P R O T E I N - B O U N D I O D I N E L E V E L S IN D A I R Y C A T T L E P L A S M A , M IL K , A N D C O L O S T R U M By ROBERT CHARLES LEWIS 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 Dairy 1952 P roQ uest Number: 10008364 Alt rights reserved IN FO R M ATIO N TO A LL USERS The quality o f this reproduction is dependent upon the quality o f the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these will be noted. Also, if m aterial had to be removed, a note will indicate the deletion. uest, ProQ uest 10008364 P ublished by ProQ uest LLC (2016). Copyright of the D issertation is held by the Author. All rights reserved. T his w ork is protected against unauthorized copying under T itle 17, United States Code M icroform Edition © ProQ uest LLC. P roQ uest LLC. 789 East Eisenhow er Parkw ay P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS The w riter wishes to express his sincere appreciation to Dr. N. P. Ralston, P ro fesso r of Dairying, for his interest and constructive suggestions regarding the conduct of this work and for his aid in the preparation of this manuscript. A large debt of gratitude is also owed Dr. E. P . Reineke, P rofessor of Physiology, for his original suggestion which led to the develop­ ment of this work and for his continued interest in its success. Thanks are due to Dr. J. A. Williams, of the Pathology Department, and Dr. L. O. Gilmore, of the Ohio State Univer­ sity, for their aid in furnishing certain plasma samples. Ap­ preciation is also expressed to Dr. Earl Weaver, Head, Dairy Department, Michigan State College, for his aid in establishing a project under which this work was financed. P R O T E I N - B O U N D I O D I N E L E V E L S IN D A I R Y C A T T L E P L A S M A , M IL K , A N D C O L O S T R U M By Robert Charles Lewis AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in p artial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1 R O B E R T C H A R L E S L E W IS ABSTRACT Induced hyper- or hypothyroidism causes marked a lte ra ­ tion in dairy cattle performance, leading one to speculate on the relation of normal variability in thyroid function to growth, reproduction, production, longevity, efficiency, and like factors which determine the value of a dairy cow. Very little information is available on the thyroid activ­ ity of dairy cattle. In other species the plasma level of p ro ­ tein-bound iodine has been found to be closely correlated with thyroid activity. Therefore, in this study an attempt has been made to determine the normal ranges of plasma protein-bound iodine in dairy animals in order to provide a basis for evalu­ ating its relation to performance. A digestion-distillation technique for the determination of protein-bound iodine in organic m aterial has been used in more than three hundred determinations of this iodine fraction in dairy cattle plasma, milk, and colostrum. The plasma level of protein-bound iodine in dairy cattle was found to show a marked rise gressively decrease with age. soon after birth and to p ro ­ Newborn calves had an average 2 R O B E R T C H A R L E S L E W IS ABSTRACT plasm a level of 8.9 m icrogram s percent, but within a few hours, after nursing, it rose to 15.0 m icrogram s percent. This rise appeared to be due to the extremely high protein-bound iodine concentration in the initial colostrum. four calves from The thyroid glands of 12 hours to 6 days old were examined m icro­ scopically and showed progressive colloid storage and cellular activity with advancing age. The average protein-bound iodine values obtained on animals of different age groups were 12.8, 7.3, 6.2, and 4.6 m icrogram s percent for animals under 48 hours, 48 hours to 12 months, 12 to 24 months, and over 24 months of age, respectively. The suggestion is made that the following ranges of plasma protein-bound iodine concentration be considered normal: calves under 48 hours old, 8.0 to 18.0 m icrogram s percent; 48 hours to 12 months, 3.5 to 12.0 m icrogram s percent; 12 to 24 months, 3.5 to 10.0 m icrogram s percent; and animals over 24 months old, 3.0 to 8.0 m icrogram s percent. No breed difference could be demonstrated between J e r ­ sey and Holstein cows maintained in the same herd. A group 3 ROBERT C H A R L E S L E W IS ABSTRACT of Jerse y cows recently brought from California were signifi­ cantly lower in their plasm a protein-bound iodine concentrations than either Je rse y or Holstein cows reared in the college herd. A number of cows under investigation as sterility cases also had significantly lower values. No significant seasonal trends in the plasma proteinbound iodine concentration of Jerse y cows could be shown a l­ though they tended to have lower values in November than dur­ ing March, June, or August. The seasonal changes were sim ­ ila r and statistically significant in the Holstein cows. No r e ­ lation of the plasma protein-bound iodine to the stage of gestation or rate of lactation could be demonstrated in the limited production data of this study. The protein-bound iodine concentration in the plasma of five p airs of identical twin heifers was found to be no more alike than that of more distantly related or unrelated animals. TABLE OF CONTENTS Page INTRODUCTION...................................................................................................... 1 REVIEW OF LITERATURE .....................................-................................ 3 The Phylogeny of the Thyroid G la n d ....................................... 3 Iodine in the Thyroid G la n d .............................................................. 5 Placental T r a n s m is s io n .................................................................. 6 Fetal Thyroid .......................................................................................... 7 Uptake and Binding of I o d i n e ................................................... 9 Distribution Between Thyroxine and Diiodo tyro sine .................................................................................. F ree Iodide, Thyroxine, and Diiodotyrosine The In Vivo Synthesis of Thyroxine . . . . 12 15 ....................................... 16 Nature of the Thyroid H o r m o n e ........................................... 16 P re c u rs o rs 18 and Synthesis of T h y ro x in e........................ The Circulating Hormone .................................................................. Rate of Thyroxine Secretion 22 ................................................... 22 Nature of the Circulating H o rm o n e ................................... 27 Iodine of the Blood and P l a s m a ........................................... 35 iv Page The Metabolism and Excretion of Thyroxine ................ 43 Endocrine and Other Relationships of the Thyroid Gland .........................................................................^ . 46 ..................................... 46 .................................................................... 50 Relation to the Anterior Pituitary Relation to the Gonads Effect on Reproduction of Farm Animals ................ Relation to Quantity and Composition of Milk 53 . . 55 Relation to the Adrenal Cortex ............................................. 60 Relation to the Adrenal M e d u lla ............................................. 62 Relation to the T h y m u s .................................................................... 62 Relation to the Parathyroids .................................................... 63 Relation to the P a n c r e a s ................................................................ 63 Relation to N u tritio n .......................................................................... 64 High protein diet High fat diet .......................................................................... 64 ...................................................................................... 65 High carbohydrate diet ........................................................... 65 ...................................................................... 65 Cabbage g o i t e r .................................................................................. 66 Relation to T e m p e r a tu r e .............................................................. 67 Relation to Light 68 Vitamin deficiency .................................................................................. V Page Antithyroid Compounds ................... 68 Thyroid H o r m o n e .................................................................................. 69 I o d i n e ................................................................................................................. 69 Thiocyanate 70 .............................................................................................. Antithyroid Substances .................................................................. EXPERIMENTAL MATERIALS AND METHODS M aterials Methods Reactions 71 ................ 72 ............................................................................................................. 72 .............................................................. 73 ............................................................................................................. 73 R e a g e n ts ......................................................................................................... 74 P r o c e d u r e ..................................................................................................... 76 Precipitation and washing ................................................... 76 D ig e s tio n .................................................................................................. 78 Distillation .......................................................... Colorimetric determination of iodine Notes on the Procedure 79 .................... ....................... EXPERIMENTAL R E S U L T S ...................................................................... Test of Method 81 82 87 .............................................................................................. 37 ........................................................... 87 ............................................................... 90 Recovery of Added Iodine Repeatability of Results Page Plasm a Protein-bound Iodine ofDairy C a t t l e ................... 92 .............................................................................................. 92 Calves Before and After F ir s t N u r s in g ........................... 105 Jerse y and Holstein C o w s ........................................................... 116 Cases of S t e r i l i t y .................................................................................. 124 Identical Twins ...................................................................................... 127 DISCUSSION................................................................................................................. 129 SUMMARY AND CONCLUSIONS................................................................ 136 BIBLIOGRAPHY..................................................................................................... 139 Age Changes INTRODUCTION The economic situation today demands that we obtain the greatest possible efficiency in the production of milk, butterfat, and meat from our dairy animals. This can not be done unless we thoroughly understand the physiology of our cattle and so s e ­ lect and manage them as to fully utilize their productive poten­ tials. A long step forward would be taken if some means could be developed whereby the future worth of an animal could be estim ated while it was still a calf. An endocrine analysis may offer some possibilities in this direction. No suitable assay method exists for some of the hormones, but the rate of se c re ­ tion of the thyroid hormone, which has been shown to be related to dairy cattle performance, is closely related to the plasma level of protein-bound iodine in many species. It has been a useful tool in estimating thyroid function in human medicine and is closely related to thyroid activity in rats, mice, dogs, ra b ­ bits, and other laboratory animals. Little information is avail­ able, however, on the plasma protein-bound iodine concentration 2 of dairy cattle and its relation to the functional condition of their thyroid glands. As an approach to this problem, the plasm a proteinbound iodine levels of dairy animals of various ages and breeds have been determined in a relatively large number of cases. It was hoped that such data would provide a basis for establish­ ing a normal range of protein-bound iodine values. If such a range could be established, the influence of various environmen­ tal factors and the relation of plasma protein-bound iodine con­ centration to performance could be determined in future studies. REVIEW OF LITERATURE The Phytogeny of the Thyroid Gland In his recent excellent review, Goldsmith (75) traced the phylogenetic development of the thyroid gland as one ascends the evolutionary tree. The most prim itive thyroid gland is found in the adult cyclostome, of which the lamprey is an example. Lower form s, in which no thyroid tissue has been identified, probably do not secrete thyroxine although the possibility has not yet been ruled out. In this connection it is well to rem em ber that even in the higher vertebrates a certain amount of extra thyroidal thyroxine is present (40, 104, 138). Iodine in organic combination is found in many inverte­ brates and, indeed, 3,5-diiodotyrosine was f ir s t isolated from a species of coral (84). In these lower forms, and in the lamprey, exogenous thyroxine is said to have no effect on maturation a l­ though there is some data to the contrary. The thyroid gland of the adult lamprey has been shown to a rise from the endostyle of the larva which itself must possess 4 some type of thyroidal activity since it is capable of accumulat­ ing radioactive iodine. The gland of the adult lamprey is not encapsulated but is made up of a small number of isolated fo l­ licles. In some elasmobranches the thyroid is encapsulated and a rise s as a solid epithelial bud ventral and caudal to the f ir s t and second gill with the follicles developing as they do in the human. The thyroids of the trout and salmon arise from the floor of the pharynx and clasp the upper end of the trachea. In the elasmobranch and in teleosts there is some variation in the degree of compactness of the thyroid tissue. L arger indi­ viduals of these species tend to have a more compact, nodular gland. The thyroid gland of the higher vertebrates a rise s from the ventral wall of the pharynx and clasps the upper end of the trachea. The gland is divided into a right and left lobe and is at f ir s t attached to the pharynx by a stalk, the thyroglossal duct. This duct atrophies during the intrauterine period, although seg­ ments may p e rsist, giving rise to thyroglossal cysts during postuterine life. 5 Iodine in the Thyroid Gland The amount, concentration, and nature of the iodide com­ pounds in the thyroid gland will obviously be somewhat dependent upon the iodine intake and physiological status of the subject. The factors which affect thyroid activity will be discussed in another section. It is the intent here to describe the conditions which exist in the normal gland. decide what is "n o rm a l.11 It is, of course, difficult to If, however, those animals on a cu s­ tom ary diet, maintained under usual conditions, and which show no gross abnormalities, are considered to be "n o rm a l,” it will be possible to describe the normal situation. In reviewing the data on the iodine content of the thyroid gland, one is struck by the general agreement among investi­ gators, even when the results of early workers are compared with the most recent reports. It is all the more amazing when one considers the techniques now used which were not available to the early workers. It is best, however, not to compare the absolute magnitude of the findings of the various investigators, since many of them, especially the early workers, were forced to use relatively impure m aterials and crude procedures. trends and relationships may be safely compared. The 6 The iodine-concentrating ability of the thyroid gland has been demonstrated many times; Salter (169) has reviewed the older litera tu re in this respect and has summarized the iodine content of various tissues of man, dogs, and rabbits as reported by several workers. In general the thyroid, followed by the an­ te rio r pituitary and the ovaries, contains a higher concentration of iodine than the other organs. Placental Transm ission Hudson (103) has shown that inorganic iodide can pass through the placenta from the mother to the fetus. Thus, the level of iodine in the fetal thyroid reflects the availability of iodine to the mother. The thyroids of hairless pigs, for in­ stance, are very low in iodine, and fetuses aborted by cretinoid mothers are nearly iodine-free (223). The ability of the thyroid hormone to pass through the placental b a rrie r from mother to fetus is not yet established. C ourrier and Aron (47) fed fre sh ox and hog thyroids to dogs and guinea pigs throughout gestation. Although the m others' thyroids were markedly altered, no gross or microscopic change occurred in the fetal thyroids. When the thyroid administration 7 was continued after delivery the thyroids of the nursing young rapidly changed. From this it was concluded that the hormone can not pass through the placenta. On the other hand, it is well known that pregnant bitches may be thyroidectomized with no ill effects appearing until after they have been delivered. The marked hyperplasia of the fetal thyroid under these circumstances points to tra n sfe r of thyroid hormone from the fetus to the mother (169). Fetal Thyroid On the basis of limited evidence, it would appear that the fetal thyroid is inactive in many species until about the middle of pregnancy. The apparent lack of thyroid activity p rio r to this time of course implies, too, that the anterior p i­ tuitary is not elaborating the thyrotropic hormone or that the thyroid is not capable of responding to its stimulation. Schultze and Turner (172) have shown that p rio r to mid­ term the thyroid glands of fetal goats do not respond to the administration of thiouracil or thiourea to the mother. After m id-term , however, the glands were greatly enlarged when these goitrogens were administered. 