CD EXCHANGE BIOLOGY LIBRARY TEbe mnfx>et*tt£ ot STUDY OF THE DISTRIBUTION OF IODINE BETWEEN CELLS AND COL- LOID IN THE THYROID GLAND A DISSERTATION SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL OF SCIENCE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGICAL CHEMISTRY AND PHARMACOLOGY BY HARRY BENJAMIN VAN DYKE Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from 'he JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XLV, No. 2, 1921 ; Vol. LIV, No. i, 1922 The AMERICAN JOURNAL OF PHYSIOLOGY, Vol. LVI, No. i, 1921 tlntversitg'of 'Gbicago A STUDY OF THE DISTRIBUTION OF IODINE BETWEEN CELLS AND COL- LOID IN THE THYROID GLAND A DISSERTATION SUBMITTED TO THE FACULTY OF THE OGDEN GRADUATE SCHOOL OF SCIENCE IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHYSIOLOGICAL CHEMISTRY AND PHARMACOLOGY BY HARRY BENJAMIN VAN DYKE Private Edition, Distributed By THE UNIVERSITY OF CHICAGO LIBRARIES CHICAGO, ILLINOIS Reprinted from The JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. XLV, No. 2, 1921; Vol. LIV, No. i, 1922 The AMERICAN JOURNAL OF PHYSIOLOGY, Vol. LVI, No. i, 1921 BfOLOGY LIBRARY G CXCHANGE . ; & Reprinted from THE JOURNAL OF BIOLOGICAL CHEZJ^TRY, Vol. XLV, No. 2, 1921 A STUDY OF THE DISTRIBUTION OF IODINE BETWEEN CELLS AND COLLOID IN THE THYROID GLAND. II. RESULTS OF STUDY OF DOG AND HUMAN THYROID GLANDS. BY HARRY BENJAMIN VAN DYKE. (From the Laboratory of Physiological Chemistry and Pharmacology, . University of Chicago, Chicago.} (Received for publication, November 29, 1920.) In the first paper of this series Tatum1 described a method whereby thyroid cells may be separated from colloid material and examined chemically. Briefly the method consists in cutting frozen sections of the thyroid gland and floating these sections on Ringer's2 solution. The colloid material immediately drops out of the acini and is presumably dissolved in the Ringer's solu- tion. The cells may then be separated by centrifugalization, dried, weighed, and analyzed. Comparisons between the iodine content of cells so separated and the iodine content of control pieces of unsectioned whole gland may indicate the distribution of iodine between cells and colloid under different functional conditions. In this paper I wish to report the results of a study, suggested by Dr. A. L. Tatum, of the distribution of iodine in the thyroid glands of normal and iodine-fed dogs as well as in human glands obtained from individuals subjected to operation for toxic goiter. The method earlier described by Kendall3 was used in making the final iodine determinations. Incidental to the determination of the iodine distribution in dog and human thyroid glands, some control experiments were performed relative to the alterability of the intracellular iodine concentration during the process of cutting. It may be argued that a portion of the iodine-containing compound diffuses from 1 Tatum, A. L., J. BioL Chem., 1920, xlii, 47. 2 Campbell, J. A., Quart. J. Exp. Physiol., 1911, iv, 1, Formula "A." 3 Kendall, E. C., J. BioL Chem., 1914, xix, 251. 325 THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XLV NO. 2 520421 326 Distribution of Ibdme -In. Thyroid Gland. II cells as they lie suspended in Ringer's solution during the sepa- ration of the colloid material. If it is assumed that such an outward diffusion takes place, it seems reasonable to expect that the iodine compound should diffuse back into the cells if we should increase the concentration of that iodine compound in the Ringer's solution in which the cells are suspended. Several experiments like those given in Table I were undertaken to determine whether or not the iodine content of the cells could thus be increased. TABLE I. Effect of Floating Cells on Pure Ringer's Solution and on Ringer's Solution on Which Cells of an Iodine-Rich Gland Had Been Floated. Animal No. Weight of whole gland used. Iodine in whole gland. Weight of cell mass used. Iodine in cell mass. Ratio of percentage of iodine in cells to percentage of iodine in whole gland. Remarks. mg. percent mg. percent 14 146.0 0.187 40.5 0.017 0.091 Cells floated on pure Ring- er's solution. 