine the mech­ anism of the tolerance In normal animals. They were unable to find any difference in the response of control and thyroidectomized dogs to oral desiccated thyroid or Intravenous thyroxine. They found no difference in the BMR or PBI of the two groups after 0.77 gm. daily of U.S.P. desiccated thyroid per os for a period of five to sixteen weeks or in PBI after 2 mg. of thyroxine daily for 13 to 37 days. Borgman (19^9) saw distinct differences in the response of intact and thyroidectomized dogs to considerably smaller doses of exogenous thyroid substance or thyroprotein (protamone). One mg. of protamone per kg. per day was needed to restore the metabolic rate of thyroidectomized dogs to nor­ mal. Intact dogs tolerated doses of 2, 1+,, and 8 mg. per kg. 51 per day for 30 days without rise in basal metabolic rate. Normal dogs were similarly tolerant to desiccated thyroid. These experiments were planned to determine whether a greater storage, excretion, or destruction of thyroxine would be found in intact dogs than in thyroidectomized ones. A study of the metabolism of I-131-labeled thyroxine was pro­ posed in an attempt to determine whether there is a differ­ ence in its behavior in these two groups. It was decided to set up the experiments in such a fashion that the thyroid­ ectomized dogs would have been pretreated with daily doses of non-radioactive thyroxine for a long enough period so that their basal metabolic rates should be normal and to compare these animals with normal ones which had been pretreated in the same fashion and also with normal ones which had not been pretreated. Since the difference between the two kinds of animal seemed to be revealed when large doses of exogenous thyroidal material were given, it was decided to pretreat the animals with a dose which was equal to the estimated daily thyroid secretion rate and then to determine the fate of the radioactivity after a dose of I-131-labeled thyroxine equivalent to ten times the pretreatment dose. Materials and Methods. Male mongrel dogs obtained through the usual channels were used in these experiments. The descriptions, weights, and estimated ages of these animals are given in table 7 of the appendix. Thanks is extended to Dr. Robert G. Schirmer for his judgment regarding the last observation. All of the dogs, as far as could be determined, were in the time of the experiment. Diet,? The supplemented by good health at dogs were fed on Borden's "Chunx" dogbiscuit Rival canned dog food. Thyroidectomy: Thyroidectomies were performed at least six weeks before the experiments by the technique described by Borgman (19ii9). When the thyroids were removed, at least two parathyroids were carefully teased from the glands and left in place* possible. Their circulation was kept intact whenever If this was not possible, the parathyroid was in­ serted into a neck muscle. Some of the dogs showed tetany after the operation and were maintained temporarily with cal­ cium gluconate in sufficient dose to control symptoms. None of the dogs showed any signs of tetany at the time of the ex­ periments . As evidence of the completeness of thyroidectomy, no accumulation of radioactivity was found in the neck region of any of the thyroidectomized dogs post mortem. Pretreatment with Thyroxine: All of the animals, except one of the groups in experiment 1, were pretreated with Lthyroxine subcut an eously in a daily dose of 6 ugm. per kg. body weight per day. The solution was prepared in 0.9^ NaCl with sufficient NaOH added so that the thyroxine was in solu­ tion. The dose was calculated from the data of Borgman (19i+9) as the minimum amount of thyroxine required to maintain a nor­ mal metabolic rate In thyroidectomized dogs. In experiment 1 , the dogs were pretreated for five days; in experiment 2 , for ten days. 53 l*ligc^qn _of Radioactive