8 The bovine fetal thyroid may begin to function at a com­ paratively e a rlie r age, since it has been found to contain m ea­ surable amounts of iodine at 60 days (231). At this time the percentage of the total iodine which was present as inorganic iodide was the same as that found in the adult thyroid. The ability of the thyroid to concentrate iodine progressively in­ creased with advancing age of the fetus. The same workers (139) found, too, that after 62 days' pregnancy the growth of the fetal thyroid, as m easured by increased thyroid weight, is nearly directly proportional to the body weight. According to Salter (169), the total iodine in the thyroid tissue of the human fetus between the third and ninth months varies from 1 to 19 m icrogram s, or a concentration of 2 to 21 m illigram s percent, of which at least a portion is present in an active form. Newborn babies have been reported as containing 2.4 to 48 m icrogram s of thyroidal iodine, varying from 2.3 to 1,450 m icrogram s percent in concentration, while the other body tissues contained a concentration of only 12 to 46 micrograms percent. The thyroids of eighteen newborn infants, most of whom were term fetuses born dead, were analyzed by P alm er et al. 9 (140), who found a range of 35 to 654, with an average of 254 m icrogram s of iodine per gram of dry tissue. The average percent of the total iodine which was present in the form of thyroxine was 20.0, which is only slightly less than the adult fig u re. The uptake of radioiodine by the ham ster fetus has r e ­ cently been studied by Hansborough and Seay (87). With a g es­ tation period of 15 days and 8 hours, no accumulation of radio­ iodine could be detected in the fetal thyroids until the thirteenth day of development. The uptake of radioactive iodine did not begin until the appearance of the follicles which occurred be­ tween the twelfth and thirteenth day. As the number of follicles increased, the accumulation of iodine also increased. Uptake and Binding of Iodine The rate of uptake of iodine by various tissues has been m easured by Perlm an and associates doses of radioactive iodine. (144), who used tra c e r Within 25 to 50 hours the thyroid gland had taken up 65 percent of the administered dose. other tissues The studied contained a maximum of 0.60 5 percent of the adm inistered iodine at this time. Whereas the amount of 10 radioiodide in the thyroid had steadily increased up to this time, the amount in the other tissues rose to an early peak which was followed by a gradual decline. The situation appeared to be one of diffusion of inorganic iodide into the nonthyroidal cells and then diffusion back into the circulation rather than an exhibition of any especial affinity of these cells for iodine. The uptake of injected radioiodine by the thyroid occurs very rapidly after its administration. Lein (114) found the most rapid uptake occurred during the f ir s t 10 minutes after injec­ tion. A small amount of acetone-insoluble labeled iodine could be detected in the gland within 5 minutes after its administration. The rate of iodine binding by surviving slices of dog, sheep, and ra t thyroids was measured by Morton and Chaikoff (137). Within 2 hours after the addition of radioactive iodine to Ringers solution containing thyroid tissue, sheep glands con­ verted 31 to 37 percent of the radioiodine into diiodotyrosine and 5 to 6 percent into thyroxine. During the same time dog thyroid tissue converted 21 to 24 percent of the iodine into diiodotyrosine and 3 to 4 percent into thyroxine while the c o r­ responding rate for ra t tissue was 60 to 71 percent and 8 to 12 percent, respectively, for diiodotyrosine and thyroxine. 11 In a recent review, Chaikoff and Taurog (37) presented data which showed that withing 15 minutes after rats are injected with a tra c e r dose of radioactive iodine (I 131 ), 95 percent of the radioactivity in the gland occurs in the organically bound p ro ­ tein. Of this amount, 80 percent is in the diiodotyrosine-like fraction and the balance in the thyroxine-like portion. The r e ­ lationship between the percentages of thyroxine and diiodotyrosine in the gland remained quite constant even though the gland in ­ creased in its total iodine content. In a later study designed to measure the maximum c a ­ pacity of the thyroid gland to take up and store iodine, Taurog and Chaikoff (194) fed groups of rats at various levels of iodine intake. With a daily iodine intake of 1 to 2 m icrogram s, the thyroid contained 21.5 m illigram s percent of total iodine (wet weight) and 5.9 milligram s percent of thyroxine iodine. When the daily intake was increased to 78 m icrogram s, the c o r r e ­ sponding values changed to 134 and 44.4 m illigram s percent. With an intake of 440 m icrogram s the concentrations were and 36,5 m illigram s percent, respectively. 131 Thus, the ability of the gland to take up and store iodine is limited. At its 12 peak the gland contained an iodine concentration about 10,000 tim es g reater than that found in the blood. Distribution Between Thyroxine and Diiodotyrosine The distribution of iodine between the thyroxine and di­ iodotyrosine fractions has been studied by Perlm an et al. (145). Labeled iodine was deposited in the diiodotyrosine fraction in about twice the amount that appeared in the thyroxine fraction. Forty-eight hours after the administration of labeled iodine as much as 16 percent of it appeared in the thyroxine and 32 p e r ­ cent in the diiodotyrosine fractions. The thyroid glands of fifty-two humans were analyzed by Leland and Foster (115), who extracted an alkaline hydroly­ sate of the glands and found about 25 percent of the gland's total iodine was in the form of thyroxine. In fifty-four patients they obtained a range of 0.33 to 4.21 milligram s of iodine per gram of dry tissue and a concentration of 0.173 to 5.93 milligrams of thyroxine in the whole gland. These values were believed to be about 15 percent low because of some loss of thyroxine dur­ ing the hydrolysis. E arlier, F oster (67) had reported that 33 13 percent of the iodine of thyroglobulin is p resent as diiodotyrosine and 16 percent as thyroxine. Blau (24) modified the Leland-Foster technique to elim ­ inate most, if not all, of the thyroxine loss encountered by these workers. He obtained a range of total iodine per gram of dry human thyroid of 0.72 to 4.04 m illigram s and a concentration of 0.013 to 0.887 milligram s of thyroxine iodine in a series of six fresh human thyroids. The thyroxine iodine varied from to 31.8 percent of the total iodine. 1.77 These values averaged about 9.7 percent higher than those obtained by the Leland-Foster method on the same glands. In a later tria l (25) five fresh human thyroids were found to contain 0.59 to 0.895 m illigram s of iodine per gram, of which 23.9 to 29.8 percent of the total iodine was in the form of thyroxine. Two fresh pig thyroids containing 0.615 and 0.695 m illigram s of iodine per gram had 29.8 and 31.8 percent of the total iodine in the thyroxine-like fraction. A desiccated human thyroid contained 1.41 milligram s of total iodine per gram and a desiccated pig thyroid, 3.45 m il­ ligram s. The corresponding thyroxine concentrations were 19.2 and 31.2 percent of the total iodine, respectively. 14 In a group of eleven ra ts, Taurog and Chaikoff (193) found from 4.1 to 7.4 m icrogram s of total iodine in the thyroid glands, of which 1.1 to 2.0 m icrogram s were thyroxine: roxine percentage of 23 to 30 percent. a thy­ In another group of ra ts, under a wide range of iodine intake, an average value of 31.0 was obtained for the average percent of thyroxine iodine in the gland. Wolff and Chaikoff (229) have reported that an average of 29.6 percent of the thyroidal iodine was in the form of thy­ roxine in eleven vertebrates, including fish, reptiles, birds, and mammals. The changes in the thyroxine concentration of the human thyroid have been studied by Glimm and Isenbruch (73). Dur­ ing the f ir s t year the concentration was 24 m illigram s percent, and during the f ir s t decade it was 25 m illigram s percent. The concentration rose to 42 m illigram s percent in the second dec­ ade, dropped to 29 m illigram s percent during middle life, and again rose to 36 m illigram s percent after the sixtieth year. Gutman and associates (85) reported that the average total weight of the human thyroid in New York was about 25 gram s, which contained 8.85 m illigram s of iodine. The range of iodine 15 concentration was from 0.05 to 0.45 percent of the dried gland. The average was 0.186 percent. The alteration in iodine content of the thyroid gland with variations in iodine intake has been illustrated by Salter (169), who found 1.2 percent of iodine, of which over 60 percent ap­ peared to be thyroxine in the partially purified, heat-coagulated thyroglobulin of Argentine sheep. Human thyroglobulin in Bos­ ton contained 0.22 percent iodine, of which 25 percent was thyroxine-like, while a human colloid goiter contained only 0.006 percent iodine and no detectable amount of thyroxine. Free Iodide, Thyroxine, and Diiodotyrosine Recently, Taurog et al (201) have reported that the in ­ organic iodine of the thyroid gland of rats varied from a con­ centration of 0.6 to 2.0 m illigram s percent and represented about 1.0 percent of the gland's total iodine. This is about 500 times more concentrated than occurs in the plasma. The free (not bound in peptide or other linkages) thyroxine and diiodo­ tyrosine found by Tong (20 3) amounted to about 0.5 percent of the total iodine of the gland. Even this represents a concen­ tration over 100 times g reater than that of the protein-bound 16 iodine in the plasma. This concentration gradient may be of some significance in the passage of thyroxine into the circ u la ­ tion. The In Vivo Synthesis of Thyroxine Nature of the Thyroid Hormone Thyroglobulin, the stored hormone-containing protein of the thyroid gland, has been reported to have a molecular weight of about 675,000 (Heidelberger and Pedersen [92]). is taken at 665,000, Brand and co-workers If the value (32) have indicated that, according to Bergman's theory, the thyroglobulin molecule contains 5,760 amino acid residues, of which 120 are cystine (240 cysteine), 60 methionine, 60 tryptophane, diio dotyro sine, and 2 thyroxine. 110 tyrosine, 10 There would also be 80 glu­ cosamine residues as well as some other carbohydrates p r e s ­ ent. The authors suggested that the molecule is composed of ten units of 576 amino acids each of which, aside from the thy­ roxine grouping, are sim ilar in amino acid composition. Salter (169), however, quoted Svedberg as stating that he had evidence that the thyroglobulin as originally determined is an aggregate. 17 Thus, we have no definite knowledge as to the actual structure of thyroglobulin. Recently, Tong and associates (203) have identified three major iodized constituents of the thyroid: thyroglobulin, thy­ roxine, and diiodotyrosine, which exist in both the free and com­ bined states. E arlier, the same workers (198) found about 15 percent of the iodine in a thyroid hydrolysate to be present as monoiodotyrosine. Gross and Leblond (83) have presented evidence for the existence of three other as yet unidentified iodine compounds labeled 1, 3, and 5. Compound 5 has been identified as elemental iodine by chromatographic techniques, but it may be an artifact resulting from the manipulations (83). Unknowns 1 and 3 appear to be organic iodide-containing com­ pounds resulting from thyroglobulin breakdown. These authors also indicate that some of the monoiodotyrosine and diiodotyro­ sine may result from thyroglobulin breakdown. Since most of these compounds have not been identified in the blood plasma, it appears that with the exception of thyroxine they are largely metabolized in the gland and are recombined into thyroglobulin or are excreted as noniodized compounds. 18 P re c u rs o rs and Synthesis of Thyroxine It has been mentioned previously that the thyroid gland has many times been shown to possess extraordinary iodine concentrating ability. This iodine has been assumed to be u ti­ lized for iodination of tyrosine and the resulting diiodotyrosine converted to thyroxine. The administration of labeled inorganic iodine and its consequent appearance in the diiodotyrosine and thyroxine fractions of the thyroid confirmed the hypothesis that inorganic iodide is the source of the organic iodine but the sequence and process of conversion has only recently been shown experimentally. Harrington and Barger (88) suggested that diiodotyrosine is the p recu rso r of thyroxine because of the sim ilarities in their stru ctures. In 1940, Block (27) was able to form thyroxine from a synthetic diiodotyrosine in vitro. Morton (137) has demon­ strated the conversion of iodide to diiodotyrosine and thyroxine by surviving slices of thyroid glands from sheep, dogs, and ra ts . In this case as much as 21 and 37 percent of added I 131 was incorporated into diiodotyrosine and 4 and 6 percent into thyroxine when dog and sheep thyroids, respectively, were in­ cubated for 3 hours. Mann (122) has presented evidence that 19 the conversion of inorganic iodide to diiodotyrosine takes place at the level of the cell membrane. From the data of Leblond and Gross (113) it appears that circulating iodide is continuously bound to protein in the cytoplasm of the cell and the thyroglobulin formed is sim ul­ taneously deposited in the follicle. Specific activity studies have shown that the monoiodo­ tyrosine found by Taurog (198) and Gross (83) and their a sso ­ ciates is not a breakdown product of diiodotyrosine, but is most likely a p re c u rso r of it. Specific activity-time curves have also shown (196) that diiodotyrosine is indeed the p re c u r­ sor of thyroxine and not the resu lt of cleavage of the thyroxine molecule. In the same manner it has been indicated that free thyroxine in the gland does not represent a step in the synthesis of thyroglobulin but, rather, is a product of colloid breakdown and is the p recu rso r of the circulating hormone (203). Little can be definitely stated regarding the mechanism by which iodinations occur in the thyroid gland and the manner in which the stored hormone is released. Dempsey (52), in 1 9 4 4 , found peroxidase activity in thyroid cells. Since peroxi­ dases catalyze the release of iodine from iodides, they may be 20 related to biological iodinations (Keston [110]) and thyroxine synthesis (Westerfeld and Lowe [224]). The release of colloid to the blood and lymphatic systems can not be explained on the basis of simple diffusion through the cell membrane because of the size of the molecules involved. F urtherm ore, little, if any, thyroglobulin is found in the blood stream . Salter and Lerm an (168), in 1936, synthesized iodoproteins, using total thyroid extracts, which resembled thyroglobu­ lin in activity. These authors suggested that an enzymatic mech­ anism involved in both synthesis of the hormone and destruction of the thyroid protein might be present. In 1940, Gersh and Caspersson (71) suggested that follicular colloid is digested enzymatically and the products taken up by the cells. that time DeRobertis and his associates Since (54, 56) have demon­ strated proteolytic activity in thyroid colloid which varies in intensity with the pH of the medium and physiological activity of the gland. As will be discussed later, these alterations in proteolytic activity may explain the known changes in thyroid activity produced by administered thyrotropic hormone, iodine, and various goitrogens. 21 The physical and chemical cytology of the thyroid gland were poorly understood p rio r to 1940. of DeRobertis, Since that time the work summarized in his recent review (58), has some­ what clarified the picture. Through use of a freeze-drying tech­ nique he (53) was able to show the product of thyroid secretion which he designated as intracellular colloid and, with Gersh and Caspersson (71), presented evidence that this m aterial con­ tains organic iodine, which is presumed to represent the thy­ roid hormone. Administration of the thyroid-stimulating hormone of the anterior pituitary causes rapid increase in the intracellular colloid (DeRobertis [55]). The colloid droplets are formed near the nucleus and, increasing in size, move toward the apex, where they are excreted into the follicular cavity. Expulsion of the colloid into the lumen is accompanied by rupture of the cyto­ plasm. L ater, the cells stop secretion toward the lumen and begin to secrete toward the base, reabsorbing the colloid p r e s ­ ent in the lumen. Gersh and Caspers son (71) suggested that the follicular colloid might be digested by enzymatic action and the products 22 absorbed by the cells p rio r to release of the hormone to the circulation. The presence of proteases in the follicular colloid has been demonstrated by DeRobertis (54). The increased col­ loid loss in toxic goiter and colloid storage in simple goiter may be explained by increased or decreased levels of protease activ­ ity. That the enzymatic activity does change under these condi­ tions has been shown by DeRobertis and Nowinski (56). The Circulating Hormone Rate of Thyroxine Secretion Dempsey and Astwood (51) developed a method for e s ti­ mating the rate of thyroxine secretion based upon the amount of hormone required to resto re the thyroid weight of thiouraciltreated ra ts to normal. Using this method, these workers found the secretion rate in rats at 25° C. to be 5.2 micrograms of 1-thyroxine per 100 grams of body weight daily. Similar r e ­ sults were obtained by Reineke and his associates (161), who reported that 4.75 m icrogram s of thyroxine per 100 grams of body weight daily were necessary to restore the basal metabolic rate of thiouracil-treated rats and that 4.8 micrograms daily resto red the gland to normal weight. Somewhat lower results 23 were obtained, by Griesbach and Purves (78), who found a daily secretion rate of 2.25 m icrogram s of d,1-thyroxine per 100 grams of body weight. When Chaikoff and Taurog (37) applied the method of Z ilversm it et^ a l. (234) to their data, the calculated time required for complete renewal of the thyroxine in the gland (turnover time) was about 24 hours. Since the average thyroxine content of the thyroid glands of the ra ts was 3.3 m icrogram s, the rate of thyroxine iodine secretion was calculated to be about 1.5 m icrogram s of d,1-thyroxine per 100 grams of body weight p er day. By specific-activity-tim e studies these authors (196) demonstrated a daily secretion rate of about 2 microgram s of thyroxine p er 100 grams of body weight. Wolterink and Lee (232) compared the thyroid activity as assayed by the thiouracil-thyroxine method with the results obtained from the rate of radioactive iodine turnover and found the two techniques gave comparable results. By the Dempsey and Astwood method a group of rats secreted 5.36 m icrogram s of d,l-thyroxine per 100 grams of body weight p er day. The turnover rate calculated on the same rate was 5.16 m icrogram s 24 d,l- thyroxine daily. This was a 4 -percent difference which was not significant. The quantity of d,1-thyroxine required to restore the thiouracil-induced enlargement of the thyroids of White Leghorn cockerels to normal size was determined by Schultze and T u r­ ner (171). The requirem ent for cockerels 2, 6, 7, 9, and 12 weeks old was 1.95, 7.55, daily. 