53.0 0.017 0.091 Cells floated on Ringer's solution on which previ- ously cells of iodine-rich gland had been floated. 20 287.2 0.030 161.5 0.005 0.167 Cells floated on pure Ring- er's solution. 151.0 0.006 0.20 Cells floated on Ringer's solution on which previ- ously cells of iodine-rich gland had been floated. It may be seen from the data given in Table I that no increase in the iodine content of cells could be brought about by increas- ing the concentration of the characteristic iodine compound in the Ringer's solution. Again the iodine concentration of the cell mass does not seem to be in the least altered whether or not the freshly centrifugalizecl cell mass is washed several times with iodine-free Ringer's solution. Moreover, in the attempt to find a suspending medium more nearly related physicochemically with thyroid cells than is Rin- ger's solution, I have used fresh dog serum and have not found the iodine content of the cells measurably different from that of control cells suspended in Ringer's solution. H. B. van Dyke 327 Finally autolysis does not seem to be much of a factor in the loss — if any occurs — of iodine compounds from cells as they lie in contact with Ringer's solution. Throughout the process of cutting and separating the cells, the reaction of the suspending medium should remain weakly alkaline — a reaction which has been shown to be unfavorable to autolysis.4 Also in experiments to be reported later in which the same technique was used under slightly different conditions, every effort was made to eliminate autolysis by cutting successively small portions of the gland and floating the cells on Ringer's solution cooled by ice. The cells from the small portions cut successively were at once centrifu- galized and dried. No change could be noticed in the iodine distribution. Results of Study of Dog Glands Taken at Random. TABLE II. Quantitative Determination of Iodine in Whole Gland and in Cells Free from Colloid Material of Thyroid Glands of Normal Dogs. Animal No. Weight of whole gland used. Iodine in whole gland. Weight of cell mass used. Iodine in cell mass. Ratio of percentage of iodine in cells to percentage of iodine in Morphology. Cells. Colloid. whole gland. mg. per cent mg. per cent 1 5 550.0 0.25 154.0 0.031 0.124 Flat. Fair. 6 388.0 0.175 265.0 0.027 0.154 ft Rich. 10 480.0 0.017 342.0 0.003 0.176 Cuboidal. Poor. 11 505.5 0.032 197.5 0.005 0.156 u " 12* 278.7 0.006 286.5 13 689.0 0.234 154.0 0.047 0.201 Flat. Fair. 14 146.0 0.187 40.5 0.017 0.091 u ft 19 474.5 0.024 116.0 " Poor. 20* 287.2 0.030 161.5 0.005 0.167 21* 604.7 0.038 189.0 0.006 0.158 23 458.2 0.003 390.4 Cuboidal. Poor. 24 318.7 0.021 295.4 " " 31 513.8 0.055 205.6 0.008 0.145 tt " 32* 235.2 0.179 103.9 0.037 0.207 33 289.3 0.011 538.3 0.002 0.182 Flat. Poor. * No histological examination. 4 Bradley, H. C., J. BioL Chem., 1915, xxii, 113. Bradley, H. C., and Taylor, J., /. BioL Chem., 1916, xxv, 261. 328 Distribution of Iodine in Thyroid Gland. II From the results given in Table II one sees that the ratio of iodine concentration in cells to iodine concentration in whole gland (and hence the ratio of iodine concentration in cells to colloid-iodine concentration) has a quite constant value. In some glands (Nos. 12, 19, 23, and 24), to be sure, the iodine con- tent of the cell mass analyzed was so low that no ratio value could be obtained. But in general, despite great variations in the iodine content and morphology of the glands analyzed, the ratio values change relatively little. These findings in the dog's thyroid gland are similar to those of Tatum1 in the beef, pig, and sheep thyroid glands. A comparison of the ratio values in dif- ferent animals is given below. Effect of Feeding Iodine. It was thought that perhaps ratio changes could be induced by the administration of iodine or iodine compounds. Capsules containing 1 gm. of potassium iodide or 2 drops of tincture of iodine in starch were fed over varying periods of time. No more than one capsule was given in 24 hours. The results of the