Thyroxine: Radioactive L-thy- roxitoj purchased from Abbott Laboratories, was prepared with carrier so that each cc. of solution contained 60 ugm. of Lthyroxine and 12 uc. of 1-131 {experiment 1 ) or 15 or 20 u c • of 1-131 (experiment 3). Sufficient NaOH had been added to put the thyroxine into solution. One cc. per kg. body weight was injected intravenously into the cephalic vein. The injection vein was not used for bleeding purposes for at least twenty-four hours. Blood Samples? At fifteen minutes, thirty minutes, one hour, and steadily increasing intervals until the conclusion of the experiment, blood samples were removed from a cephalic vein of the front leg or a saphenous vein of the hind leg. Each sample totaled approximately four cc. and was drawn into a syringe moistened with heparin. Urine and Feces Samplesr During the first four to six hours of the experiment the dogs were under light nembutal anesthesia. Urine was collected by means of a polyethylene tube which had been inserted into the bladder through the urethra. A urine sample, which consisted of all the urine which had flowed from the catheter during the period, was obtained at the time of each blood collection. The animals were placed in metabolism cages at the end of four to six hours. Prom this time on urine was collected for each period from a container underneath the cage. feces, whenever available, were collected from the wire screen in the bottom of the cage. The Sh The data on total urinary and fecal output result from the following procedures. In the case of the urine, an ali­ quot of known weight was taken from the whole sample for each time period. These aliquots averaged about %00 mg. and were measured on the gramatic balance into the dishes used for counting. The weight of the whole sample was measured on a triple-beam balance. The procedure with the fecal samples was the same except that, prior to the removal of the aliquot, the feces were placed in a Waring Blendor and mixed thor­ oughly with a minimum amount of water. The weight of this mixture was determined by difference on a triple-beam balance. The weight of fecal samples taken for counting also averaged about 500 mg. Total weights of the urine samples from which aliquots were taken ranged from If. to 500 gm.; of fecal samples, from 150 to 750 gm. In the experiment as a whole, ratios of total sample to aliquot ranged from to 750. Certain of the dogs had well formed feces and in ad­ dition tended to be neat housekeepers of their cages. Others either had feces which were less well formed or tended to step on them indiscriminately. Data on urine and feces for only those dogs in which separation was judged to be good have been reported here. Dog No. 16 escaped from his cage the first night of the experiment. External Thyroid Counts: His data are not reported. In experiment 2 external thy­ roid counts were performed using a count rate meter and an end window G-—M tube covered with an aluminum shield. In order to quiet the dogs they were anesthetized with the 55 ultra-short acting barbiturate, sodium symptomatically in intravenous dose of approximately twelve mg. per kg. pentothal,given The rate recorded was the highest which could be obtained when the tube was just touching the neck. A count over the thigh of the animal was used as background rate. Statistics: The curves for the plasma 1-131 levels at different times after radioactive L-thyroxine were analysed by a method which divided them into the sum of several ex­ ponential functions. Points which visually seemed to be in a straight line on a semi-logarithmic scatter diagram of plasma level against time were selected in amanner described later.The equation of such a line is: log y where « log a + bt y = % of dose of I-13l/gm. plasma t s time in hours and a and b are constants. The constants log a and b can be