11.35, 14.4, and 16.5 micrograms If these amounts are assumed to represent the normal rate of thyroxine secretion, the rate of secretion p er 100 grams of body weight decreased with age during this time. Very sim ­ ila r values were obtained by Reineke and Turner (163) when they m easured the rate of thyroxine secretion in groups of twoweek-old White Plymouth Rock chicks at intervals throughout the year. Females varied from a d,l-thyroxine equivalent of 0.75 to 2.7 m icrogram s and males from 0.9 to 2.45 m icrogram s daily, depending upon the season of the year. Since d-thyroxine has little or no activity (162) the 1-thyroxine values would be one-half those listed. The thyroid glands of chicks were destroyed through adm inistration of radioactive iodine by Winchester et al. (227), who then adm inistered thyroxine in graded doses to ascertain 25 the amount required to give normal growth. This occurred when thyroxine was adm inistered at the rate of 3.8 microgram s per 100 gram s of body weight daily. Turner (209) has shown that the rate of thyroxine s e c re ­ tion decreases in older hens. Two-year-old White Leghorns secreted the equivalent of 12 m icrogram s of d,1-thyroxine daily, or 0.6 of a m icrogram per 100 grams of body weight, as de­ term ined by the method of restoring thyroid weight after thiouracil administration. Male White Pekin ducklings were shown to secrete * m icrogram s of d,l-thyroxine, and females, 14.0 micrograms daily at 3 weeks of age, by Biellier and Turner (23). to 2.85 and 2.67 m icrogram s p er 13.9 This amounted 100 grams of body weight. At 12 weeks the males were secreting 60 micrograms daily, and the fem ales, 61.4 m icrogram s. These values correspond to 3.18 and 3.39 m icrogram s of d,1-thyroxine daily per 100 grams of body weight. Hoffman (100). secreted per Slightly higher secretion rates were found by In his study, 1- to 3-week-old White Pekin ducks 18.7 m icrogram s of thyroxine daily, or 3.8 m icrogram s 100 grams of body weight. In both cases the rates of 26 secretion calculated for ducks was considerably higher than those which have been reported for the chick. The hourly rate of thyroxine secretion in dogs was found by Mann and co-w orkers (122) to be about 1.55 percent of the hormone contained in the gland. Taurog and his associates (195) found a turnover rate of 50 to 100 m icrogram s each 24 hours. This represented a rate of hormone secretion capable of completely replacing the protein-bound iodine in the c irc u ­ lation every 4 to 7.5 hours. Using the Dempsey and Astwood method (51), Schultze and Turner (172) have reported the changes in thyroxine s e c re ­ tion rate with increasing weight of goats. At two months of age goats weighing 22 pounds secreted 1.80 m icrogram s of d,l-thyroxine daily per 100 grams of body weight, while at 45 pounds the rate was 3.13 m icrogram s, and at 76 pounds the secretion rate was 2.69 m icrogram s per 100 grams of body weight daily. In 1935 the requirem ent for thyroxine to restore the normal basal metabolism of human myxedematous patients was reported by Thompson et al. (202) to be 0.25 to 0.35 milligram s daily. 27 It is well known that the rate of hormone secretion may­ be influenced by many factors. However, under standardized conditions the gland is thought to show little diurnal variation in its rate of secretion. The uptake of iodine and the release of hormone to the circulation appear to be simultaneous, con­ tinuous, and to occur at a constant rate (59). Nature of the Circulating Hormone It was at f ir s t believed that thyroglobulin is the circ u ­ lating form of the thyroid hormone (15, 93). This view was discarded as a result of the work of Trevorrow (204), who found that the iodine of blood possesses alcohol solubility prop­ ertie s sim ilar to iodide and thyroxine but unlike thyroglobulin of the gland, and Lerman (116), whose immunological studies failed to show any thyroglobulin in the serum or urine of hyper­ thyroid, normal, or hypothyroid patients. Until recently, however, investigators have been reluctant to state that thyroxine p er se is the circulating form of the thyroid hormone. (197), were: The reasons, according to Taurog and Chaikoff M(l) the failure of some investigators to account completely for the biological activity of thyroglobulin by its 28 thyroxine content, (2) the delayed response of animals to injected thyroxine, and (3) the failure of thyroxine to act in v itro." In 1935 Harington (89, 90) postulated that the circulating hormone is a peptide containing both thyroxine and diiodotyro­ sine but la te r he concluded that the peptide hypothesis unneces­ sarily complicated the picture and that the circulating hormone is thyroxine (91). The development of more sensitive methods for the chemical analysis for iodine (10, 12, 13, 43, 192), the use of radioactive iodine in fractionation studies in conjunction with chemical analysis (197), and the recent use of filter-p ap er chromotography (80, 112, 199) have aided m aterially in estab­ lishing the nature of the circulating thyroid hormone. Trevorrow (204), Mann and her co-workers (121), and Bruger and Member (35) demonstrated that "organic" iodine in plasma can be precipitated with the Smogyi zinc sulfate precipitating reagent (183), while the mains in the supernatant liquid. "inorganic" fraction r e ­ Many workers had previously attempted to fractionate blood on the basis of its solubility in methyl alcohol, ethyl alcohol, or acetone. Trevorrow (204) and Boyd and Clarke (31) have demonstrated that fractionations based 29 on this solubility are invalid since the amount of iodine ex­ tracted varied with the conditions of the extraction. F or the sake of clarity, and in conformity with present usage, the terminology used here in referring to the blood io­ dine fractions will be those suggested by Salter (169). He has proposed that the blood iodine should be separated into an in­ organic and a precipitable (or protein-bound) fraction. The precipitable fraction may then be separated into a thyroxinelike and a diiodotyrosine-like fraction. Leland-Foster (115), in 1932, showed that n-butyl alco­ hol completely extracts all of the thyroxine from thyroid pro ­ tein which has undergone strong alkaline hydrolysis. Taurog and Chaikoff (197) have employed this technique to fractionate the iodine of plasma. Since this iodine is easily extracted with butyl alcohol, while thyroglobulin must firs t be hydrolyzed, it is apparent that the circulating hormone differs from that of the gland. It does not mean, however, that the circulating hormone is necessarily free from attachment to protein. As a m atter of fact, the results of Trevorrow (204) and Riggs and his associates (167) would indicate that the circulating hormone is attached to protein. Riggs et al. concluded from dialysis 30 and sedimentating rates that circulating thyroxine must exist in a molecule of about the same size as the plasma albumin or in sm aller molecules which are attached to the albumin. Taurog and Chaikoff (197) confirmed these findings and those of Bassett, Coons, and Salter (18) that most of the organic io ­ dine of plasm a is associated with the albumin fraction. The concentration of organic iodine, however, was highest in the quantitatively sm aller gamma-globulin fraction. These authors also added to the evidence for circulating thyroxine through showing that crystalline thyroxine c a r rie r ex­ hibited a constant specific activity upon repeated re c ry sta lliz a ­ tions after addition to the butyl alcohol extract of the plasma of ra ts injected with I 131 . In addition, radioactive iodine in the butyl alcohol extract was distributed between two unmiscible solvents almost exactly as added thyroxine c a rrie r was, but quite unlike an added thyroxine peptide c a rrie r. Wilmanns (226), however, is reported as having treated whole blood with hot butyl alcohol and found that only 65 percent of the iodine could be extracted, and of this only 28 percent could not be r e ­ extracted with 1 N. sodium hydroxide. He concluded, th e re ­ fore, that blood contains two organic fractions: free thyroxine 31 (28 percent) and a stable protein-bound iodine fraction (34 percent) (197). Since Harington in 1935 (91) postulated the presence of both thyroxine and diiodotyrosine, the presence of a diiodotyro­ sine-like fraction has been confirmed by Trevorrow (204) and Morton and associates (135). More recently, using a chromato­ graphic technique in conjunction with tra c e r doses of labeled iodine, Taurog and his co-workers (199) have presented f u r ­ ther evidence that thyroxine is the major organic-iodine-con­ taining fraction in the plasma. In some cases a faint band c o r­ responding to diiodotyrosine and a faint darkening at the sol­ vent front could also be seen. Taurog questioned, however, whether this technique could distinguish thyroxine from one of its sm all peptides. Thus, the possibility of a circulating thy­ roxine peptide has not yet been ruled out. These results were confirmed by Laidlau (112). Gross et al. (80, 83) were able to show only thyroxine and iodide in ra ts. In iodine-deficient rats, compound 1, to which reference has previously been made as a component of the thyroid gland, was also found. These workers believe that free diiodotyrosine does not normally appear in the circulation. 32 In the hypophysectionized ra t Morton et al. (136) have shown that about 80 percent of the radioactive iodine taken up by the gland appeared in the diiodotyrosine-like fraction. The diiodotyrosine-like fraction of the blood plasma was likewise g re a ter than normal while the thyroxine portion of the thyroid and plasm a decreased. The exact nature of the circulating iodide compounds must thus remain a question. It is well established that inor­ ganic iodide and thyroxine are found in the circulation. The evidence indicates that the thyroxine is loosely combined with the plasm a protein, but not through a peptide linkage. The identity of other organic iodides in the plasma must await f u r ­ ther work. Under certain pathological conditions, diiodotyro­ sine may be excreted by the thyroid gland. in the normal animal is an open question. does occur the amount is small. Whether this occurs Undoubtedly if it Recent work has indicated that other organic iodides may be present under certain condi­ tions but their existence has not been confirmed. The distribution of iodine between the blood plasma and red blood cells has also been questioned. Silver (182) found very little protein-bound iodine in the erythrocytes, while 33 MeClendon and F o ste r (128) reported that fully half of the p ro ­ tein-bound iodine of the blood is present in the red blood cells; however, the analytical methods used were subject to e rro r through im purities. Trevorrow (204), on the other hand, stud­ ied a series of analyses and found the total iodine of blood and plasm a to be distributed in proportion to the water content of the cells and plasma. Recent work by Rail and co-workers (150) has shown that, in vitro, chlorides and iodide pass rapidly across the red cell membrane and there is no reason to expect the situation to be altered in vivo. Scott (176) has shown through radioactive stud­ ies that inorganic iodide rapidly penetrates the red cell. How­ ever, the cell membrane appears to be impermeable to large protein molecules containing labeled iodine. It is interesting to note that the initial red cell-plasm a iodine ratio varies from 0.42 to 0.52 and is the same in hyper­ thyroid, euthyroid, and hypothyroid patients immediately after the oral administration of 100 to 150 m icrocuries of radioactive iodide. When the time required for the ratio to drop from 0.5 to 0.25 is calculated, a significant difference between the three thyroid states may be found. (A ratio of 0.25 suggests that 34 one-half of the iodine in the blood stream is protein bound and can not penetrate the red blood cells.) Human hypothyroid p a ­ tients required 100 hours, euthyroid patients, 40 hours, and hyperthyroid patients needed 12 hours to reach this ratio. Thus, the red blood cell-plasm a iodide ratio after administration of radioactive iodide appears to be a good indicator of thyroid statu s. At any rate, Salter (169) has recommended the use of plasm a values rath er than those obtained from whole blood because of the greater spread of values and consequent g reater sensitivity when plasma is used. Furtherm ore, whole blood is unsuited for certain methods of iodine determination because of the presence of certain substances, such as iron, which may interfere with the final colorim etric determination. There is some evidence that the thyroid may elucidate some substance or substances other than the iodinated com­ pounds previously discussed. gastric Truesdell (205) has reported that secretion of dogs with the Pavlov pouch may be reduced through feeding whole thyroid. Katz is reported to have c o r­ related hypothyroidism with peptic ulcers in the human. Wat- man and Nasset (220, 221) have reported that thyroidectomy 35 significantly reduces the survival time of guinea pigs injected with histamine. Thiouracil administration has no effect on the survival time and thyroxine or diiodotyrosine are ineffective in combatting formation of the peptic ulcer which form s in the stomach or duodenum, its perforation, and the resulting p eritionitis. Since the animal is hypersensitive when made hyper­ thyroid either in the presence or absence of the gland, it would appear that the effect is not one of antagonism or detoxification of histamine. The actual presence or nature of a thyroidal secretion affecting gastric secretion is not confirmed, but an area for future investigation is indicated. Iodine of the Blood and Plasm a L iterally thousands of determinations have been made of the iodine fractions of the blood, plasma, or serum of human thyroid patients since 1920, when Kendall and Richardson (109) established that norm al blood contains iodine in a characteristic concentration. The early results establishing the relation b e­ tween the level of blood iodine and thyroid status has been reviewed by Curtis et al. (48), who concluded that there is good agreem ent between the blood iodine level and thyroid function. 36 The total iodine content of the whole blood of normal human patients was found by Perkin e_t al. (142) to range from 2.4 to 18.5 m icrogram s per 100 gram s of blood, while a some­ what narrow er range was obtained by Davis and co-workers (50) on patients in Chicago. In thirty-four determinations on twenty- eight people, they obtained a range of total iodine of 8.5 to 16.2 m icrogram s percent. cent. The average was 11.9 m icrogram s p e r ­ A slightly g reater variation was obtained with women than with men. The eighteen women varied from 8.5 to 16.2 m icrogram s percent. gram s percent. The range in men was 9.5 to 14.5 m icro ­ Since none of the patients showed evidence of thyroid disease and none received iodized salt, the greater variability of the women was attributed to their changing men­ strual state. The effect of various thyroid conditions on the blood iodine level was studied by McCullagh and McCullagh (129). The blood iodine of ten patients hospitalized with nonthyroidal diseases averaged 10.2 m icrogram s percent as compared with the normal level 10.0 microgram s percent. Violent exercise reduced the level to 6.8 m icrogram s percent within 2 hours. Ten hyperthyroid patients varied from 11.1 to 49-8 micrograms 37 percent and six cases of hypothyroidism averaged 7.5 m icro ­ gram s percent, while of six cases of hypometabolism not r e ­ lated to the thyroid, two patients showed normal levels and the rem ainder varied from 6 to 11.2 microgram s percent. In six cases of hypermetabolism with no evidence of thyroid disease, the level was between 6.1 and 10.3 micrograms percent. The blood iodine in thyroid, cardiorenal disease, and leukemia was investigated by Turner and associates (210) in New York City. Twenty males ranged from 3.8 to 8.6 m icro­ gram s percent, averaging 5.9, while a like number of females averaged 6.8, with a range of 3.5 to 10.4 micrograms percent. In twenty hyperthyroid patients the blood iodine was elevated in fourteen and normal in six. in five hypothyroids. The level was normal or low The blood iodine was low in eight of twelve cases of myeloid leukemia and normal in the other four. The range was average of 3.4. 