examination of the glands of animals so treated are given in Table III. The ratio obtained from analyzing the cells and whole gland of the thyroid glands of animals which had received varying amounts of iodine or iodine compounds over periods of no less than 3 days is practically the same as that of the animals which had received no iodine. One gland (that of No. 16) gave an unusually high value which I cannot explain. In view of the variations in mor- phology and iodine content of the glands of the normal series together with the quite constant ratio exhibited by that series, it is not surprising that the ratio still remains constant despite the feeding and consequent absorption of iodine. From a con- sideration of the glands examined it appears that the ratio is not altered by the feeding of iodine as potassium iodide or free iodine over periods of time ranging from 3 days to 3 weeks; and yet the total iodine content of these glands undoubtedly is greatly increased during that same period. H. B. van Dyke 329 TABLE III. Quantitative Determination of Iodine in Whole Gland and in Cells Free from Colloid Material of Thyroid Glands of Iodine-Fed Dogs. Period of feeding. Ratio of percent- !Ani- mal No. Form of iodine. Daily. On alter- nate Total. Weight of whole gland used. Iodine in whole gland. Weight of cell mass used. Iodine in cell mass. age of iodine in cells to percent- age of iodine in days.* whole gland. days days days mg. per cent mg. per cent 2 KI 14 0 14 98.2 0.31 77.7 0.045 0.145 7 KI 14 7 21 147.0 0.12 113.0 0.031 0.258 8 KI 14 7 21 124.5 0.87 125.0 0.080 0.092 9 KI 14 2 16 353.5 0.251 116.0 0.040 0.159 16 KI 12 0 12 651.4 0.319 316.1 0.117 0.367 17 KI Tincture of I. 12 12 0 0 24 646.5 0.617 302.3 0.083 0.134 27 (( it T 3 0 3 556.1 0.302 332.8 0.046 0.152 29 l( (( T 3 0 3 195.2 0.314 158.6 0.039 0.124 30 l< (( T 3 0 3 163.3 0.357 127.5 0.041 0.115 After feeding daily. Human Glands. The distribution ratio of iodine between cells and colloid mate- rial was determined in thirteen human glands obtained from operative cases.5 In Table IV are given the results of the anal- yses of the human glands together with the clinical diagnosis made in connection with ten of the cases. It will be seen that most of the thyroid glands reported were clinically diagnosed as toxic goiters. Considerable variations in the ratio value occur and do not appear to be related either to the iodine content of the gland or to its morphology. The ratio value of No. 22 is inexplicably high. In the human gland series, however, as in other gland series previously reported, the ratio variations are of a much smaller magnitude than the varia- tions in total iodine content. So here too the ratio is fairly constant despite variations in morphology and iodine content. 6 Through the courtesy of Dr. C. B. Davis and Dr. A. D. Bevan of the Presbyterian Hospital, Chicago, and of Dr. A. J. Ochsner of Augustana Hospital, Chicago. 330 Distribution of Iodine in Thyroid Gland. II TABLE IV. Quantitative Determination of Iodine in Whole Gland and in Cells Free from Colloid Material of Human Thyroid Glands. Series No. Weight of whole gland used. b *0 X! 1 a 'i! •g-a Weight of cell mass used. 1 a II 3s Ratio of percent- age of iodine in cells to per- centage of io- dine in whole gland. Morphology. Diagnosis. Cells. Colloid. mg. per cent mg. per cent 22 284.7 0.081 260.8 0.049 0.605 Cuboidal. Poor. Colloid cystic goiter with toxic symp- toms. 25* 600.2 0.274 308.6 0.105 0.383 Flat. Rich. Mild exophthal- 411.0 0.276 240.7 0.098 0.355 mic goiter. 35 671.8 0.286 179.2 0.051 0.178 Flat. Rich. Toxic thyroid. 36 439.7 0.158 219.9 0.020 0.127 Cuboidal. Fair. " goiter. 37f 667.5 0.092 258.4 0.027 0.293 Colloid goiter. 38 578.6 0.079 191.7 Trace. Cuboidal. Poor. « « following par- enchymatous hyperplasiaof exophthalmic goiter. 39f 516.0 0.279 297.6 0.095 0.341 40 469.2 0.345 225.5 0.066 0.191 Flat. Rich. Exophthalmic goiter. 41 559.2 0.280 187.0 0.067 0.239 (i « 48* 549.9 0.043 312.4 0.005 0.116 Cuboidal. Poor. 607.3 0.044 311.3 0.005 0.114 49* 590.7 0.137 256.0 0.026 0.189 Cuboidal. Fair. Exophthalmic 204.5 0.027 0.197 goiter. 