found by formulas from statistics to be: ^ E “T log a = ( £ l o g r ) N where ET s the E n - b(£ t ) I number of points £ ( t x log y) - (£ t ) A - £ t 2 - ( £ t)2 N D - ^log y 2 - (^.log y ) 2 ET (4.log N yl 56 The error in the whole line is cr . The probability is 0.68 that all the points lie withbi±c^e 0f the line log y * log a t b t . = /D - bE V N - 2 The error in the slope of the line isC£. The probabili­ ty is 0.68 that the slope falls within b ± cr^. b a &e v^zr The data in tables 5 and 6, in which these regression lines are reported, are in terms of log10, which was used in their calculation. in terms of loge . Properly these results should be expressed However, since the statistics are equally valid whichever base is used (Baten, 1953 )> l°Sio was em“ ployed for ease in calculation. The means have been con­ verted to loge and the formula for each line expressed at the bottom of the tables in the "exponential form. Experimental and Results. Experiment l : This was a preliminary experiment in which three groups of animals were studiedr two intact dogs, two thyroxine pretreated intact dogs, and three thyroxine pretreated thyroidectomized dogs. Thyroxine pretreated ani­ mals were given a daily subcutaneous dose of 6 ugm. of Lthyroxine per kg. Blood plasma levels and urinary output of radioactivity were studied. A semi-logarithmic plot of per cent of dose of 1-131 per mg. of plasma against time, graph 3* appendix, suggests 57 that m this experiment the blood, levels of activity were lower in tlfce normal animals than in the thyroidectomized ones. The urinary output data on these animals, table 6 of the appendix, show that there was probably no difference in output among the groups up to two days. In the four animals which were studied up to seven days, there was greater uri­ nary output in the thyroidectomized than in the normal dogs. Counts of the thyroid uptake in the intact animals had not been made in this experiment, and it is possible that this difference is represented by uptake of radioactivity by the thyroid gland. This study revealed the necessity for carrying out an experiment in which data were collected until seven days, 168 hours, after injection of thyroxine. It also demon­ strated that, if any Interpretations of the dynamics of the circulatory fall in radioactivity were desired, it would be necessary to have several values between 90 and 172 hours. It seemed that sufficient observations had been made at early times. The preliminary data also revealed the need to deter­ mine thyroid uptake at the conclusion of the experiment. It also suggested the advisability of developing a satisfactory method for counting fecal output so that it too could be measured. Examination of blood curves and urinary output data showed no apparent difference between the control animals which had been pretreated with thyroxine and those which had 58 not* It was decided to include only two groups in the next experiment: Intact dogs and thyroidectomized dogs, each of which had received pretreatment with thyroxine. Since radio­ active thyroxine by then was being made on a regular schedule by the Abbott Laboratories and could be obtained within a known length of time, it was decided to pretreat the animals with thyroxine for a slightly longer time which finally re­ sulted in a ten day pretreatment period in place of the five days used in experiment 1 . Experiment 2 Z This experiment was performed in order to evaluate the need for considering thyroidal recirculation of 1-131 in the normal dogs. Three dogs were given approxi­ mately 100 microcuries of carrier-free 1-131 subcutaneously and counts over the thyroid gland were begun on the third day. Albert (1951) used a similar method for determining the biological decay of thyroidal radioiodine in rats. The m o ­ bility of the skin and musculature of the