1.3 to 7.4 micrograms percent, with an In lysophoid leukemia the level was normal or elevated. The age differences in blood iodine have been reported, for children, by Fashena (64). Those under 24 hours old a v e r­ aged 4.7 m icrogram s percent with a range of 1 to 11.0 microgram s 38 percent. Up to 13 years the average was 6.6 m icrogram s p e r ­ cent, ranging from 3.0 to 12.0 microgram s percent. P erkin and Brown (143) studied the effects of iodine adm inistration and sex difference on the blood iodine of dogs. When 72 m illigram s of iodine daily were fed, the blood iodine varied from 75 to 2,000 microgram s percent. The blood level of males was greatly depressed following complete thyroidectomy but there was no apparent effect on the females. However, following bilateral oophorectomy of the thyroidectomized females, the level fell to one comparable with the males. As indicated by the work of Turner (210) and Salter (169, 170), the total blood iodine, under certain circumstances, fails to reflect the actual thyroid status. Clarke and Boyd (41), for instance, were unable to show any seasonal variation in thyroid activity of pigeons and chickens through the blood iodine, although it is known that the thyroid activity does vary in these birds. Because of this occasional lack of sensitivity, in recent times the protein-bound iodine fraction of the plasma, or blood has been used. serum, Salter and co-workers (170) correlated the relation of protein-bound iodine of the blood to the final 39 diagnosis of thyroid status in one hundred of their cases. In about one-third, in which thyroid status had been suspected, the basal metabolic rate was not compatible with the bedside diagnosis. In every case the protein-bound iodine level con­ firm ed the diagnosis. In mild hypothyroidism, where the m eta­ bolic rate had not fallen to an abnormal level, this measure was especially useful in establishing the need for thyroid th e r­ apy. Total iodine levels were not sufficient in these cases. For example, one patient showed a total iodine level of 7.1, but a protein-bound iodine level of 2.2 microgram s percent, as compared to the normal averages of 6.3 and 4.8 micrograms percent, respectively. In the one hundred cases the basal metabolic rate and protein-bound iodine agreed in seventy-one, and in twenty-nine the protein-bound iodine was more reliable. The range of protein-bound iodine values of blood does not differ greatly from total iodine of the blood in human p a ­ tients on a low iodine diet. In livestock receiving iodized salt or feed which contains variable amounts of iodine the difference between the protein-bound iodine and total iodine may be more variable. In any case, however, the protein-bound iodine is somewhat more sensitive to changes in thyroid secretion rate 40 than is the total iodine. Taurog and Chaikoff (194), for exam­ ple, have found the correlation between the plasma protein-bound iodine and thyroxine iodine to be 0.84. The plasma p rote in­ bound iodine is dependent upon and limited by the gland’s abil­ ity to produce thyroxine. The level of the protein-bound iodine in plasm a falls rapidly after thyroidectomy. A noticeable de­ crease occurs within 4 hours and a minimum value is reached by the third day. Injection of the thyrotropic hormone raises the plasm a protein-bound iodine (36). Representative plasma protein-bound iodine levels have been reported by several investigators. McClendon and F oster (128) found the level in two cows to be 6.4 and 6.8 micrograms percent, respectively; humans ranged from 5.8 to 9.4; a horse, rabbits, and a cat gave values of 9.2, 6.6 to 11.1, and 7.6, respectively. Connor et al. (43) found the level in human serum to range from 4 to 6 m icrogram s percent, with an average of 4.8. The nonprotein-bound iodine in these cases ranged from 1 to 3 m icrogram s percent. Long and co-workers (120) have recently studied the plasm a protein-bound iodine of a large number of dairy and beef cows. They averaged 3.15 m icrogram s of iodine per 100 41 m illiliters were: shire, of serum. The values, arranged according to breed, Jersey , 4.11; Guernsey, 3.51; Brown Swiss, 3.37; Ayr­ 3.19; Holstein, 2.73; and beef breeds, 2.19 micrograms percent. Some of these differences were significant. were age differences also. There Calves averaged 4.8 micrograms percent, 3- to 4-year-old cows averaged 3.1, and 7- to 8-yearold animals averaged 2.6 m icrogram s percent. A considerable amount of data on patients at the Iowa State University Hospital has recently been presented by Barker and his collaborators (13). The range of values of 942 d eter­ minations of plasm a protein-bound iodine on 694 patients clus­ tered between 3 and 7 percent. There was no sharp line sep­ arating the normal and hyper- or hypothyroid patients. D eter­ minations made on sixty-eight apparently euthyroid students, technicians, and staff and faculty members ranged from 3.4 to 8.0; the mean was 5.1 m icrogram s percent. At that institution the normal range of values has been considered to be from 4.0 to 8.0 m icrogram s percent. If, however, the range had been lowered to 3.5 m icrogram s percent, of eighty-nine patients on whom basal-m etabolism and iodine-uptake data were available, only one hypothyroid and one thyrotoxic patient would have been 42 included in the normal group. In contrast, several BMR's were at considerable variance with the general clinical opinion. resu lts are These supported by a previous report by Talbot et al. (191). The relation of obesity to thyroid function, as determined by the plasm a protein-bound iodine, was studied by Williams (225). Of twenty-four obese patients, the plasma protein-bound iodine was less than 4 m icrogram s percent in eleven and above this amount in thirteen. Thus, the relation between obesity and the plasm a protein-bound iodine is not great. Care must be exercised in interpreting the results of plasm a protein-bound iodine determinations, since recent iodine therapy or topical application of iodine to the patient will cause abnormally high results (11). Evidence of a nonthyroidal iodine fraction in the plasma protein-bound iodine of cattle has been presented by Reece and Man (156), who found the serum -precipitable iodine of nonpreg­ nant, lactating Jerseys averaged 4.6 micrograms percent, while the butanol-extractable iodine averaged 2.5 micrograms percent. Similar results were obtained with Brown Swiss cattle, although the serum -precipitable iodine concentration was somewhat less for the la tte r breed. 43 The Metabolism and Excretion of Thyroxine The metabolism of thyroxine remains nearly as much of a m ystery today as it was 20 years ago. Although the evidence just discussed points toward thyroxine as being the circulating thyroid hormone, even this fact is not established with certainty. The metabolic effect of thyroxine is well known, but the re a c ­ tions by which the cellular oxidations are increased have not been demonstrated. The paths by which thyroxine is excreted have been shown, but the excreted metabolites have yet to be identified. Recent work by Gross and Leblond (81), which confirms previous reports by Monroe and Turner (134), Kellaway et al. (107), Taurog and co-workers (200), and the early work of Barnes (14), has thrown some light on the paths by which the circulating hormone is transported to the body cells and finally excreted, but does not attempt to show how the metabolic ef­ fects of thyroxine are produced. T racer amounts of radioactive iodide injected into rats were found in the stomach in a butanol-insoluble form within 2 hours after administration. After the iodide had time to be taken up by the thyroid and released again as thyroxine (24 to 44 72 hours) the activity was found distributed throughout the plasma and tissues of the body. The radioactivity of both the plasma and tissues decreased with time, but the rate of decrease was g reater in the plasma. This can perhaps be explained by the conversion of thyroxine into its metabolites by the tissues. The radioactivity was ini­ tially in a butanol-soluble form, but the butanol-insoluble activity increased with time. The tissue decrease in activity was paralleled by a fall in the activity of the whole body. Thus, in this case it was calculated that the thyroxine content of the body was completely renewed each 25 to 45 hours. The liver was particularly rich in its thyroxine content, probably because of its role in the deactivation of thyroxine. The mechanism by which the liver deactivates thyroxine is not known. That simple deiodination c£ thyroxine (Salter [169]) with elimination of the iodide by the kidney and inactive residue through the bile is not sufficient explanation is indicated by recent reports (82, 200). The deamination of thyroxine followed by reamination to form the inactive d-thyroxine is a possibility 45 (60), or a breaking of one or both of the tyrosine rings may occur (20). In the light of this and other work it appears that thy­ roxine is norm ally destroyed in the liver and the degradation product excreted through the bile, although some thyroxine may be excreted through the intestinal wall (79). large amounts of thyroxine are present, When abnormally some may appear in the bile in active form, but once the level falls to normal the degradation process converts all, or nearly all, to the usual metabolites. The elimination of thyroxine and its metabolites from the body appears to be largely through the urine and feces: the urine containing iodide and the feces carrying off the m e­ tabolites of thyroxine, iodide, and under some conditions, thy­ roxine itself (82). Very little thyroxine is excreted in the urine (62, 79, 81), and apparently the kidney has no significant role in the excretion of the hormone per se. No doubt, too, a certain amount of the iodide is returned to the circulation and carried to the thyroid gland where it is reincorporated into thyroxine. 46 While no explanation of the ability of thyroxine to p ro ­ duce a sustained ris e of oxygen consumption can be given, it appears to be the end resu lt of a series of reactions. This is deduced from the fact that the hormone is rapidly eliminated from the body, but its metabolic effects continue for an extended period. Endocrine and Other Relationships of the Thyroid Gland Relation to the Anterior Pituitary It is well known that the anterior lobe of the pituitary produces a hormone which stimulates the thyroid gland to s e ­ crete its hormone. Albert (2), in his recent review, defined the thyrotropic hormone as "a substance from pituitary tissue which, when given parenterally in proper dosage to various v ertebrates, induces specific effects on the thyroid consisting of secretory alterations of the cytological components of the follicular cells, hypertrophy and hyperplasia of the epithelium, vacuolization and resorption of colloid, loss of hormonal iodine, and increase of vascularity and of the size of the gland." Although the stimulating effect of anterior pituitary ex­ tra c ts on the thyroid had been known for many years, it was 47 not until 1929 that Aron (7) and Loeb et a l. (117, 118) demon­ strated the thyroid-activating principle of the pituitary gland. The site of the production of the thyrotropic hormone in the pituitary has not yet been well established. Purves and G ries- bach (149) have recently presented evidence that two types of glycoprotein-containing cells are present in the anterior pitui­ tary, one of which they believe secretes the thyrotropic hor­ mone. E a rlie r, these workers had presented evidence that the basophilic cells of the anterior pituitary were involved in the secretion of thyrotropin (78); others, however, have shown good reasons to believe that the acidophila may also be involved (74, 104, 178, 179). The process by which the thyrotropic hormone activates the thyroid gland is unknown, but the effects of its presence or absence are well known. Hypophysectomized animals have a reduced thyroidal ability to take up inorganic iodide from the blood (72, 216), and decreased conversion of inorganic iodine to thyroxine (4, 216), but the prim ary effect seems to be one of preventing the release of thyroxine by the gland (155, 216). These effects of a deficiency of thyrotropic hormone are then 48 manifested by lowered levels of circulating hormone (72) and lowered metabolic rate (66). Although the thyroid gland is generally said to be con­ trolled by the anterior pituitary gland through the thyrotropic hormone, in a manner of speaking, it is self-controlling. The rate of secretion of the thyroid-stimulating hormone is d ete r­ mined by the tite r of thyroid hormone in the circulation (78). When the amount of circulating thyroxine is low, through r e ­ duced activity, thyroidectomy, or iodine deficiency, the pituitary responds with typical changes in the basophilic and acidophilic cells and increased weight (74, 78). In addition, thyroxine ad­ m inistration reduces the activity of the pituitary (16, 69, 17 8). It has been postulated, too, that the thyroid gland may be partially self-regulatory in that thyroxine and, to a le s s e r extent, inorganic iodide are capable of depressing the rate of thyroxine secretion directly as well as through its action on the pituitary gland (3, 230). DeRobertis and Nowinski (57) have indicated that this effect may resu lt from inhibition of the proteolytic enzyme system by the iodine. The metabolism of the thyrotropic hormone once it has perform ed its normal task of activating the thyroid gland is not 49 well understood. Obviously it must be eliminated per se or inactivated and then eliminated. Although the methods of thyro­ tropin assay are crude, the evidence points towards the second conception as indicating the fate of this hormone. In vitro studies by Raws on ejt a l. (153) and Albert et al. (1) have definitely shown that thyroxine and iodine can inactivate the thyrotropic hormone under these conditions. In addition, in vivo, thyroxine and po­ tassium iodide have been shown to depress the response of the thyroid to thyrotropin (5, 45), although inorganic iodide does not prevent the cellular proliferation of the thyroid produced by this hormone. The mechanism by which the thyrotropic hormone is in­ activated is not known. It has been suggested that a prosthetic group may be removed from the hormone or that thyrotropin may somehow be bound (153) or that a chemical change, prob­ ably oxidative in nature, takes place (1, 154). It has been suggested that the control of the pituitary secretion of thyrotropin may depend upon nervous impulses. This, however, has been disproven by the work of Hektoen et al. (94), Marine and Rosen (126), Gorbman (76), and B arrnett and Greep (16), who, through attempted nerve stimulation, thyroid 50 transplants, pituitary transplants, and pituitary stalk section, were unable to show any nervous relationship in thyroid activa­ tion. In spite of the role of the thyrotropic hormone in thy­ roid control, secretion of thyroxine does not entirely cease on hypophysectomy. The thyroid is able to respond to the stimu­ lus of low iodine intake through increased cell height and vas­ cularity (39) and thyroxine secretion remains at about 12 p e r ­ cent of its form er level (152) even after hypophysectomy. Relation to the Gonads Little is known regarding thyroid-testes relationship in comparison to the thyroid-ovarian relations. T r> the rat, thyroid feeding increases the testis weight (95), while testosterone causes thyroid hypertrophy (96) and increases the uptake of iodine (133). It is likely, however, that in the male the thyroid hormone affects testis development only through its relation to cellular metabolism. In females the relation of the ovary to the thyroid is essentially one of antagonism as indicated by the well-known fact that goiter occurs much more frequently in women than men. 51 F u rth erm o re, thyroid enlargement and increased hormone s e ­ cretion takes place during pregnancy and lactation (49, 169). Iodine excretion is increased during menstruation (42) and blood iodine is not greatly lowered in thyroidectomized females unless they are simultaneously castrated (143). Turner and Cupps (206) found the thyrotropic hormone content of the female albino ra t to be only about 50 percent as high as in the male during growth. The concentration rose during the la tte r p a rt of pregnancy and increased further d ur­ ing lactation. The rise in plasm a neutral fat, calcium, and phosphorus which is caused by estrogen administration is prevented by simultaneous injection of thyroxine (130). In 1910 Hoskins (101) reported that feeding thyroid to mother guinea pigs resulted in reduced ovarian weight of the offspring. comes from Supporting evidence Tyndale and Levin (213), who observed that thyroxine prevented ovarian response to menopausal urine. Since the r e ­ sponse was prevented in hypophysectomized animals, the inhib­ iting effect was direct and not mediated through the pituitary. Herring (95), on the contrary, reported increased ovarian size in ra ts fed thyroid m aterial. 52 An effect of estrogen on thyroid activity was shown by KLarp and Kostkiewicz (106), who caused colloidal goiter by folliculine administration. by Gardner (70). Similar results were recently reported Small doses of estrogen increase the output of thyroxine by the thyroid of rats and mice, while massive doses depress iodine turnover, according to Wolterink et al. (233). According to Money et a l. (133), some estrogens increase and others decrease iodine accumulation in the ra t thyroid. Diet seemed to have an effect on whether enhanced accumulation took place. Pregnancy urine extracts as well as the follicular hor­ mone have also been successfully used in treating human hyper­ thyroidism (186). The relations between the thyroid and ovary are com­ plicated and reciprocal. Some of the inhibiting properties of the thyroid on ovarian function are mediated through the pituitary and others are direct repressions. It is believed, too, that the ovary may depress thyroid function through preventing the f o r ­ mation or release of the thyrotropic hormone. The effect may also be a direct one produced by release of the ovarian iodine into the blood stream in quantities sufficient to depress thyroxine release. Under normal conditions, however, there is little 53 evidence that physiological concentrations of female sex h o r­ mones affect thyroid status. Effect on Reproduction of Farm Animals The relation of hypo- or hyperthyroidism to reproduction in farm animals has not been thoroughly studied, particularly as fa r as the female is concerned. not show visible symptoms of estrus Thyroidectomized cows do (33, 185); however, ovula­ tion does occur and fertilization and pregnancy can take place in myxedematous animals. Only casual observations have been made regarding the effects of thyroprotein administration on dairy cattle. Van Landingham et^ a l. (2 1 9 ) have reported that cows made hyperthyroid in this way are slow to come into heat. The effects of thyroid deficiency or hyperfunction are better known in male than female farm animals, but even here there is no agreement. Thyroidectomized bulls are entirely lacking in libido (147), but spermatogenesis is apparently normal and semen ob­ tained by massaging the ampullae is unaffected. Reineke (164) fed thyroprotein to fourteen aged bulls and obtained increased 54 vigor and speedier ejaculation from this treatm ent in ten of the sire s. Schultze and Davis (173) obtained better conception rates with five of seven bulls treated with thyroprotein. This effect of thyroprotein is probably a result of the increased metabolic rate, since metabolism declines with age (34); and dinitrophenol, a metabolic stimulant, has the same effect on male sexual behavior as thyroprotein (147). In addi­ tion, thyroidectomy, and perhaps naturally occurring thyroid deficiency, reduces the pituitary gonadotropic hormone level in goats (158) and response to gonadotropins in rats (132). F u r­ therm ore, added thyroxine increases the metabolism of semen (123, 174, 175) and perhaps the conception rate. Unfortunately there is no known information available on the normal thyroxine tite r of semen and its fluctuations with thyroid function. In ram s, the tem porary infertility found during hot sum­ m er weather (131) appears to be identical with that caused by thiouracil (29), and may be prevented or cured by administration of thyroactive substances. Thyroidectomy, Berliner and Warbritton (19) observed, causes a decrease in sperm numbers and an increase in ab­ normal form s. The testes were edematous, the interstitial 55 tissue decreased, and the seminiferous tubules sloughed and were pyconocic. These observations, and the fact that thyroxine secretion falls during hot weather (51) point towards a direct relationship between the thyroid hormone and reproduction in ram s. The effect of thyroid status on egg production of poultry is not clear. The Missouri workers (207, 20 8) have reported increased egg production when thyroprotein was added to the poultry ration. However, neither the experimental nor control groups did well, as evidenced by the fact that in one experiment the experimental group averaged 40.6 percent and the control group 22.6 percent production during their second year. One wonders if the effect of thyroprotein would have been the same with high-producing birds. As a matter of fact, Hutt and Gowe (105) were unable to perceive any particular effect of thyropro­ tein beyond a slight initial decrease in egg production. Relation to Quantity and Composition of Milk Since the development by Reineke and Turner (159) of an artificial thyroprotein containing thyroidal activity in good amount, a great amount of interest has developed regarding the relation 56 of thyroid activity to the quantity and composition of milk from dairy cattle. Swett et a l. (190) have reported that the thyroid gland weight of the breeds of cattle varies inversely with the quantity of milk produced, at lea st in so far as the Holstein, Ayrshire, Guernsey, and Jerse y breeds are concerned. The thyroid is not essential for the initiation of lactation or milk production, but, following thyroidectomy, lactation ceases about six months after parturition (184). Incomplete thyroidectomy tem porarily lowered production, but it gradually returned to normal. The milk content of fat, lactose, and nitrogen, and its specific grav­ ity appeared to remain unchanged. Oral administration of thy­ roid m aterial prevented these effects of thyroid deficiency. There have been many reports on the effects of feeding thyroactive m aterial to cattle, a number of which have recently been reviewed by Blaxter and his collaborators (26). In gen­ e ral, cattle respond to thyroxine administration by increased milk and butterfat production, especially after the normal peak of lactation has been reached. The increase in butterfat comes about not only as a resu lt of increased quantities of milk, but also through a rise in the percent of butterfat. 