51 731.4 0.283 294.4 0.072 0.254 Flat. Rich. « « 60 370.0 0.152 228.0 0.015 0.099 it « Cystic goiter with toxic symptoms. * Duplicate determinations made, t No histological examination. H. B. van Dyke 331 Comparison of Ratio Value in Different Animals. A comparison of the ratio values of the thyroid glands of the different animals so far examined is given in Table V. The few abnormally high and unexplained ratio values are not included in the table. From what data are available there appear to be some differ- ences in the thyroid glands of different animals in the numerical value of the ratio of the percentage of iodine in cells to that in whole gland. The ratio value of iodine distribution for dog thyroid glands seems to be consistently lower and more constant than that for the thyroid glands of the other animals studied. TABLE v. A Comparison of the Value of the Ratio of the Percentage of Iodine in Cells Free from Colloid Material to the Percentage of Iodine in Whole Gland in Different Animals. Animal. Extremes of iodine content of whole gland. Ratio values. Extremes. Mean. Average. Beef* ... per cent 0.023-0.468 0.011-0.870 0.043-0.345 0.377-0.810 0.089-0.442 0.21 -0.48 0.091-0.258 0.099-0.384 0.20 -0.34 0.23 -0.41 0.35 0.175 0.242 0.27 0.32 0.36 0.154 0.22 0.27 0.33 Doe Man Pig*f Sheep* * Tatum.1 t Only two glands analyzed. SUMMAKY. 1. The method described by Tatum1 was used to determine the ratio of the percentage of iodine in cells to the percentage of iodine in whole gland in the thyroid glands of normal and iodine- fed dogs as well as in human thyroid glands obtained from opera- tive cases. 2. Evidence is presented indicating that the concentration of intracellular iodine is independent of the suspending medium, whether that is pure Ringer's solution, Ringer's solution containing iodine-rich colloid material, or homologous blood serum. 332 Distribution of Iodine in Thyroid Gland. II 3. The ratio value was found to be relatively constant despite great variations in the morphology and iodine content of the glands examined. The ratio value for the dog's thyroid gland seems quite constant and is much lower than that of the beef or sheep. Reprinted from THE AMERICAN JOURNAL OF PHYSIOLOGY Vol. 56, No. 1, May, 1921 A STUDY OF THE DISTRIBUTION OF IODINE BETWEEN CELLS AND COLLOID IN THE THYROID GLAND III. THE EFFECT OF STIMULATION OF THE VAGO-SYMPATHETIC NERVE ON THE DISTRIBUTION AND CONCENTRATION OF IODINE IN THE DOG'S THYROID GLAND HARRY BENJAMIN VAN DYKE From the Laboratory of Physiological Chemistry and Pharmacology, University of Chicago Received for publication February 5, 1921 For many years it has been held that the thyroid gland is supplied with true secretory nerves. In support of this assertion there is con- siderable anatomical evidence and some physiological evidence. Of late the nerves which anatomists have traced into the thyroid gland and have considered to be possibly secretory in function have been declared to be branches of the cervical sympathetic nerve. And recent physi- ological work has tended to confirm this view. Only twelve years ago Wiener (1) published the report of experiments from which he concluded that extirpation of the inferior cervical ganglion produces a marked atrophy of the thyroid gland on the side of the extirpation. Wiener maintained that no comparable effect on the lobe of the thyroid gland on the side of the operation could be produced by vagotomy or by removal of the superior cervical ganglion. More recently Rahe et al. (2) announced that they were able to produce a quite marked diminution in the iodine concentration of the lobe of the thyroid gland on one side by stimulating the thyroid nerves in several different ways. They stimulated the nerves of the superior thyroid artery, the intact vago- sympathetic nerve as well as the vago-sympathetic nerve near the level of the superior cervical ganglion after ligating the nerve low in the neck and cutting the nerve central to the point of stimulation. They found that the most marked loss was brought