d o g ’s neck seems to make external counting more variable In dogs than it is in other species such as the rat. These data did not show a linear trend in a semi-logarithmic plot until the seventh day after 1-131. Counts were then followed without further treatment of the dogs up until the fourteenth day; on the fourteenth day an intravenous dose of 60 ugm. of L-thyroxine per kilogram was given to the dogs and counts were continued until the tv/enty-second day, at which time the experiment had to be abandoned because the counts were too low to be measured. A semi-logarithmic plot of counts per second against time is 59 recorded in graph 2p of the appendix* These counts have been corrected for p h y sical decay and have been adjusted so that the count of each dog on the seventh day is recorded as 100 per cent* These dogs have an estimated half time for thy­ roidal radioactivity of eight days* It was hoped that the thyroid secretion would have been completely suppressed by the 60 microgram dose of L-thyroxine for the period of seven days during which the experiments were performed* This was not unreasonable since the dose represented ten estimated daily secretion rates. The individual observations were too variable to permit an estimate of the length of time that thyroid secretion was suppressed in each dog individually but a plot of the mean values of all three dogs— these are average logarithms— suggests that secretion was inhibited for at least three days. Experiment 3 * Two groups of animals were studied here: six normal dogs and six thyroidectomized dogs. Both groups were pretreated for ten days with a daily subcutaneous dose of 6 ugm. of L-thyroxine per kg. Blood levels, urinary out­ put and fecal output of radioactivity were studied for a period of approximately 172 hours. Thyroid uptake was mea­ sured in the intact dogs at the conclusion of the experiment. The total output of radioactivity in feces and urine is recorded In tables 3 und Ip. for the intact dogs. Thyroid uptake is also Included These data are only for those animals in which urine and feces were judged to be well separated. Table 3: Dog No. Total urinary and fecal output and thyroid u p ­ take of 1-131 in normal dogs after radioactive L-thyroxine• Experiment 3* Urinary 1-131 Output % of Dose Fecal 1-131 Output % of Dose Thyroicl 1-131 Uptake % of Dose Total Accountable 1-131 % of Dose 3 14-3.1 59.7 3*6 105.9 10 3k- 5 62 .8 8.6 105.9 12 37.6 3^.3 6.1 78.0 56.3 J+2.8 U-2.9 24-9.9 23 13 103.3 20 Average 5.6 98.3 61 Table 1+: Total urinary and fecal output of 1-131 in thyroid ectomized dogs after radioactive L-thyroxine. Experiment 3* Dog No, Urinary 1-131 Output % of Dose Fecal 1-131 Output % of Dose 1+6.5 36.O Total Accountable 1-131 % of Dose 2 1+ 16 — — 82.5 — 17 31.1 87.5 1 58-5 112.9 15 Average — k 5 .k -14-8.9 -91+. 3 62 The method of analysis of the blood curves in experiment 3 is outlined in graphs 6 and 7 and the results obtained in tables 5 and 6* The third exponential function was the first to be determined. A semi-logarithmic plot of the plasma 1-131 levels against time was examined for each dog. As many points at the end of the curve as appeared to be in a straight line were selected by visual inspection. The best semi-logarithmic straight line through them was determined by the method of least squares. A similar procedure was used to obtain the other two regressions. cess was carried out. Graphs 6 and 7 indicate how this pro­ The method of analysis used is a stan­ dard one for this kind of data well described by Jones (1950). Tables 3 an<^ U- indicate that there was no difference seen between total fecal output of radioactivity in these dogs. Likewise the sum of the thyroid uptake and urinary output of the intact dogs was equivalent to the urinary output of the thyroidectomized dogs. Statistical analysis by means