57 The effects of continued administration of thyroidal m a­ te ria l is not yet known, although this type of experimentation has been underway for a decade. The Bureau of Dairy Industry of the United States Department of Agriculture, however, has advised against this type of stimulation since cows fed in this way required more nutrients and did not respond to the stim ­ ulation as well in succeeding lactations as they did in the first. In addition, calf losses were somewhat higher in their experi­ mental groups (157). The feeding of thyroidal or other iodine-containing feeds increases the iodine content of the milk; thyroxine, however, is not secreted per se in the milk (160). Matthews and co-workers (127) have reported an average milk iodine content of 80 m icro grams percent from cows receiving 3.2 m illigrams percent of iodine as potassium iodide in the ration. This amount was seven to twenty-six times that of milk from cows on a sim ilar ration which did not include an iodine supplement. The effect of iodine supplementation was least during the late spring and early fall. B artlett et^ al. (17) found the normal iodine content of milk to be 1.5 m icrogram s percent, but when 1 gram of iodine 58 in the form of iodinated protein was fed daily for 8 days the value rose to 125 m icrogram s percent. Remington and Supplee (165) studied pooled milk from several points in South Carolina and reported a variation in iodine content with the region. Milk obtained from the Piedmont a re a was considerably higher than that from the coastal region. Samples from New York and Wisconsin were lower than those from the southern state. Little seasonal variation was found in the Carolina samples, although they were significantly lower in April and May than at other times. The seasonal variation was somewhat g reater in the New York and Wisconsin samples. The iodine content of skimmed human milk and human and dairy-cow colostrum was investigated by Turner (212). Skimmed human milk ranged from 6 to 23 micrograms percent, with an average of 12.4 m icrogram s percent. The average value was higher in goitrous than nongoitrous areas, possibly because of compensatory enlargement of the m other's thyroid. The io­ dine concentration tended to decrease after the third month of lactation. The iodine content of both human and cow colostrum is considerable higher in- iodine, particularly the f ir s t day or two 59 after parturition than la te r in lactation. This initial high iodine content may be of importance to the young in meeting the stre sse s of its new environment. The effect of hyperthyroidism or thyroprotein adm inistra­ tion on milk and butterfat composition is not well established. The composition of the milk fat appears to be unchanged, a l­ though there is a tendency for a rise in the unsaturated fatty acids and a fall in the saturated acids for a short time after treatm ent begins (99). The solids-not-fat increase (65). The percentage of lactose was reported to increase by Ralston ejt al. (151) and several other workers, but Archibald (6) found a decrease. Many workers have studied the protein content of milk from hyperthyroid cows, but again there is disagreement as to what effect, if any, this condition has on the milk nitrogen. Ralston (151) and Archibald (6) and their associates reported declines in the nitrogen or protein content; however, Van Landingham and his co-workers (218) and Hibbs and Krauss (97, 98) reported no change in this constituent. No large amount of work has been carried out on the vitamin content of milk from hyperthyroid cows. The indications 60 now are that Vitamin A and carotene are not affected (97); Vitamin C may be decreased (17, 217), although Hibbs and K rauss found it unchanged; thiamine content may be unchanged or decreased (98, 108); and riboflavin is lowered (108). No information is available on the other vitamins. It may be seen, then, that the changes in milk composi­ tion are not great and its nutritional value is on the whole un­ changed by thyroxine administration. Relation to the Adrenal Cortex Hoskins (102) f ir s t showed an antagonism between the adrenal cortex and the thyroid gland by demonstrating that hyperthyroidism in guinea pigs caused adrenal hyperplasia. The effect of hypothyroidism produced by thiouracil and thy­ roidectomy on adrenal activity has recently been studied by Freedman and Gordon (68), who found that thiouracil caused pronounced atrophy of the adrenal glands and an initial increase in their ascorbic acid concentration which soon returned to normal. Thyroidectomy, on the other hand, produced less atrophy but a g reater adrenal inhibition, as indicated by a final decrease in ascorbic acid concentration. This effect of 61 hypothyroidism has been shown to be prim arily one of failure of the pituitary to release the adrenal corticotropic hormone, although its rate of production may also be reduced (86). P e rry (146) has demonstrated that cortisone and the adrenal corticotropic hormones reduce the thyroxine content of the thyroid probably through direct reduction of the rate of iodine uptake. The effect did not appear to involve the thyrotropic hormone since no atrophy of the thyroid occurred. Paschkis et al. (141) were unable to show any signifi­ cant effect of desoxycorticosterone acetate and adrenal cortical extract on radioactive iodine uptake by the thyroid. Severe stre s s , which is also probably mediated by the adrenals, has been shown to reduce iodine uptake by the thyroid. Bogorach and Tim ira (30) suggested that this may be caused by reduced secretion of the thyrotropic hormone which decreases the thy­ ro id ’s ability to trap iodine. Since, however, the prim ary ef­ fect of the thyrotropic hormone has been demonstrated to be on the release of thyroxine from the thyroid, it would appear that the stre ss mechanism is more likely to be one of direct inhibition of the thyroidal concentration of iodine. 62 Relation to the Adrenal Medulla The adrenal medulla appears to have only a minor ef­ fect on thyroid activity. Adrenaline administration produces a tem porary rise in blood iodine followed by a fall to a subnormal level. The source of this iodine has not been proven, although there is some evidence that it comes from the thyroid gland. Its clinical or physiological significance, if any, is not known (169). Relation to the Thymus The antagonism between the thyroid and thymus is well known, although its significance is not. In 1924, Marine et al. (125) showed a relationship between the two glands. Thyroidec­ tomy hastened the involution of the thymus in rabbits, while hyperthyroidism caused hypertrophy of the thymicolymphatic tissue. Likewise, thymus extract inhibits the response of rab ­ bits to thyroxine administration (169). Boatman and Campbell (28) were recently unable to show any effect of thymus feeding on radioactive iodine uptake or histological appearance of the thyroid glands of rats. Cosma 63 (46), however, found increased thyroid activity in thymectomized animals. Relation to the Parathyroids When large doses of thyroxine are administered to an animal the parathyroid glands tend to hypertrophy, and calcium excretion is greatly increased. thyroxine induces tetany. In hypoparathyroid animals Thyroidectomy, on the other hand, reduces calcium turnover and causes involution of the parathy­ roids in rabbits (169). The effect of thyroxine on calcium and phosphorus metabolism thus appears to be a reflection of the activity state of the endocrine glands involved, and not a direct relationship between calcium and iodine balances. Relation to the Pancreas The aggravation of diabetes in hyperthyroidism and its alleviation by thyroidectomy in man establishes a link between the thyroid gland and carbohydrate metabolism. This effect of the thyroid is due to (1) increased oxidation of carbohydrate and (2) increased rate of hepatic gluconeogenesis (21). 64 Ligation of the dog pancreas causes an increase in col­ loid and iodine content of the thyroid (189). ■ki&k in. diabetes Blood iodine is (211) unless the plane of nutrition is low. Intravenous adm inistration of insulin causes a tem porary drop in the circulating iodine. The pancreas itself has little affinity for iodine (169). Relation to Nutrition The relation of nutrition to goiter has recently been r e ­ viewed by G reer (77), who has classified the early work into four divisions: n (l) The effects of high protein diet; (2) high fat diet; (3) high carbohydrate diet; (4) vitamin deficiency and e x c e s s .'1 L ater work is referred to as cabbage goiter, since most recent work has been with cabbage and related foods. High protein diet. Early reports from England and G er­ many, and later in this country, have shown that diets high in meat, especially liver, tended to produce enlarged thyroids. Whether such glands are over- or underfunctional is not known, and since such factors as tem perature are not known to have been controlled, further studies utilizing present knowledge might well be undertaken. 65 High fat diet. conflicting. The results on high fat diets have been Enlarged thyroids in dogs and reduced growth of tadpoles has been attributed to the large amount of fat injested. On the other hand, rats and rabbits have given no response to fat. Reduced food intake on high fat diets with consequent iodine deficiency could, in some cases, account for any thyroid en­ largem ent observed. High carbohydrate diet. The last report is over a quar­ te r of a century old, but a few experiments tended to show thyroid hypertrophy on high carbohydrate diets. These reports are difficult to evaluate, however, since the iodine content and palatability of the rations varied markedly. Vitamin deficiency. Much of the early work showed de­ ficiency of Vitamins A, B, and C caused thyroid enlargement but, as with most of this work, too little was known about thyroid physiology and the vitamins themselves for the results to be conclusive. More recently, Sherwood and Luckner (181) found that administration of Vitamin A causes an increase in the stroma and epithelium of the acini, and that colloid is reduced. Carotene 66 produces sim ilar, but not identical, changes. Cooper and co­ workers (44) reported that hyperthyroid chicks have a higherthan-norm al requirem ent for Vitamin A. Sure and Buchanan (187) have presented data showing that crystalline Vitamin B ^ on ra ts . Vitamin B ^ counteracts the toxic effect of thyroxine has also been reported to protect rats against massive doses of thyroxine (188), although Ershoff (63) could not confirm this. Liver concentrate rich in the antiper- nicious anemia factor was effective in this case, but whether the active principle is Vitamin B ^ alone is not certain. and Lardy (22) indicated that Vitamin B Betheil is a growth factor for J- w hyper thyroid ra ts. Cabbage goiter. Cabbage, spinach, carro ts, and various related vegetables have shown marked goitrogenic properties for many animals, including goats, sheep, and man. The active principles appear to be related to the thioureas and their a c ­ tion on the gland resemble this goitrogen. Soybeans are also goitrogenic (180), but iodine administration counteracts the con­ dition . On the other hand, legume and fresh-cut green grass, c a rro ts, and oats have been claimed to have antigoitrogenic 67 properties which prevent the action of the vegetable goitrogens. It has been suggested, that these m aterials may contain some thyroxine-like m aterial. However, the presence of these anti - goitrogens has not yet been proven. More recently, Remington et al. (166) found the antigoitrogenic activity of dried milk, oysters, and haddock to be proportional to their iodine content, while Irish moss is less so than its iodine content would indi­ cate . Relation to Temperature The decrease in thyroid secretion rate at elevated tem ­ p eratu res was f ir s t described by Dempsey and Astwood (51). The effects of such declines on farm animals have already been mentioned in connection with summer sterility in ram s. It is also evidenced by the well-known decline in egg production during the summer months. Seidell (177), in 1913, reported the iodine content of the thyroid gland to be higher from June to November than during the winter and early spring months. This high iodine level in the gland presumably indicates a lower rate of hormone secretion into the circulation. 68 Relation to Light Light and darkness are known to affect thyroid activity in many species. Puntriano and Meites (148) recently reported thyroid atrophy and reduced activity when mice were exposed to continuous light. This effect of light is thought to be m edi­ ated by way of the pituitary and does not represent a direct action of light on the thyroid gland. It is probable that the observed seasonal variations in metabolism and breeding patterns of domestic and wild animals are largely due to the interaction of tem perature and light on the thyroid and pituitary glands. Antithyroid Compounds The modes of action of various antithyroid compounds have recently been reviewed by Astwood (8), who classified them into four groups: "(1) Thyroid hormone; (2) Iodine (3) Thio- cyanate ion (4) Antithyroid substances proper, compounds which interfere with thyroid hormone synthesis." 69 Thyroid Hormone It has previously been mentioned that the thyroid h o r­ mone can, to some extent, regulate the rate of hormone release by the gland. This is accomplished through reducing the rate of thyrotropin secretion by the pituitary gland and perhaps through a direct effect by desensitizing the thyroid gland to the action of the thyrotropic hormone. Iodine Iodine has long been utilized to reduce thyroid activity in Graves disease, although the mechanism of its action is not known. It has been suggested (54) that its effect may result from iodination of the proteolytic enzyme which breaks down thyroglobulin and releases thyroxine to the circulation. On the other hand, Marine and Lenhart (124) showed that iodine admin­ istration to goitrous dogs had the same effect as thyroxine in­ jections. Hypothyroid rabbits became hyperthyroid when iodine was adm inistered to them (222). Thus, iodine may act differ­ ently under different circum stances of thyroid activity. These effects of iodine are unexplained, but may be related to the 70 observations that iodine reduces the thyrotropic hormone of the norm al pituitary (119), and that iodine reduces the resp irato ry metabolism of thyroid tissue when exposed to the thyrotropic hormone (214). Astwood (8) has suggested that, in Graves disease, a normal diet is iodine deficient. Iodine administration would then act to reduce the goiter simply by supplying a normal amount of iodine, which would be followed by inhibition of the hyperplasia that occurs in iodine deficiency. Thiocyanate Thiocyanate administration produces all of the symptoms of myxedema, but they can be prevented through thyroxine ad­ m inistration (9). It has been shown that the thiocyanate ion is unique in that the uptake of iodine is reduced (228), whereas most goitrogens affect the conversion of iodide to thyroxine or the release of the hormone. In addition, Vanderlaan and Van- derlaan (215) have shown that inorganic iodide already present in the thyroid is discharged upon thiocyanate administration. The mechanism of the effect of thiocyanate is not known. At f ir s t it was thought that thiocyanate might be selectively adsorbed 71 by the thyroid. However, no accumulation of this ion has been found (8). Antithyroid Substances The anti thyroid substances include a large number of compounds, the most active of which contain either a thiocarbonamide grouping (thioureas) or aminobenzene grouping (sul­ fonamides). Both of these compounds are thought to exert their influence through preventing the oxidation of inorganic iodides which is necessary before they can be converted to diiodotyrosine and thyroxine. Again, the mechanism is not known. DeRobertis (58) suggested that thioureas inhibit the peroxidase system of the thyroid and that sulfonamides have a competitive action by which the iodine combines with it rather than with tyrosine. EXPERIMENTAL MATERIALS AND METHODS M aterials Plasm a, milk, and colostrum samples used in this study were for the most p a rt obtained from cows and calves of the Michigan State College dairy herds maintained at East Lansing. A lim ited number of plasma samples were obtained from cows in farm herds which were examined as sterility cases by Dr. J. A. Williams, of the college Department of Pathology. Dr. L. O. Gilmore, of the Ohio State College Agricultural Experi­ ment Station, also kindly supplied plasma from cows after calving, and their calves before and after firs t nursing. The blood samples were drawn from the jugular vein directly into a 15-m illiliter centrifuge tube containing 4 drops of 20-percent sodium oxalate as an anticoagulant. The blood was centrifuged as soon as possible after withdrawal and placed in a re frig e ra to r until the determination of iodine could be made. 73 Methods The protein-bound and inorganic iodine determinations were made according to the method described by Barker (10, 11), with slight modifications. The reagents and procedure will be described in detail for convenient reference. Reactions This method involves the separation of the protein-bound iodine fraction from the inorganic iodine by precipitation and washing. The precipitate is then digested and oxidized with chromic oxide and sulfuric acid, leaving the iodine in a highly oxidized inorganic state. The exact form of the iodine is not known, but is thought to be iodic acid. The iodic acid is then reduced with phosphorous acid and the volatile iodine (hydrogen iodide or elemental iodine) is distilled off and collected in a sodium hydroxide-arsenious acid solution. The colorim etric determination of iodine is based on its catalytic effect on the decolorization of eerie arsenious acid. sulfate by 74 Reagents Distilled water: Once-distilled water was used in making up all solutions, as well as in other phases of the work. (43) used water once redistilled over glass, while Barker Taurog and Chaikoff (42) took the precaution of redistilling over glass from alkaline solution. Satisfactorily low blanks were obtained in this laboratory, however, when once-distilled water taken from a Stokes still was used with no further purification. Tap water itself gave very low iodine values. 