about by the stimulation of the intact vago-sympathetic nerve. Watts (3) undertook to find out whether or not the results obtained by Rahe, Rogers, Fawcett and Beebe might be due to vasomotor 168 DISTRIBUTION OF IODINE IN THYROID GLAND 169 changes in the gland on the stimulated side. Watts likewise found that he could reduce the iodine content of the right or left lobe of the thyroid gland of the dog by stimulating the "cervical sympathetic iso- lated from the vagus sheath" and the "nerve filaments accompanying the superior thyroid vessels." However Watts maintained that he could cause some diminution in the iodine content simply by periodi- cally reducing the blood flow through the gland by "occluding the main thyroid artery" the nerves of which had been dissected away. Hence he concluded that all of the effects of stimulation on the iodine content can be accounted for by the coincident vasomotor changes which he showed to be present. Positive evidence of the secretory effect of sympathetic stimulation has been reported by Cannon and his co-workers (4) in several communi- cations. Working with cats they sutured the phrenic nerve with the cervical sympathetic nerve and observed following the operation in- creased basal metabolism, respiratory hippus and falling hair which they interpreted as the results of hypersecretion of the thyroid gland caused by the periodic bombardment of the gland by impulses carried from the respiratory center to the gland's secretory nerves. Cannon and Cattell (5) adduced additional evidence as to the role of the sympa- thetic nerves in experiments dealing with the electrical condition of the gland. Following the stimulation of the upper thoracic sympathetic nerves or the injection of epinephrin they were able to show a definite action current in the thyroid gland after a latent period of five to seven seconds. Recently Cannon and Smith (6) maintained that gentle mas- sage of the thyroid gland or stimulation of the cervical sympathetic nerve increases the rate of the denervated heart. The denervated heart is said not to be affected when the cervical sympathetic nerve is stimulated after removal of the thyroid gland. Moreover Levy (7) ob- served that the pressor effect of epinephrin after a variable latent period is increased by the stimulation of the cervical sympathetic nerve. He declared that stimulation of the cervical sympathetic nerve has no such effect after thyroidectomy. However the conclusions based on the experiments mentioned above have not been universally accepted. Burget (8) was unable to alter the thyroid gland noticeably either by uniting the phrenic and cervical sympathetic nerves or by removing a section of the cervical sympathetic nerve. Marine, Rogoff and Stewart (9) sutured together the phrenic and cervical sympathetic nerves in several cats. They demonstrated a functional union between the phrenic and .cervical sympathetic nerves 170 HARRY BENJAMIN VAN DYKE but observed no exophthalmos, tachycardia or respiratory hippus in their animals. There was no apparent difference either grossly or his- tologically in the lobes of the thyroid gland on the operated and the non- operated sides. Troell (10) reported that he was unable to produce either exophthalmos or respiratory hippus by suturing the proximal end of the phrenic nerve to the cervical sympathetic nerve. Employing cocaine as a sensitizer for sympathetic nerve endings, Mills (11) did not observe, following the repeated injection of cocaine, any alteration in the amount or nature of the thyroid secretion as judged by what is known of the gland's histology. Finally Rogoff (12) records one experi- ment in which he drew blood from a vein of the left lobe of the thyroid gland and at the same time stimulated the cervical sympathetic nerve on that side in the hope of increasing the secretory activity of the stimu- lated lobe. From the right lobe he also collected blood by way of a vein. While drawing the blood he massaged the right lobe to some extent but did not stimulate the right cervical sympathetic nerve. He found that specimens of dried