of a nt n test of the data regarding blood levels of radioactivity showed no difference between the groups in log a^, b^, log a^, or b^. dogs the mean of log ap was significantly less In intact than thyroidectomized dog^ and in the intact dogs the mean of b 2 was significantly more than in the thyroidectomized. Discussion of Results. To interpret the results in experiment 3, it is necessary to consider some of the things which are known about the metab olism of thyroxine. -P o> O' G ** O © cp •H G o o ♦ o 00 ^ © Gw bOG t © P G rH • 0 -0 G c\]© o G »o o ^ rG -P • C\J ii o o r— 10 © © pm 4J3 G ii O b0 O rH © Cd ^ O -P G CO O Cti 5 ■G *P O C ,G © © O © G o -p rG WO -P G G © •P £ hO O rH *H £ G ©

© *H h|n p co co in *H A •H © A © £ C *H C O u 2 o K p P± 1A cA O♦ a O A o I vD cd cd A *H i— IP p a o A o OA C O o C O^ p © a © £ co cd Sh A to a © © o C Ou o 0 © P co A to o p P A *h cd C\J vD O CA CA « O rH I O o O CO P A A © p fi *P A h?. -1.294 - 1.328 -1.1+71 -1.512 - 1 .1+31 -1.107 -0.299 -0.508 -1.12k -O .136 -0.1+58 -0.328 0.070 0.025 O.I 36 0.052 0 .0I4.6 0.251 0.262 0.195 0.017 0.0057 0.013 1.0 0.59 0.27 2.2 0.66 0.92 -1.357 -O.I+76 log a . First Exponential 3 10 12 23 13 20 4 3 3 4 1+ k Mean O.OOlIi. O. 9I4- Second Exponential 3 10 12 23 13 20 hr 3 5 3 6 Mean - 1 .21+97 -1.1193 -1 .21+30 -1.2217 -1.1+11+7 -1.1363 -0.0311 -0.0557 -0.03556 -0.01+68 -0.0250 -0.0302 -1.2308 -0 .0371+ 0.081+0 0.00866 0.0201 0.0325 0.0568 0.0193 0 .0031+ 9.7 0.00058 5.4 8.5 0.00057 6.4 0.0019 0.0011 12.0 O.OOO 63 10.0 8.7 Third Exponential 3 10 12 23 13 20 Mean 7 8 7 10 5 5 - 1.9689 -1.8520 - 2 .IO 63 -1.71+02 -2.3066 -2.2221 -0 .001+53 -0 .00I+70 - 0 .001+63 -0.00397 -0.00393 - 0.003172 -2.0327 -0.004155 y = 0.01+4 e- 1 *1^ +■ 0.059 0.0575 0.0311 0.0228 0 .0321+ 0.0356 0.0310 O.OOO 63 0.00028 0.00027 0.00008 0.00053 0.00047 66 64 62 76 67 77 68.7 e-°*o86t 0.0093 e-°*0°96t Table 6. Dog No. Analysis of blood, levels of radioactivity of thy — roidectomized dogs* Experiment 3* N b cr e -1.337 -I.l4.3lj-I.I4.88 -1.288 -1.593 -1.301 -0 .14714.! -0.352 - 0 .2 2 6 -0.313 -0.299 -0.31+.7 0.053 0.062 0.075 0.031 0 .224.1 0.019 -1. lj.07 -0.335' Xo r a ^b ti hr. First Exponential 2 k 16 17 1 1? k k 5 k 1*b Mean 0 *0 3 9 0 .0 2 1 0 .0 3 2 O.O 2I4. 0 .1 8 0 o .o lj5 0 . 6 Ll 0 .8 6 1.3 0 .9 6 1 .0 0 . 8? 0.9b Second Exponential 2 5 b 14- 16 17 1 15 3 3 24 b Mean -1.3027 -1.0626 -1.3918 -1.3817 - I . 5 0 3 IJ. -1.3&14-7 - I .33245 -0.0333 -O.O 35I4-0.0311 -0.0282 - 0.0269 -0.0392 0 .0 6 8 6 0.0795 0.0559 0.0333 0.0206 0.0318 0.0019 0 .0 0 3 3 9 .0 8.? 0.00070 9 .7 0 .0 0 1 3 0 .0007? O.OOli*. 1 0 .2 11.2 7.7 -0.0323 9 .k Third Exponential 2 k 16 17 1 1? Mean 7 6 8 8 7 9 -1.8902 -2.006? -2.2003 -1.9007 - 2.2032 -1.9212 -0.00?ljJL -0.00377 -0.00?12 -0.0032!j. 0.0302 -o .o o ? ? k 0.0671 0. 03?6 0. 032k 0. 03?6 0.00031 0.00093 0.00037 0.00033 0.00037 ?6 72 ?9 93 -O.OO 678O 0.0269 0.00022 L}1|. -2.0203 -0.00lj.978 5b 63.0 y = 0.039 e-°'77t +- 0.0t|.6 e“0»°7!4-t +. 0.0095 e“°*01l6t 67 Gross and Leblond (1950) were the first to study the metabolism of physiological doses of thyroxine. They studied doses of 0.007 ugm. of 1-131 labeled L-thyroxine, 0.07 ugm. and 20 ugm. in 80 to 120 gm* rats. At 2l\. and 72 hours they found most of the injected dose in the urine and feces. They showed that with increasing dose of thyroxine, the per cent of dose excreted in the feces increased. Urinary excretion of radioactivity after L-thyroxine is chiefly inorganic in form although small amounts of organi­ cally bound iodine may be present. After administration of carrier-free iodide to myxedematous humans maintained at euthyroid level, Albert and Keating (19J+9) found 80 per cent of the dose in the urine and only 0.8 per cent in feces. By butanol extraction Myant and Pochin (1950) found an average of 9 per cent (5*7 “ 23 .8 ) of the urine of five