70-percent sulfuric acid: 780 m illiliters of concentrated sulfuric acid (C.P.) is slowly added, with cooling, to 600 m illi­ lite rs of water. 7 N. sulfuric acid: 196 m illiliters of concentrated sul­ furic acid (C.P.) is added to 600 m illiliters of water and the volume made up to one lite r. 3.5N. sulfuric acid: furic acid 49 m illiliters of concentrated (C.P.)is added to about 300 m illiliters of water sul­ and the volume made up to 500 m illiliters. 0.25 N. sulfuric acid: furic acid (C.P.)is added to volume made up to one liter. 7 m illiliters of concentrated sul­ 600 m illiliters of water and the 75 60-percent chromic oxide: 600 grams of chromic trioxide (CrO^) (C.P.) is dissolved in water and the volume brought to one lite r. 50-percent phosphorous acid: The contents of a one- pound ja r of phosphorous acid (C.P.) is weighed and dissolved in an equal weight of water. Sodium hydroxide-arsenious acid: 1.5 grams of arsenious acid (As^O^) (C.P.) is dissolved in 20 m illiliters of 5 N. sodium hydroxide (4 grams of NaOH in 20 m illiliters of water) and the volume made up to 100 m illiliters with water. Arsenious acid: 3.71 grams of arsenious acid is d is­ solved in 50 m illiliters of 1 N. sodium hydroxide (2 grams of NaOH in 50 m illiliters of water). Add 200 m illiliters of water and neutralize with 70-percent sulfuric acid (about 2.5 m illi­ lite rs). Add 54 m illiliters of 70-percent sulfuric acid and make up to 500 m illiliters. Dissolve 3.125 grams of sodium chloride (C.P.) in the above solution. Ceric sulfate: is 12.65 grams of eerie ammonium sulfate s tirre d into 500 m illiliters of water plus 230 m illiliters of 7 N. sulfuric acid. Make up to one liter. 76 Smogyi precipitating reagent: 12.5 grams of zinc sulfate (ZnSO^ - 7H^O) (C.P.) is dissolved in 125 m illiliters of 0.25 N. H^SO^ and the volume made up to 1 lite r. The £>&iogyi reagent and the 3-percent sodium hydroxide are so balanced that 50 m illiliters of the zinc sulfate solution requires 6.7 to 6.8 m illiliters of the sodium hydroxide to show permanent pink to phenolphthalein. Sodium iodide stock: Dissolve 1.181 grams of desiccator- dried sodium iodide (C.P.) in 1 lite r of water. lilite rs of this solution to 1 lite r. Dilute 100 m il­ This solution will contain 118.1 m illigram s of sodium iodide per lite r, or 100 micrograms of iodine per m illiliter. This stock solution is stored in a brown bottle and kept under refrigeration. Standard iodide so­ lutions containing 0.02 to 0.10 microgram of iodine per m illi­ lite r (or other concentrations as required) were obtained by proper dilutions of the stock solution. Procedure Precipitation and washing. A 2-m illiliter aliquot of plasma, milk, or colostrum was pipetted into a 50-m illiliter round-bottom pyrex centrifuge tube with an Ostwald-Folin 77 pipette. Sixteen m illiliters of Smogyi's acid zinc sulfate reagent (185) was added from a burette, and to this 2 m illiliters of 3- percent sodium hydroxide was slowly added from a pipette while the centrifuge tube was gently shaken. It was found that drop- wise addition of the sodium hydroxide and constant agitation of the centrifuge tube helped to obtain complete precipitation of the zinc-protein combination. With each serie s of protein precipitates, a blank p re ­ cipitation was made for use in preparing the standard curve described late r. The blank precipitate was made by adding 2 m illiliters of sodium hydroxide to 16 m illiliters of the p r e ­ cipitating reagent. The zinc-protein precipitates were centrifuged for ap­ proximately 10 minutes to obtain good compaction of the p r e ­ cipitate. The supernatant liquid was poured off and washed three times by re suspending the precipitate in 10 m illiliters of water and centrifuging after each washing. The blank precipitate and sim ilar precipitates used in the determination of total iodine were at f ir s t washed three times in the same manner. Tests showed, however, that the washings did not alter the blank value so, to conserve time, 78 the washings were la te r omitted on blank and total iodine de­ term inations. Digestion. Three m illiliters of chromic oxide were pipetted into a 250-m illiliter flask. The precipitate was d is­ solved in 5 m illiliters of 70-percent sulfuric acid and poured into the flask. The centrifuge tube was rinsed with four addi­ tional 5-m illiliter portions followed by one rinse with 5 m illi­ lite rs of distilled water. The blank precipitate and the precipi­ tates for determination of total iodine were sim ilarly tr a n s ­ fe rre d to the flask. In the case of total-iodine determinations, the 2 m illi­ lite rs of plasma, milk, or colostrum were now added directly to the flask. Considerable difficulty was encountered in digesting the colostrum and some milk samples, especially Jersey milk, due to the large amount of organic m aterial present. At firs t the milk and colostrum samples were centrifuged and determination made on the skimmed portion. tained on these Very low iodine values were ob­ samples and it appeared that some of the iodine was lost in the fatty portion which rose to the top. Later it was found that satisfactory results could be obtained by increasing 79 the amount of chromic acid used in the digestion. In obtaining the data reported here on milk and colostrum, 3 m illiliters of chromic acid were used on most milk samples and 6 m illiliters were routinely used with colostrum. In the few cases where these amounts were not sufficient for crystalization of the chromic oxide after digestion, the amounts of chromic oxide were further increased. No evidence was found that in c re a s­ ing the amount of chromic oxide had any untoward effect on the reaction. A few glass beads were added to the flask, a therm om e­ te r inserted, and the contents heated to 165° C. The flame was immediately removed when the desired temperature was reached, and the flask was set aside to cool. When the temperature fell below 100° C., 15 m illiliters of water and a few more glass beads were added and the digest reheated to 165° C. Then the flask was set aside to cool. Distillation. Distillation of the iodine was carried out in B a rk e r's modification of the Chaney-Riggs - Talbot still. The digest was tra n sfe rre d to the 250-m illiliter distillation flask by 25 m illiliters of water added in 5-m illiliter portions. glass beads were added and the still assembled. A few One-half of 80 a m illiliter of the arsenious acid-sodium hydroxide solution added through the top of the still so that it filled the region of the stopcock in the trap and 5 m illiliters of 50-percent phosphorous acid was pipetted into the bowl of a side-arm thistle tube. A Bunsen burner with a moderately low flame was placed under the distilling flask and the condenser inserted. In assembling the distillation apparatus it is essential that all glass joints be well lubricated with water to prevent freezing. Heating of the digest was continued until the condensed vapor started dripping into the return tube. The stopcock on the sid e-arm thistle tube was turned to perm it the phosphorous acid to drip into the reaction flask. It was not found necessary to blow the phosphorous acid out of the thistle tube, as described by B arker and Taurog and Chiakoff. Care was taken, however, to close the stopcock immediately after passage of the la st of the phosphorous acid. Heating was continued for 7 minutes after the addition of phosphorous acid was completed. At this time the flame was removed and the distillate immediately drawn into a test tube graduated to 25 m illiliters. tillate normally amounted to 7 to 9 m illiliters. This d is­ 81 The condenser was raised and the still rinsed with three 4 -m illiliter aliquots of water added in 2-m illiliter portions. These rinsings were added to the original distillate and the total volume made up to 25 m illiliters. The test tubes were sealed with parafilm and placed in a refrig erato r until the colorim etric determination of the iodine. Colorimetric determination of iodine. Four m illiliters of the distillate were pipetted into an Evelyn colorim eter tube; 1 m illiliter of water and 0.5 m illiliter of arsenious acid were added. Standard tubes for preparation of a standard curve were prepared by pipetting 4 m illiliters of the blank distillate into each of five colorim eter tubes. One m illiliter of water was added to the f ir s t tube and 1 m illiliter of sodium iodide solu­ tion containing 0.02, 0.04, 0.06, and 0.0 8 microgram of iodine per m illiliter were added to the other four tubes. These iodine dilutions were prepared by appropriate di­ lution of the stock solution and were found to be stable for periods of several months when kept refrigerated in tightly closed brown bottles. One m illiliter of eerie sulfate was added to the colorim ­ e te r tubes on a 30-second time schedule and the tube was 82 immediately placed in a water bath at 36.5° C. The tubes were incubated for variable lengths of time, depending upon the num­ ber in the serie s. No incubation time of less than 10 minutes or more than 20 minutes was used. About 20 seconds before the expiration of the incubation time the tubes were removed, wiped dry, and read in an Evelyn colorim eter at the same 30-second interval. The standard curve was plotted on two-cycle semilog paper and iodine values of the unknowns were determined from this standard. It was nec­ e ss a ry to multiply the results by the factors 6.25 and 50 to con­ v ert them to m icrogram s per 100 m illiliters of plasma. Notes on the Procedure Two general techniques have been recently used for the determination of iodine in plasma or tissue. One, described here, is a digestion-distillation procedure, while the other is an alkaline-ashing method. Both methods were studied while the w riter was attempt­ ing to m aster the technique of the microdetermination of iodine. It became apparent, however, that the ashing method could not be easily utilized in this study. 83 The ashing procedure of Barker and Humphrey (12) has much to recommend it. It appears to be simpler and less time-consuming than the distillation method and is said to give essentially the same re su lts. However, in order to utilize an Evelyn colorim eter it is necessary to increase the dilution of the dissolved ash. Whether due to this or some other factor, the author was unable to obtain satisfactory or consistent iodine values by this method in a long series of experiments. Two contributing difficulties were the lack of an ashing furnace which could be used only for this work, and the difficulty with which the transference of carbon particles into the colorim eter tubes was avoided. While no implications as to the accuracy of the ashing method for iodine determination are intended, it is the author's opinion that any deviation from the procedure as described by Barker et al (12, 13) will markedly affect the resu lts. The distillation method which was used in this work is not difficult to use after it has once been mastered. It is, however, a difficult technique to learn unless someone who is fam iliar with the procedure is present to oversee the details. 84 B arker (10) and Taurog and Chaikoff (192) have described the digestion phase as ending when the sulfuric acid fumes sta rt to form . Chaney (38) and Riggs (167) used digestion tem pera­ tures of about 200° C. in our hands. Neither of these were very satisfactory It was soon found that the heating time required for producing visible fumes varied with the type of digestion flask used. When a digestion tem perature of 200° C. was used, the chromic oxide-sulfuric acid-protein precipitate mixture turned green and, on cooling, no crystals of chromic oxide formed. The recovery of iodine from this digest was very low. In order to standardize the procedure, various tem pera­ tures from 140° C. to 200° C. were tried and the recovery of added iodide measured for each. The percentage recovery in­ creased to 170° C. and fell after a temperature of 180° C. was reached. Since the percentage was a little less than 100 at 160° C., and above 100 percent at 170° C., it was decided to use a digestion tem perature of 165° C. The recovery of sodium iodide, thyroxine, and radioactive iodine added to plasma, as described in the next section, were very satisfactory at this tem p eratu re. 85 In general, the digestion method appears to be much less sensitive to alterations in the amount or concentration of the reagents used than the ashing procedure. It has been observed above that the distillation technique is difficult to learn without instruction from someone who is well versed in the procedure. However, the mechanics of making the determinations have been successfully taught to several students in our laboratory who had little chemical background. Thus, for routine work, where some supervision can be given, skilled technicians are not necessary. Previous workers (38, 192) have mentioned encounter­ ing lots of phosphorous acid which appeared to be incapable of releasing iodine from the digest. On one occasion in this study a lot of phosphorous acid was used in which some bottles were active while the others were inactive. Acting on a suggestion by Dr. G. M. Curtis, of the Ohio State University, various amounts of manganese dioxide were added to the digest before the distillation step. It was thought that the manganese dioxide might catalyze the reduction of the digest by the inactive phos­ phorous acid, but it was found that the apparent iodine recovery varied with the amount of manganese dioxide added. No solution 86 of the problem of inactive phosphorous acid is known except that le ss trouble is said to occur if technical instead of C.P. grades of reagent are used. EXPERIMENTAL RESULTS Test of Method Recovery of Added Iodine The efficiency of this procedure in recovering iodine from the plasma of dairy cattle was estimated by determining the recovery of inorganic and radioactive iodine (I 131 ) and thyroxine added to precipitated and washed plasma protein. Table I shows that the recovery of the added iodine and thyroxine was accomplished very efficiently. In a series of experiments the recovery varied between 97.5 and 105.0, with an average of 100.9 percent. The percent recovery was checked with radioactive io­ dine added to the washed precipitate in the same manner. The range of recovery which was somewhat greater than that de­ term ined colorim etric ally can be (Table U) attributed, in part at least, to mechanical difficulties in preparing the distillate for m easurem ent of its radioactivity. Even so, the recovery of radioactive iodine averaged 98.5 percent. 88 TABLE I RECOVERY OF SODIUM IODIDE AND THYROXINE ADDED TO PLASMA PROTEIN No. of Trials M icrograms of Added Iodine M icrogram s of Recovered Iodine Percent Recovery 1 .04 .044 105.0 3 .06 .061 101.7 3 .08 .078 97.5 2 .10 .102 102.0 .066 98.5 1 Average .067 (thyroxine) 100.9 ± 2.7 89 T A B L E II RECOVERY OF RADIOACTIVE IODINE ADDED TO PLASMA PROTEIN Number in Serie s Average Counts per Second Trial No. Sample i Standard Distillate a 1 1 81.0 83.4 103.0 2 Standard Distillate 6 63.3 58.2 91.9 Jo Standard Distillate A 71.9 72.3 100.6 Percent Recovery 98.5 ± 4.8 Average 3. Each serie s was determined in triplicate. 90 Repeatability of Results During the course of this work, it was the practice to occasionally in sert duplicate or triplicate plasma samples into a series in order to provide a check on the repeatability of iodine recovery in routine analysis. The results of one such check, more extensive than most, are shown in Table III. Even during routine iodine determinations, the results were reason­ ably consistent and the variation less than 10 percent. It has been our experience in several hundred analyses of plasma protein-bound iodine that the method used gives ac­ curate and repeatable results and does not require the services of a skilled laboratory technician. However, untrained p erson ­ nel require constant supervision until they learn the mechanics of the procedure. Furtherm ore, they must be im pressed with the need for accuracy in every step; particularly in the colori­ m etric determination of iodine. 91 T A B L E III RESULTS OF NINE CONSECUTIVE PROTEIN-BOUND IODINE DETERMINATIONS ON ONE LOT OF PLASMA Sample No. Average Colorimeter Reading PBI Y % 1 18.5 5.1 2 18.6 5.2 3 17.3 4.3 4 19.0 5.5 5 17.9 5.0 6 18.9 5.3 7 18.6 5.2 8 18.1 5.0 9 19.0 5.5 5.1 ± .37 92 P lasm a Protein-bound Iodine of Dairy Cattle Age Changes Between June 12, 1951, and March 3, 1952, 296 d e te r­ minations were made of the plasma concentrations of proteinbound iodine of 177 cows and calves in the Michigan State College dairy herds. The resu lts, arranged according to the ages of the animals, are shown in Table IV. The over-all average of the 296 determinations was 6.8 m icrogram s percent. An inspection of these data reveals, how­ ever, that an average value, not qualified by a consideration of age, has little meaning. These data show that the organic iodide fraction of plasma tends to decrease with age. However, for convenience, the age changes can be grouped into four periods, each with a characteristic iodine concentration. Thus, in this study, calves averaged 12.8 micrograms percent of plasma p ro ­ tein-bound iodine during the f ir s t 2 days after birth. the re s t of the f ir s t During 12 months the average concentration was 7.3 m icrogram s percent. From 13 to 24 months the average was 6.2 m icrogram s percent, while the level in cows over 24 months old was 4.6 m icrogram s percent. It is therefore pointless 93 T A B L E IV AGE CHANGES IN THE PLASMA PROTEIN-BOUND IODINE CONCENTRATION OF DAIRY CATTLE Number of Animals Number of Dete rminations 22 22 14.8 24 - 48 hours 8 8 10.8 3 -4 days 8 9 7.9 5 -7 days 9 9 7.6 24 34 6.9 Age Under 24 hours 8 days - 1 month Average r % 1 -3 months 18 18 6.2 4 -6 months 11 17 8.1 7 - 12 months 21 27 7.3 13 - 10 27 6.8 months 16 33 5,7 25 - 36 months 12 31 4.7 37 - 48 months 4 7 4.6 49 - 72 months 8 33 4.6 73 months or over 6 21 4.4 177 296 6. 