blood from each lobe were potent when fed to tadpoles. But he could detect iodine chemically only in the blood from the non-stimulated lobe. Moreover the non-stimulated lobe had a lower iodine content than the stimulated lobe. In connection with some studies on the distribution of iodine in cells and colloid in the thyroid gland I attempted to alter acutely the total iodine content of the gland by stimulating the vago-sympathetic nerve of the dog. Some inconsistencies in the results in the early part of the work forced me to investigate the matter more carefully and to repeat the work of Rahe et al. (2) and of Watts (3). Methods. Dogs were used in all of the experiments. All except those whose numbers are above that of no. 78 were given daily feedings of iodine over a period of one to eleven days. The daily feeding con- sisted of a capsule containing two drops of tincture of iodine in starch. In the animals fed iodine the stimulation of the vago-sympathetic nerve was undertaken from two to ten days after the last feeding of iodine. Throughout the experiments the animals were lightly anesthetized with ether. Platinum wire electrodes were applied to opposite sides of the carefully isolated vago-sympathetic nerve and shielded from all sur- rounding tissues by glass. In all of the experiments except those re- corded in table 5, a tetanizing current from three to six times as strong as that sufficient to bring about a pupillary dilatation and apparent protrusion of the bulbus oculi was employed. The regulation of the strength of the current was made possible by a rheostat inserted in the DISTRIBUTION OF IODINE IN THYROID GLAND 171 circuit. Again in all of the experiments except those to be found in table 5 the stimulating current throughout the period of stimulation was made for about 0.8 second at intervals of 1.6 seconds by means of a clock and ratchet device. The strength of the current used and the rate at which the current was made in the experiments of table 5 are described below. TABLE i Variations in the iodine content of neighboring specimens of the same lobe of the thyroid gland ANIMAL NUMBER NUMBER OF FEEDINGS OF IODINE LOBE OF THYROID GLAND WEIGHT OF SAM- PLE OF DRIED GLAND ANALYZED IODINE IN DRIED GLAND ANALYZED DIFFERENCES IN THE CONCENTRA- TION OF IODINE IN NEIGHBORING SPECIMENS OF THE SAME LOBE gram per cent per cent Left 0.1192 0.1378 0.331 0.424 0.093 68 11 Right I 0.1525 0.1668 0.418 0.455 0.037 / / 0.6931 0.366 0.005 Left 0.6420 0.371 0.034 I 0.6846 0.405 0.039 74 6 ( 0.4549 0.370 0.006 Right \ 0.4931 0.364 0.019 1 0.5533 0.383 0.013 Left 0.4454 0.5298 0.121 0.132 0.011 88 0 • Right | 0.7857 0.4528 0.119 0.129 0.010 Left 0.7241 0.7138 0.158 0.176 0.018 105 0 Right | 0.4489 0.4516 0.163 0.148 0.015 After a number of experiments had been performed it was found that there is considerable variation in the iodine content of adjoining pieces of the same gland. In table 1, for example, are given a few analyses of neighboring specimens of the same gland. The differences in neigh- boring portions of the glands are somewhat greater in iodine-fed animals. 172 HARRY BENJAMIN VAN DYKE TABLE 2 Quantitative determination of iodine in whole gland and in cells free from colloid material of thyroid glands of dogs whose isolated vago-sympathetic nerve had previously been stimulated for approximately three hours ANIMAL NUMBER LOBE STIMU- LATED LENGTH OF PERIOD OF STIMULATION WEIGHT OF WHOLE GLAND USED IODINE IN WHOLE GLAND WEIGHT OF CELL MASS USED IODINE IN CELL MASS RATIO OF PER CENT OF IODINE IN CELLS TO PER CENT OF IODINE IN WHOLE GLAND APPARENT GAIN (+) OK LOSS ( — ) IN CONCEN- TRATION OF IODINE IN WHOLE GLANU OF STIM- ULATED LOBE gram per cent gram per cent per cent 42 Left 2 hrs. 15 min. < L 0.1792 R 0.1693 0.531 0.560 0.2533 0.1360 0.371 0.303 0.699 0.541 -0.029 45 Left 3 hrs. 0 min. < L 0.4602 R 0.5336 0.148 0.102 0.5117 0.4750 0.011 0.014 0.074 0.137 +0.046 46 "Left 3 hrs. 30 min. -^S^ •i I S i o|| | 1 0 11 t 1 •8 nt of iodin elapsing pletion of removal •8 s in whole t t of cell m: • a 1 a rtl g a g ® a^Ja 0 'S'o a s c i 1 '3