human subjects, after radioactive L-thyroxine, to be due to thyroxine-like material. The fecal excretion of radioactivity after injection of labeled L-thyroxine is largely organically bound. After carrier-free 1-131 Albert and Keating (19^9) found 0.8 per cent in feces at 72 hours, after L-thyroxine, 23 per cent. Taurog, Briggs and Chaikoff (1951, 1952) found two organic forms of iodine in bile, thyroxine itself and a thyroxine containing compound which they suggested is a glucuronide of thyroxine. Radioactive L—thyroxine is absorbed from the gastro-intestinal tract but not completely so (Albert et al* 1952b). The plasma radioactivity after radioactive L-thyroxine has been found to be chiefly organic in form, Myant and Pochin (1950), estimating from known renal iodide -131 clear­ ance, calculated that a mean of less than ten per cent of plasma radioactivity, at 1 , 6 , and 211 hours after a physiolo­ gic dose of radioactive L-thyroxine, was in the form of io­ dide ♦ Albert and Keating (1952) studied the metabolism of radioactive L-thyroxine in rats by a method similar to the one used here except that each time period was represented by a group of rats. rats. They gave 0.6 ugm. L-thyroxine to 35-55 gm. They obtained three regression lines by analysing the blood concentration of radioactivity in a manner similar to that used here with half-times of O .36 hr., 15 h r s ., and 62 hrs. They believed that the first one represented equili­ bration of the radioactivity with a larger volume of dilution almost entirely within the gastro-intestinal tract, that the second represented disappearance of the radio-thyroxine from its initial volume of dilution into the excreta, as well as into tissues, and the third indicates that 1-131 is leaving blood by some other route than urine and feces into some larger equilibrium volume within the body. O'Neal (1953) studied plasma PBI in thyroidectomized dogs after injection of 500 ugm* and 5000 ugm. of thyroxine. By a method of analysis similar to that used here they found two regressions with half-times of 0.97, 2.15, 1*15 an<^ 27*7, 18 .3 , and 27*7 hours after 500 ugm. They found an increased 69 rate of disposal aftqr 5000 ugm, so that at 50 hrs. 300 ug. was left as compared to 113 ug, after 500 ug, dose. A comparison of Albert and KeatingTs results in rats with the data obtained in this work shows a good agreement in the half times obtained: ti in Hours Rats O'•, 69 Thyroidx 0 62 8.7 Dogs 1 15 ___ Normal 0.9U 1 0.36 ^ 9-k 63 The first half-time obtained by 0 TNeal was similar to the one obtained here in dogs. The second was in accord with neither of the other half-times obtained here; it is suggested that this may have been because of their relatively large dose (500 u g . ). Since fecal output after radioactive thyroxine is known to be organic in form and is the only organic output of great quantitative significance, it was believed that if a greater output of fecal activity was found in the intact than in the thyroidectomized animals it could be taken to indicate that the intact animal excretes a greater amount of each dose of thyroxine as such than does the thyroidec tomi zed animal. As can be seen by examination of tables 3 and 1}., this hypothesis was not realized. As far as can be told from these data, if a difference between the two groups exists it is not in the output of the radioactive thyroxin© as such. 70 The control data on thyroid, output of* radioactivity J in 4 experiment 2 showed that the recirculation of iodine -3 31 in the intact dogs was at most a small per cent of dose. The maximum uptake of the thyroid gland in these dogs was ten per cent. If it is assumed that thyroid output was inhibited for three days and then proceeded at a rate such as to have a halftime of eight d ays, the maximum recirculation of radioactivity would have been 30 per