8 18 months 19-24 Total or Average 94 to give an average plasm a protein-bound iodine value without specifying the age of the animal concerned. Although these averages show distinct age differences in the plasm a iodine concentration, there is a considerable amount of overlapping in the range of values one obtains in the different age groups. Figures 1 through 5 show the range of values and the frequency of each in the 177 animals tested and in each age division. In Figure 1 the range of 248 plasma protein-bound iodine concentrations obtained on 130 dairy animals over 1 week old are given. Although the values varied from 2.1 to 17.2 m icro­ grams percent, more than 80 percent of the concentrations were under 8.0 m icrogram s percent. When the distribution of values is broken down into the four age groups mentioned above, the effects of age can be seen. The values of calves under 48 hours old seldom fall below 8.0 m icrogram s percent (Figure 2) or rise above about 18.0 m icro ­ gram s percent. regular. Within these lim its the distribution is fairly An abrupt decrease in the average plasma protein- bound iodine concentration occurs soon alter the second day. This is illustrated in Figure 3, where it is shown that 84.0 95 Figure 1. The distribution of 248 plasma protein-bound iodine determinations on 130 dairy animals over 1 week old. The values ranged from 2.1 to 17.2 m icrogram s percent with 80 percent of them under 8.0 m icro ­ grams percent. 96 50.0 - PERCENT 40.0 - 300 - 200 - 10.0 - 2 .0 - 3 9 4.0 - 5.9 6.0 - 7.9 8.0 - 9.9 I0.0-JI.9 12.0-13.9 MICROGRAMS PERCENT I4.0-IS.9 16.0-17.9 97 Figure 2. The distribution of thirty plasma protein-bound iodine determinations on thirty calves under 48 hours old. The concentrations varied from 6.0 to 29.7 m icro ­ grams percent and averaged 12.8 m icrogram s percent. 98 25.0 PERCENT 20.0 15.0 10.0 5.0 J 0.0 6 .0 - 7.9 8.0 “ 9.9 10.0-11.9 (2.0 -13.9 14.0-J5.9 16.0-17.9 22.0-23.9 28.0-29.9 MICROGRAMS PERCENT 99 Figure 3. The distribution of 114 plasma protein-bound iodine determinations on ninety-one calves between 48 hours and 12 months old. The values ranged from 2.7 to 18.0 micrograms percent and averaged 7.3 m icro grams percent. 100 30.0 - P E RCE NT 25.0 - 20 0 - 15.0 - 10.0 - 2.0 - 3.9 4. 0 - 5 .9 6 .0 -7 9 8.0-9.9 10.0-11.9 12.0-13.9 14.0-15.9 16.0-17.9 18.0-19.9 M I C R O G R A MS PERCENT 101 Figure 4. The distribution of sixty plasma protein-bound iodine determinations on twenty-six heifers old. 13 to 24 months The range of values was from 3.0 to 15.3 m icro- grams percent with an average of 6.2 m icrogram s percent. PERCENT 102 3QO 20.0 10.0 n 0.0 0.0 2.0 - 3.9 4.0 - 5.9 6.0 - 7.9 8.0- 9.9 J2.0-I3.9 14.0“ /5.9 MI CROGRAMS P E R C E N T 103 Figure 5. The distribution of ninety-two plasma protein-bound iodine determinations on thirty cows over 24 months of age. The values averaged 4.6 m icrogram s p e r ­ cent with a range of 2.1 to 16.5 m icrogram s percent. 104 60.0 - 50.0 ~ PERCENT ♦ 0.0 - 30.0 - 20.0 10.0 “ “ 2 .0 -3 .9 4 .0 -5 .9 6.0 - 7.9 8.0 -9 .9 (6.0-17.9 M I C R O G R A M S PERCENT 105 percent of the determinations on calves between 48 hours and 12 months old were less than 10.0 m icrogram s percent. In addition, the values fell as low as 2.7 micrograms percent. The downward trend continued in heifers between 12 and 24 months of age (Figure 4) with 85.0 percent of the values below 8.0 m icrogram s percent. The concentration of cows over 24 months of age was generally still lower (Figure 5). In this group, nearly 90 percent of the values were less than 6.0 m icro­ grams percent. In view of these data, it is suggested that a certain range of plasma protein-bound iodine concentrations be consid­ ered normal for animals of each of the different age groups. These suggested ranges are shown in Table V, and were selected so as to include 85 to 90 percent of the determinations in each case. Calves Before and After F irs t Nursing Since the protein-bound iodine concentration in the plasma of young calves was considerably higher than in older animals, a more intensive study was made of this age group. 106 TABLE V NORMAL RANGE OF PLASMA PROTEIN-BOUND IODINE CONCENTRATIONS OF DAIRY ANIMALS AT DIFFERENT AGES Age No. of Animals No. of D eterm i­ nations Avg. r% Suggested Normal Range r% % of Determi­ nations Included Under 48 hours 30 30 12.8 8. 0 - 18.0 86.7 48 hours 12 months 91 114 7.3 3.5 - 12.0 87.7 13-24 months 26 60 6.2 3.5 - 10.0 85.0 Over 24 months 30 92 4.6 3.0 - 8.0 87.0 107 As shown in Table VI, the level of organic iodine in the newborn calf before it has nursed is approximately the same as is found in older calves. Once the calf has had an oppor­ tunity to nurse, however, the organic iodine of the plasma in­ creases markedly. Two other phenomena associated with calv­ ing which the w riter feels are of significance are that colostrum contains a much larg er amount of iodine, particularly organic, than milk (see Table VII) and that the plasma protein-bound iodine of the dam is significantly lower on the day of calving than either before or after parturition (Table VIII). F u rth er­ more, colostrum is known to contain large amounts of globulin and albumin, while milk contains only traces of these proteins ( 6 1 ). T-n view of this, it is believed that the high postnursing protein-bound iodine concentration in the plasma of dairy calves can be explained by the following: Shortly before calving, the mammary gland is unusually permeable to globulin and albumin. This sudden drain depletes the circulating protein of the cow and also her circulating thyroid hormone, which is attached to the albumin and globulin fractions of the blood. As the calf nurses, it injests enough iodine to raise its own level of organic 108 T A B L E VI PLASMA PROTEIN-BOUND IODINE LEVELS IN CALVES BEFORE AND AFTER FIRST NURSING Micrograms Percent P.B.I. Calf No. Before Nursing After Nursing Percent Increase A 7.1 8. 2 15.5 B 11. 1 12.5 12. 6 C 5.9 13.9 101.7 D 9.3 14.9 60.7 E 14.1 29.7 110.6 F 6.9 15.6 1 26. 1 G 7.7 10.5 36.4 Average 8.9 15.0 66. 2 109 T A B L E V II THE IODINE CONCENTRATION OF MILK AND COLOSTRUM Milk Sample No. FBI r % Colostrum Total Iodine r % FBI 7 % Total Iodine r % 1 4.5 12. 2 - 20.2 2 6. 1 13.6 - 28.8 3 - 8. 0 25.0 30.9 4 - 8.1 21.9 - 5 5.4 7.2 * * 6 - 9.0 * * 7 - 9.9 * * 8 7.2 9.3 9 6. 1 10 - - 6.7 * Iodine concentrations above 35.0 micrograms p e r ­ cent. 110 T A B L E V III CHANGES IN PLASMA PROTEIN-BOUND IODINE LEVELS OF COWS AT CALVING TIME PBI Level in Micrograms Percent ow No. Before Calving At Calving After Calving 5 3.8 2.6 4.1 15 4.1 1.4 5.0 17 3.8 2.5 3.7 101 4.5 2. 0 4.8 115 4.1 3.0 6.7 128 4.1 3.5 3.7 132 6.3 2.3 5.4 134 5.1 2.9 3.9 4.5 2.5* 4.6 K Average * Highly significantly lower than levels before or after calving (T = 5.30). Ill iodine to a relatively high level. It is well known that the colostrum quickly changes to normal milk and, as that occurs, an equally marked fall in the organic iodide in the calf's plasma takes place due to excretion or thyroidal storage of the iodine. While the above is conjecture, it is supported by the histological appearance of the thyroid gland during the firs t few days of extrauterine life. Plate 1 shows the histology of the gland at 12 hours, and 3, 4, and 6 days of age (see also IX). Table At 12 hours after birth there was little evidence of colloid storage or secretory activity of the gland. small and relatively free of colloid. The follicles were The secretory cells were of the columnar type associated with a relatively inactive gland. These results do not agree with those of Koneff and his associates ( 1 1 1 ), who found a marked increase in colloid storage as fetal calves neared term. Since only one calf was sacrificed in the p resent study, it is quite possible that its thyroid gland was unusually inactive or deficient in iodine. the other hand, there may be a reduction in stored hormone at birth. At three days, a marked shortening of the secretory cells and filling of the follicles with colloid had taken place. On 112 Plate 1. Sections of thyroid glands of calves at various times after birth. A. The thyroid gland of a calf 12 hours old. The follicles were few and nearly devoid of colloid. The secretory cells were columnar and poorly defined. The general appearance of the gland was one of inactivity. B. A section from a calf 3 days old. The follicles were somewhat more numerous and turgid than those in the younger calf. The amount of col­ loid in the follicles had increased. The se c re ­ tory cells were more cuboidal. C. A section from a 4-day-old calf. The numerous follicles were well filled with colloid. Little un­ differentiated tissue remained in the gland. D. A section from the thyroid from a 6 -day-old calf. The follicles were filled with deep- staining col­ loid. The secretory cells were well defined and no undifferentiated tissue remained in the gland. All glands presented a normal gross appearance. Magnification: Each division on the scale equals m icrons. 10 114 T A B L E IX PLASMA PROTEIN-BOUND IODINE CONCENTRATION OF CALVES WHOSE THYROIDS WERE EXAMINED HISTOLOGICALLY Concentration of Iodine Age 191 23.8 12 hours* 193 9.8 6 hours 15.5 30 hours Calf No. r% 3.1 149 22.7 8.3 192 80 hours* 6 hours 3 days 11.7 4 days* 3.7 6 days * * Time of thyroidectomy. 115 Presum ably this indicated a g reater secretory and storage ac­ tivity on the p art of the gland. By the fourth and sixth days the follicles were numerous and distended with colloid. The glands gave every indication of being in a normal, active condition. Whether the organic iodine obtained from the colostrum is actually thyroxine is not presently known. Thyroxine has been the only major form of organic iodine found in the circulation of ra ts, dogs, and other laboratory animals, but no information is available on dairy cattle. It is known that iodine can be organi­ cally combined in the blood without passing through the thy­ roid and, since cattle rations are often supplemented with iodized salt, they may represent an exception. Normally, the mammary gland is impervious to thyroxine, but it is unreasonable to ex­ pect this to be true at a time when albumin and globulin appear to penetrate it in large amounts (61). At any rate, the colostrum appears to supply massive amounts of precipitable iodine to the young calf, which stores it in its own thyroid gland. The physiological significance of these data is obscure. At present, one can only suggest that they represent one of the 116 protective mechanisms whereby the calf is prepared to meet the s tre s s e s imposed upon it during its adjustment to the new environment. Jerse y and Holstein Cows Plasm a samples were obtained on four different occasions from seven Jerse y and nine Holstein cows from one of the col­ lege herds. The collection dates (June, August, November, and March) correspond roughly to the four seasons, although they were not selected with this in mind. over All of the animals were 18 months old and, with two exceptions, were in milk on one or more of the sampling dates. Cow K 104 had milked previously, but was dry and open during this time, and K 131 calved for the f ir s t time after the last bleeding date. The results of the protein-bound iodine determinations are shown in Table X. The statistical analysis summarized in Table XI showed no significant difference between cows or breeds. No significant seasonal differences were found for the Jerse y cows, although the November level was lower than in other months. The concentration of the Holstein cows was also lower at the November and March samplings than at the 117 TABLE X PLASMA PROTEIN-BOUND IODINE LEVELS IN JERSEY AND HOLSTEIN COWS OVER EIGHTEEN MONTHS OLD Sampling Dates Herd No. Breed Age in Mo s. 6-12 1951 r% K 5 K 10 K101 K104 K105 K115 K132 J J J J J J J K 15 K 17 K 18 K 19 K 20 K109 K128 K131 K134 Average H H H H H H H H H r% 72 72 54 52 47 34 5.1 4.8 4.8 4.2 6.9 3.6 5.1 4.8 6. 0 5.7 5.1 4.5 22 6. 0 6. 6 5.19 5.27 ± 50 Average 8-14 1951 72 66 68 48 55 44 23 19 19 46 0.87 0.66 5.7 3.0 5.1 3.0 3.9 4.2 5.7 7.5 8.4 4.8 5.4 5.17 ± 3.12 8.1 3.0 5.1 5.7 5.4 7.2 6.3 5.67 ± 1.86 11-12 1951 r% 3-18 1952 Average r% r% 3.8 4.9 4.5 3.6 5.0 4.1 4.4 4.1 4.4 4.8 5.4 4.9 6.7 5.4 4.45±0.28 4.58±0.13 5.55±0. 8 8 4.65±0.27 5.02±0.06 5.02±0.05 5.60 ±0.6 6 4.33 ± 0.23 5.10 ± 4.97±0.76 3.3 3.8 3.8 4.4 3.0 4.2 4.1 5.2 5.1 4.10 ± 0.48 0.62 4.1 3.7 4.7 4.6 6. 0 5.0 3.1 4.8 3.9 4.50±0.56 3.98±0.73 5.42±2.66 3.75±0.57 4.50±0.31 4.78±0.34 4.58±1.04 6.18±1.35 5.92±2.82 4.44 ± 4 .84±1.93 0.58______________ 118 T A B L E XI SIGNIFICANCE OF VARIATIONS IN PLASMA PROTEINBOUND IODINE LEVELS OF JERSEY AND HOLSTEIN COWS Degrees of Freedom Jerseys Cows Season 6 Holsteins Cows Season 8 Combined Breed Season Sum of Square s Mean Square 00 00 Source of Variation F 1.35 2.03 5.29 3.96 1.32 2.78 4.51 2. 01 3 22.24 13.53 1 . 26 . 26 .13 3 15.18 5.06 2.49 3 * Significant at the 5-percent level. 3.27* 119 June and. August bleedings. The seasonal variation was s ta tis ­ tically significant in this breed. The considerable variation in iodine concentration of the same cow on different dates would lead one to wonder if the variability was due to a real differ­ ence, or if it was m erely a reflection of the inadequacy of a single sample for expressing a cow’s protein-bound iodine level. In order to test this, samples were drawn from a cow on 6 con­ secutive days, and the iodine concentration determined (Table XII). From these data it may be observed that a single plasma sample is sufficient to determine the approximate level for a cow, but two or more samples should be taken for exact infor­ mation. The iodine values reported here are somewhat higher than those reported by Long et al. (120) and Reece and Man (156). The form er workers reported that Jersey and Holsteins averaged 4.11 and 2.73 micrograms percent, respectively, while the la tte r found Jerse y cows averaged 4.6 micrograms percent. In the present study the average levels for Jersey and Holstein cows were 4.97 and 4.84 micrograms percent, respectively. The Ohio workers reported their breed differences to be statistically significant. 120 T A B L E X II DAY-TO-DAY CHANGES IN PLASMA PROTEIN-BOUND IODINE CONCENTRATION IN A JERSEY COW Sampling Date May Colorimeter Reading Micrograms Percent PBI 8 16. 2 3.7 9 16.3 3.7 10 16. 0 3.6 11 15.3 3.3 12 16.9 4.2 13 16.0 3.6 Average 3.65 ± .32 121 The fallacy of comparing animals of different ages has previously been pointed out, and insufficient data is available for determining the ages of the animals used in these studies. In spite of this, it is evident that the concentrations reported by the Ohio workers are decidedly below those obtained in this laboratory. Whether these differences were due to the herds studied or were inherent in the techniques used is not known. However, one plasma sample was exchanged between the two laboratories for comparative analysis. Our value was approx­ imately 10 percent higher than the one reported by the Ohio workers. The possibility of a difference in the organic iodine con­ centration in the plasma of cows of the same breed in differ­ ent herds is illustrated by the results obtained when plasma iodine determinations were made on a group of Jersey cows recently moved from California to the Michigan State College campus. These cows were brought to the college farm in August, I 9 5 J, and were sampled on September 24 and November 6 . plasm a levels of the cows over are shown in Table XIII. The 18 months of age in this herd The concentration of organic iodine 122 T A B L E X III PLASMA PROTEIN-BOUND IODINE LEVELS OF A HERD OF JERSEY COWS BROUGHT FROM CALIFORNIA Sampling Dates Herd No. Age in Months Average 9-24-51 11-6-51 r% r % r% 332 77 2. 6 4.3 3.4 C30 71 3.6 4.3 4.0 C47 59 1.5 2.4 2. 0 C51 55 2.7 3.9 3.3 C62L 44 4.8 2.6 3.7 C62R 43 5.4 3.3 4.4 C79 31 3.3 4.0 3.6 C84 24 3.3 3.2 3.2 C85 24 1.5 4.2 2.8 C89 22 3.0 4.7 3.8 C98 20 3.0 4.4 3.7 327 78 2. 6 3.3 3.0 349 64 2. 0 3.4 2.7 358 54 2. 0 3.3 2.6 3.0 ± .90 3.7 ± .19a Average 49.0 3.3 ± .38b a Differences between dates were not significant. b Significantly different from cows in Table IV. (t for November 6 and 12 = 2.07. t of over-all averages = 2.93.) 123 in the plasm a was unexpectedly low at the firs t sampling, and was only a little higher in November! not significant. The differences were The average value from both samplings was highly significantly less (t = 3.92) than the over-all average of the Jersey and Holstein cows previously discussed. When the samples of November 6 were compared with the values ob­ tained on November 12 (Table IX), the difference was less, but was still significant at the 5-percent level (t = 2.0 7). Whether the difference between the two herds was due to genetic or environmental factors is not known. It might be postulated that cows from areas bordering the seacoast (where the iodine content of the feed is entirely adequate to meet the animals' needs) have thyroid glands which are not immediately capable of secreting normal amounts of hormone when the cow is moved to an iodine-deficient area and placed on a ration containing no supplemental iodized salt, as these were. After a time hypertrophy of the gland occurs and the amount of thy­ roxine secreted increases, thus causing a rise in plasma pro- tein-bound iodine. On the other hand, the possibility exists that the stre ss of shipment for a long distance produced a temporary depression 124 of thyroid, activity which, after a period of adjustment to the new environment, will return to normal. It is hoped that another series of determinations can be run to see if, after several months of adjustment, the plasma iodine levels of the California Jerseys have reached those of comparable cows from the main college herd. Until that is done, the possibility of true genetic differences between these herds is not ruled out. fornia Jerseys are The Cali­ relatively highly inbred, and it is not unlikely that the endocrine system has been affected in the process. With regard to the effects of pregnancy and lactation on the organic iodine fraction of plasma, no relationship could be established with the limited data of this study. Cases of Sterility During the course of this work, a group of cows being studied as sterility cases by members of the college's pathology and physiology departments were sampled. All of these cows had histories of many breedings without conceiving, but none of them exhibited organic or pathological indications of inability to be impregnated. Some of the cows had been brought to the college and placed on a good ration as a prelim inary to further 125 treatm ent; others were sampled on the farm. Of nineteen cows, four had been pronounced pregnant by a veterinarian when sam ­ pled. The average values obtained are shown in Table XIV. The values obtained are markedly lower than those of the Hol­ stein and Je rse y cows in Table IX (t —3.23, which is highly significant). The values are very sim ilar to those obtained on the Jerse y cows from California. No one has yet shown any close connection between thy­ roid activity and reproduction of dairy cattle. Even thyroidec- tomized cattle have been capable of reproduction, even though estrus was not observed. It has been mentioned previously, however, that the ovaries appear to exert a marked influence on plasma proteinbound iodine. In particular, the plasma level is maintained in thyroidectomized females (143), and limited estrogen adminis­ tration increases thyroxine secretion (233). In view of these relationships, it is possible that, in some cases of sterility due to hypoovarian function, the usually low plasma proteinbound iodine concentration is due to hypogonadism rather than to hypothyroidism per se, although it is not known if alterations 126 T A B L E XIV M I C R O G R A M S P E R C E N T O F P R O T E I N - B O U N D IO D IN E IN T H E P L A S M A O F " S T E R I L E " CO W S Animal No. Breed Age in Years No. of Determi­ nations Avg. Y % Notes API G 4 1 3.9 Open S2 J 3 4 3.9 Open S5 G 3 2 4.3 Pregnant S6 H 4 1 2.7 Pregnant S7 H 5 1 3.9 Pregnant S9 H 2 1 4.2 P r e gnant S10 J 3 3 4.0 Open S ll J 3 1 5.4 Open S 12 H 4 3 3.5 Open S13 H 2 4 4.4 Open S14 J 2 2 3.4 Open S15 H 2 5 4.8 Open S16 J 3 1 3.6 Open 87IX J 2 1 3.3 Open 908X H 4 1 4.5 Open 9 14X H 2 1 3.0 Open 915X-1 H 2 1 3.0 Open 9 15X-2 H 2 1 3.3 Open 915X-3 H 2 1 2.1 Open Average 3.7 ± .93* * Highly significantly different from cows in Table IV (t = 3.23). 