cent of that present or a maximum of three per cent of dose. Although there is a statistically significant difference between normal and thyroidec tomi zed dogs in respect to log and b^, the final levels of radioactivity in the blood of the two groups are the same. Thus It would appear that the over-all result of giving radioactive L-thyroxine to these dogs has been about the same in the two groups. There Is nothing in these data which could account for the approximately two-fold difference in response to thyroid substance seen by Winkler and his co-workers (19^3) in man or to thyroid substance and protamone by Borgman (19lp9) in dogs. Although Winkler et al. (191*3) found a lessened response to intravenous thyroxine In euthyroid subjects as compared to athyreotic they state that this effect was not as marked as the one seen after thyroid substance; therefore, it may be that the tolerance of normal individuals Is a phenomenon which occurs only after oral thyroid substance or thyroprotein and not after intravenous thyroxine. 71 CONCLUSIONS The £111*66 hour thyroid uptake of 1—131 was increased in adrenalec tomi zed rats after 1*25 mg. of desoxycorticosterone acetate per 100 gm. body weight given intraperitoneally im­ mediately after intravenous injection of 20 uc. of 1-131 with 8.5 ugm* of carrier 1-127 Nal. The plasma level and the urinary output of radioactivity were not changed by the treat­ ment. The one and one-half hour and three hour uptake of 1-131 in adrenalectomized rats were increased after 10 cc. per 100 gm* body weight of 0 , 9 % NaCl given under the same conditions* In this case the blood level of radioactivity was decreased, and the urinary output of radioactivity was Increased. The arterio-venous 1-131 difference for the thyroid gland of the rat was measured and found to be 10%> under the conditions of the experiment. The estimated absolute thyroid circulation in the rat was calculated to be 23 mg. of blood per mg. of gland per minut e . A comparison was made of the metabolism of 60 ugm* 1-131 labeled L-thyroxine in normal and thyroidectomized dogs after ten days pretreatment with 6 ugm. per day of non-radioactive thyroxine. No difference was seen In total fecal output of radioactivity up to seven days after injection of the labeled thyroxine. Combined thyroidal-urinary 1-131 output of normal dogs equalled urinary output of thyroidectomized dogs. The plasma levels of radioactivity of both groups were described 12 by the sum of three semi-logarithmic regression lines having half-times of 0.9ll> 8*7> and 69 hours in intact dogs and 9*14., and 63 hours in thyroidectomized dogs; 8.7 hours was statistically significantly less than 9*k- hours. Appendix Table 1: Day Factors for correction for the physical decay of 1-131, based on an 8,00 day half-life. Hour Correction -t k 8 12 16 20 1 28 32 36 1.02 1.03 l.Olj. 1.06 1.08 - 1.09 1.11 1.12 1.1k 1 .1 5 1.17 2 3 lL +t 0*878 0 . 866 0.853 1 .1 9 0.8kl 52 1.21 1.22 1.2k 1.26 0.829 O .817 1.28 6 0.806 0 .7 9 k 1.30 0.782 0.771 76 1.32 6.760 80 8k 88 92 96 1.3k 1.35 1.38 1.39 l.ki 7 0.7k9 0 .73 9 0.725 0.717 0.707 1 Hour Correction -t 0 .98 5 0.971 0.958 o .9 kk 0.930 0.917 0 .9 0 k 0.891 hB 56 60 6k 68 72 Day 8 +t Too "T.k3 ' 6.6 f T 10k l.k6 0.687 108 l.lj.8 0.677 0.668 1.50 112 116 0.658 1.52 120 0.6k9 b5h l2k ' 1.56 6.639 128 1.58 0.631 1.61 132 0.621 1.63 136 0.612 1.66 O .603 II4.O 1.68 o.5?k . ikk o.55T~ ll+F 1.71 152 0.578 1 .7 3 156 0.570 1 .7 5 1.78 160 0.561 16k 1.81 0.553 168 1.83 o.5k|_ . . '1.56" 0.538 1J 2 0.530 1.89 176 180 0.521 1.92 1.9k 0.515 !8k 188 0.507 1.97 0.500 2.00 192 Appendix Table 2: Factors for correction for beta self-absorption, based on data using I-131-labeled rat liver. mg. 20 25 30 35 ko k5 5o 55 60 65 70 75 80 85 90 95 100 105 110 115 120 130 iko i5o 160 170 180 190 200 mg. /cm.2 Correction .5.3 6.6 8.0 9.3 10.6 11.9 13-3 lk. 6 15.9 17.3 17.6 19.9 21.2 22.6 23.9 25-2 26.5 27.9 29.2 30.5 31.9 3k. 5 37.2 39.8 k2.5 k5.i k6.2 5o.k 53.1 1.06 1.08 1.10 1.11 1.13 1.15 1.17 1.19 1.21 1.22 1.2k 1.27 1.29 1.31 1.33 1*35 1.37 1.39 l.kl i:$ 1.51 1.56 1.61 1.65 1.70 l.7k 1.78 1.82 Appendix Table 3: Rat No. 1 2 3 k 5 6 7 8 9 10 11 12 ik 15 16 Body weights, thyroid weights, and thyroid 1-131 uptakes of control rats. Experiment 5* Time of Sacrifice Minutes 6 6 6 30 30 3° 60 68 66 95 97 133 120 126 Body Weight gm. kok kk2 516 k°o ki8 k68 3k0 392 kk2 371 k9k ki6 382 362 k7k Thyroid Weight mg. 21.5 25.2 30.2 2k. k Thyroid Uptake % of Dose 0.11 0.17 0.18 0.91 23.2 27.8 17.6 25.9 23.2 18.2 23.9 23.2 0.58 0.85 0.59 1.25 O .83 0.99 1.60 I .83 18.5 21.5 23.3 2.13 1.55 2.61 Appendix Table Ip Body weights, thyroid weights and thyroid I uptakes of* saline-treated rats, Experiment 1'ime Hat No. of Sacrifice Minutes 1 2 6 6 6 30 3 4 5 Body Weight gm. ^yroid Weight mg. ^Thyroid Uptake % of Dose 22.0 23.1 0.18 0.07 0.15 O.I16 0 .08 358 30.7 19.1+ 22.5 19*1+ 23.1 kok 26.1 1.65 1.07 1+.02 1+52 4-8U3b o k id 8 30 30 75 58 10 69 388 20.3 1.51 12 102 100 132 18.6 1.98 k28 Ik. 6 382 16.8 117 356 lj.86 19.5 35*2 1*37 1.77 1*1+7 1.32 6 7 13 34 15 16 128 14.68 0.71 Appendix Table Arterial, venous and thyroid venous blood levels of radioactivity of control and saline-treated rats. Experiment 5« Control rats have been listed first. Rat ■No. 7 8 15 16 7 8 13 1& 16 ^Fime of Sacrifice Minutes 60 68 133 120 126 75 58 100 132 128 Systemic Arterial 1-131 ^bose/mg. Systemic Venous 1-131 $bose/mg. 'fhyroid Venous 1-131 ^Dose/mg 0.000598 0.000^89 0.00071)9 0.0005l).6 0.000521 0.000502 0.000340 0.000500 0.000529 0.000502 0.000581 0.000517 Q.000317 0.000371 0.000li.73 0.000306 0.000313 0.000216 0.000389 O.OOO 464 0.000319 0.000215 0.000333 O.OOOij.30 0.000216 0.000261). 0.000209 Appendix Table 6t . Urinary 1-131 output, intact control dogs (ij. and 7 ) , intact thyroxine-pretreated dogs (5 and 8 ) and thyroidectomized thyroxine-pretreated dogs (3 . 6 , and 9). Experiment 1 . Day Controls 7 k 0-1 1-2 28.6 1 *2 2-3 0-7 0-2 (Mean) 8 5 21.2 12.8 35.8 32.2 5k. 1 3k. 0 3 15.6 16.1 16.2 10 •0 17.2 2.19 O. 824.3 3~k k-7 0-2 k.05 28.1 Thyroidectomized 6.7 2.?1 1.76 3k. 0 31.9 ^6.If. 33.0 26 . 24. 6 9 16.8 19.1 12.6 6.13 1.95 23.5 18.1 9.35 6.31 3.32 35.8 ija.6 60.5 67.2 3k* 6 Appendix Table 7 : Weights, descriptions and estimated ages of paired dogs. Experiment 3 . Dog No. Weight kg. Description Age Intact Thyroidx 8.3 9.2 Tan and white Brown 1 yr. 2 yr. 7*3 6 .Ip Black, tan, white Black and tan 2 yr. k Intact Thyroidx 12 16 Intact Thyroidx 9.3 8.3 Black Brown and white 2| yr. 7 mo • 23 Intact Thyroidx 8.3 9.0 Black and white Black 1 yr. It yr. Intact Thyroidx 13.9 Black Black li yr. L|_—6 yr. Tan Brown 8 mo. 1 yr. 3 2 10 17 13 1 20 15 Intact Thyroidx 13.6 5.9 8.8 k yr. Appendix o I o f-i rH o 62 o 00 o 1 5k 11 1 0 0 0 1° Weight In mg. 00 Plasma Dry 0! 1 <2} ! ;o 1 38 O 1 1 1 1 1 1 t O CP 0 0 O c 0 0 0 0 0 0 <9tS 8 o o O O O O o o o! o 30 k*5 0 0 5-0 5-38 6-0 Beta Count of Sample/Q.arniria Count of Sample Graph 1: Scatter diagram of beta-gamma counting of rat plasmas labeled with 1-131Beta counts were corrected for self­ absorption. Appendix 18 _ . 6 o rH 1 16 in o *r~i CO cti P hO CA -P o OO O O o CT'CO O - o lA O Abq qqxxg jo quoQ taO O rH I—I -P I—I < i M Sh cd Q o "LA CO IA OJ vO Ipr CO Thyroid output of 1-131 in dogs. Experiment 2. 1-131 levels in thyroid gland have been adjusted so that amount present on sixth day equals 100 per cent. CO Graph o o rH CA l£l“I P T O ^ T4i REFERENCES 1. Albert, A. 1951* The In Vivo Determination of the Bio­ logical Decay of TKyroidal Radioiodine. Endocrin- QlQSZ Wt 331-338* Thyroid Gland. ------ 2. A!bert,^A ^ ^ 1952. Ann. Rev. Physiol, lip: 3* Albert, A. and F. 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