127 in ovarian activity, short of oophorectomy, are capable of in ­ fluencing the plasma iodine concentration. Identical Twins Five pairs of twin heifers believed to be identical on the basis of markings, hair sworls, and blood type were sam ­ pled on several occasions. It was thought that the plasma protein-bound iodine levels of the identical twins might be quite sim ilar. However, the results, summarized in Table XV, show that the values obtained on these animals were no more alike than those from unrelated animals. Each p air of twins, with the exception of P a ir 4, were on identical rations and maintained under the same environ­ mental conditions. The w riter knows of no data on identical twins of any species in which their rates of thyroxine se c re ­ tion are compared. It appears that their identical inheritance does not necessarily extend to the functioning of the endocrine system which is subject to modification by environmental in­ fluences. 128 T A B L E XV PLASMA PROTEIN-BOUND IODINE LEVELS OF IDENTICAL TWIN HEIFERS Sampling Dates Ani­ mal No. Twin P a ir Breed Age in Mo s. 6-12 6-26 1951 1951 7-10 1951 Y% r% Y % 7-24 1951 8-22 Y% r% 1951 Avg. r% T 1 1 H1 22 6.9 5.7 6.6 3.0 3.6 5.2 T 2 1 H 22 4.2 12. 6 7.2 5.1 4.5 6. 6 T 3 2 H J2 16 15.3 8.1 9.9 6. 0 7.5 9.4 T 4 2 H J 16 12.9 8.1 7.2 5.7 3.0 7.4 T 5 3 H J 17 4.8 - 6.6 3.6 5.1 5.0 T 6 3 H J 17 7.5 6. 6 7.8 4.5 4.2 6.1 T 7 4 3 G H 32 4.5 9.0 5.4 2.1 2.7 4.7 T 8 4 G H 32 4.2 16.5 5.4 2.7 3.6 6.5 T 9 5 H 22 - - - - 4.8 4.8 T 10 5 H 22 - - - - 3.3 3.3 1 Holstein. 2 3 H olstein-Jersey cross. Gue rnsey-Hoi stem cross. DISCUSSION Reece and Man (156) have recently cast doubt upon the validity of the plasma level of protein-bound iodine as a m ea­ sure of thyroid activity in dairy cattle by showing that two breeds of cows with different protein precipitable iodine levels had the same concentration of butanol extractable iodine in the plasma. The organic iodine level in the plasma has been thoroughly demonstrated to be a reliable indicator of thyroid activity in humans, dogs, rats, rabbits, and other laboratory animals, and it is difficult to understand why cattle should be an exception. Even though cattle rations are frequently supplemented with iodized salt, it seems unlikely that the amount usually supplied would cause any significant rise in the nonthyroidal plasma protein-bound iodine. In this connection, no effect could be ob­ served when cows in this study which had not previously r e ­ ceived iodized salt were supplemented with it. This is, of course, considered to be an iodine-deficient region, and the amount of iodine in the supplemented diet may have been no 130 more than adequate. Perhaps the results would have been dif­ ferent if the supplemental iodine had been in excess of the anim als' needs. There is little doubt but what, if sufficiently large amounts of iodine are injested, it is possible to build a concentration of nonthyroactive organic iodine in the plasma sufficient to mask any malfunctioning of the thyroid gland. Ob­ viously, the relation of the protein-bound iodine in dairy cattle plasm a to the circulating thyroid hormone must be thoroughly in ve s tig ate d. Nevertheless, it has been clearly shown that the plasma protein-bound iodine concentration of cattle in the herds studied generally fell within certain well-defined ranges. There would seem to be no good reason why these ranges should not apply to cows in other herds as well. Until evidence to the contrary is forthcoming, it is suggested that cows on sim ilar levels of iodine intake who deviate markedly from these tentative ranges do so because of thyroid hypo- or hyperfunctioning. On the basis of the data reported here, it is proposed that the following ranges of plasma protein-bound iodine con­ centration be considered normal for dairy cattle: 48 hours of age, calves under 8. 0 to 18.0 micrograms percent; animals 131 between 2 days and 12 months old, 3.5 to 12.0 micrograms percent; animals 13 to 24 months of age, 3.5 to 10.0 micrograms percent; and cows over 24 months old, 3.0 to 8.0 micrograms percent. It is nearly impossible to determine a normal range for young calves because of the variable effect of colostrum. Their iodine levels may be even less than 8.0 m icrogram s p e r ­ cent if the blood sample is taken before the calf has nursed, and after nursing it may exceed 2 0 . 0 microgram s percent if the colostrum is rich in iodine. The iodine concentration in the plasma above 8. 0 to 12. 0 microgram s percent appears to have little or no relation to thyroid activity at this age. Since no studies of the effect of hypo- or hyperthyroid­ ism on the plasma level of protein-bound iodine in cattle have been conducted, it is impossible to relate these pathological conditions to the suggested normal ranges. It may be found that these ranges can be considerably enlarged before thyroid dysfunction is encountered. It is known that cows receiving thyroprotein (protamone) at the rate of 1 gram daily per 100 pounds of body weight show protein-bound iodine concentrations of more than 20.0 micrograms percent. Undoubtedly, as the 132 relationship of the protein-bound iodine in the plasma to thy­ roid activity is better understood, some revision of these ranges will be advisable. In spite of the well-known effect of thyroxine or thyro­ protein administration on milk and butterfat production, no r e ­ lationship between the organic iodine level of the plasma and production of the cows studied was found. Neither did there appear to be any close relationship to the stage of pregnancy. This is not to say that there is no correlation, since the num­ b ers were too limited and the range of production too small for the data to be in any way conclusive. The question of genetic differences in thyroid activity of cattle is intriguing. If the differences in the plasma protein- bound iodine between cows are related to their productive abil­ ity, it may at some future date be possible to select potentially high-producing cows from their protein-bound iodine levels as calves. Or, perhaps one of the causes of the phenomenon of "nicking ’ 1 is the fortunate mating of a bull and cow whose r e la ­ tively high degree of thyroid functioning is transm itted to their offspring. 133 The little effect of season on the iodine values found in this work is not surprising, in view of the small amount of hot summer weather which occurs in this area. There is little reason to doubt that the hot summer months of more southerly states would cause a depression of thyroid activity, and in those areas seasonal changes in the plasma protein-bound iodine may be expected. The recent use of thyroprotein (protamone) as a stimu­ lant to milk production has been a cause of concern to some because of the possibility of certain unscrupulous dairymen using it on test cows. Such practices may easily be detected through determination of the plasma or milk iodine. The con­ centration of iodine in both milk and plasma is far greater than would be expected in untreated animals. Furtherm ore, the iodine level does not fall to normal for a period of days or weeks after administration of the drug ceases. The areas of future work in this field seem well de­ fined. F ir s t, it is essential that the protein-bound iodine com­ pounds in dairy-cattle plasma be identified and the relation of the plasm a organic iodine to thyroid activity established. If the relationship is close, then the factors affecting the plasma 134 protein-bound iodine concentration and the relation of this iodine level to performance of the animal must be thoroughly examined. Until this is done, the value of the plasma concentration of protein-bound iodine as a measure of thyroid activity in dairy cattle will be open to question. However, regardless of the final conclusion regarding the value of the plasma protein-bound iodine as a measure of thyroid function in dairy cattle, it will be interesting to pursue this work further and investigate the relation of the organic iodine level to the performance of the animal. The relation of nutrition and many other environmental factors to the protein-bound iodine concentration of cattle must be studied. The correlation between this iodine level in the plasma and the rate of growth or fattening, breeding efficiency, milk production, and even longevity would be valuable. The effects of estrus, gestation, production, tem perature, light, sex, and breed on the plasma protein-bound iodine should be known in order to properly evaluate the data obtained from these studies. It is one of the functions of the research er in dairy production to devise means whereby the dairy animal can produce 135 food with g reater efficiency. The plasma level of protein-bound iodine may become a useful indicator of the probable p erfo rm ­ ance of an individual animal. vestigations Until this is resolved, these in­ should be given every encouragement. SU M M A R Y A ND CO NCLUSIONS The plasm a concentration of protein-bound iodine of dairy cattle has been shown to vary with the age of the animal. after birth, the level rise s Soon as a resu lt of the injestion of colos­ trum , which initially contains a large amount of iodine, most of which is in organic combination. This is accompanied by a corresponding drop in the protein-bound iodine level of the dam's plasma. After the second day, the iodine level of the calf's plasm a falls to one 18 months. which is maintained for the f ir s t year to A small decrease is observed between 18 months and 2 years, and a further fall occurs during the productive life of the animal. It is suggested that normal ranges of protein-bound iodine be set as follows: Calves under 48 hours old, 8.0 to 18.0 m icrogram s percent; animals up to 12 months of age, 3.5 to 12.0 micrograms percent, those 13 to 24 months old, 3.5 to 10.0 microgram s percent; and cows over 24 months old, 3.0 to 8.0 micrograms percent. These ranges will probably require adjustment as more data become available. 137 Considerable variation in the organic iodine levels of cows sampled at different times of the year were found, a l­ though the seasonal changes were significant only for the Hol­ stein breed. The plasm a protein-bound iodine levels of the Jersey and Holstein cows in the same herd were not significantly dif­ ferent. The iodine levels of cows from other herds were dif­ ferent. Several cows with histories of poor conception rates were found to have low plasm a protein-bound iodine concentra­ tions. The significance of this is not known. Identical twin heifers were no more alike in their plasma organic iodine levels than nonrelated animals. No relationship of the plasma protein-bound iodine con­ centration to gestation or lactation could be found from the limited data of this study. Initially colostrum is high in protein-precipitable iodine, due probably to the infiltration of plasma proteins into the ud­ der. This provides the newborn calf with a relatively rich source of iodine, which photomicrographs indicate is stored in the thyroid gland. The physiological importance of this iodine 138 is not known. It is suggested that this is one of the means whereby the calf is prepared for adjustment to its new environ­ ment. Normal milk is much lower in total iodine than colostrum, although the inorganic fraction remains relatively constant. The need for further work in identifying the organic iodine compounds in the plasma and establishing the relation­ ship of the plasma protein-bound iodine to thyroid function in dairy cattle is emphasized. BIBLIOGRAPHY Albert, A Rawson, R. W., M errill, P ., Lennon, B., and 1946 Riddell, C. Reversible Inactivation of Thyro­ tropic Hormone by Elemental Iodine. 1. Action of Iodine. J. Biol. Chem., 166:637-647. The Biochemistry of the Thyrotropic Hormone. Ann. N. Y. Acad. Sci., 50:466-490. 1949 9 1951 ... 1951 and Tenney, A. Effect of Iodide, Thiouracil and Thyroxine on Disappearance of Thyroidal jl31^ P roc. Soc. Exp. Biol, and Med., 77:202203. 9 and Lorenz, N. Effect of Hyp ophy sec tom y on Intrathyroidal Metabolism of I ^ ^ . Proc. Soc. Exp. Biol, and Med., 77:204-205. The Effect of Thyro­ Anderson, E. M., and Evans, H. M. tropic Hormone Combined with Small Amounts 1937 of Iodine Upon the Function of the Thyroid Gland. Am. J. Physiol., 120:597-603. Some Effects of Thyroprotein on the Archibald , J. G. Composition of Milk. J. Dairy Sci., 28:941-947. 1945 Aron, M. Action de la Prehypophyse sur la Thyroide Chez le Cobaye. Compt. rend. Soc. de. Biol., 1929 102:682-684. Mechanisms of Action of Various Anti­ Astwood, E. B. thyroid Compounds. Ann. N. Y. Acad. Sci., 1949 50:419-443. B arker, M. H. The Blood Cyanates in the Treatment of 1936 Hypertension. J. A. M. A., 106:762-767. 140 10. B arker, 1948 11* S. B. Determination of Protein-Bound Iodine. J. Biol. Chem., 173:715-724. 1948 » and Lipner, H. J. In Vivo Iodination of Tissue P rotein Following Injection of Elemental Iodine. Science, 108:539. 1950 , and Humphrey, M. J. Clinical Determination of Protein-Bound Iodine in Plasm a. J. Clin. Endocrinology, 10:1136-1141. 1951 , and Soley, M. H. The Clinical Determination of Protein-Bound Iodine. J. Clin. Invest., 30:55-62. 12. 13. 14. Barnes, 1933 B. O. The Excretion of Iodine in Experimental Hyperthyroidism. Am. J. Physiol., 103:699-703. 15. Barnes, 1933 B. O., and Jones, M. Studies on Thyroglobulin. III. The Thyroglobulin Content of the Thyroid Gland. Am. J. Physiol., 105:556-558. 16. B arrnett, R. J., and Greep, R. O. Regulation of Secre1951 tion of Adrenotropic and Thyrotropic Hormones After Stalk Section. Am. J. Physiol., 167:569575. 17. B artlett, S., Rowland, S. J., and Thompson, S. Y. Iodinated 1949 Protein Feeding and Milk Composition. Proc. Xllth International Dairy Congress, 102-109. 18. Bassett, A. M., Coons, A, H., and Salter, W. T. Protein1941 Bound Iodine in Blood. V. Naturally Occurring Fractions and Their Chemical Behavior. Am. J. Med. Sci., 202:516-542. 19. B erliner, V., and Warbritton, V. The Pituitary and Thy1937 roid in Relation to Sperm Production in Rams. Amer. Soc. Animal Prod. P ro c., 137-142. 141 20. Bernheim, F ., and Bernheim, M. L. C. The Metabolism 1944 of Tryamin, 1-Tyrosine, and Phenol by Rat Tissues in Vitro. J. Biol. Chem., 153:369-373. 21. Best, C. H., and Taylor, N. B. The Physiological Basis 1945 of Medical Practice, 4th Ed. Williams and Wilkins Co., Baltimore. 22. Betheil, J. J., and Lardy, H. A. Comparative Effective 1949 ness of Vitamin B j^ j Whole Liver Substance and Extracts High in APA Activity as Growth P ro ­ moting Materials for Hyperthyroid Animals. J. Nut., 37:495-509. 23. B iellier, H. V., and Turner, C. W. The Thyroxine Se1950 cretion Rate of Growing White Pekin Ducks. Poultry Sci., 29:248-257. 24. Blau, N. F. The Determination of Thyroxine in the Thy1933 roid Gland. J. Biol. Chem., 102:269-278. 25, . The Determination of Thyroxine in Thyroid Substance. J. Biol. Chem., 110:351-363. 1935 26. Blaxter, K. L., Reineke, E. P., Crampton, E. W., and 1949 P etersen, W. E. The Role of Thyroidal Mate­ rials and of Synthetic Goitrogens in Animal Production and an Appraisal of their P ractical Use. J. Anim. Sci., 8:307-352. 27. Block, P. A Note on the Conversion of Diiodotyrosine 1940 into Thyroxine. J. Biol. Chem., 135:51-52. 28. Boatman, J. B., and Campbell, M. The Effect of Thymus 1951 and Muscle Feeding Upon Growth and the Con­ centration of Radioiodine I 131 in the Thyroid and Other Tissues of the Rat. Endocrinology, 49:422-424. 142 29. Bogart, R., and. Mayer, D. T. Environmental Temperature 1946 and Thyroid Gland Involvement in Lowered F e r ­ tility of Rams. Mo. Agr. Exp. Sta. Res. Bui. 402. 30. Bogoroch, R., and Timira, P. The Response of the Thy1951 roid Gland of the Rat to Severe Stress. Endo­ crinology, 49:548-556. 31. Boyd, E. M., and Clarke, E. L. The Fractionation of 1942 Cattle Blood Iodine with Alcohol. J. Biol. Chem., 142:619-622. 32. Brand, E., Kassell, B., and Heidelberger, M. On the 1939 Structure of Thyroglobulin. J. Biol. Chem., 128:xi. 33. Brody, S., and Frankenbach, R. F. Age Changes in Size, 1942 Energy Metabolism and Cardio-Respiratory Ac­ tivities of Thyroidectomized Cattle. Mo. Agr. Exp. Sta. Res. Bui. 278. 34. 35. 36. ___________ . Bioenergetics and Growth. 1945 ing Co., Baltimore. Reinhold Publish- Bruger, M., and Member, S. On the Fractionation of 1943 Iodine in Blood. J. Biol. Chem., 148:77-83. Chaikoff, I. L., Taurog, A., and Reinhardt, W. O. The 1947 Metabolic Significance of Protein-Bound Iodine of Plasm a: A Study of Its Concentration under Various Conditions and of its Rate of Formation as Measured with Radioactive Iodine. Endocrin­ ology, 40:47-54. 37# I 9 4 9 . Studies on the Formation of Organic ally-Bound Iodine Compounds in the Thyroid Gland and Their Appearance in Plasm a as Shown by the Use of Radioactive Iodine. Ann. N. Y. Acad. Sci., 50:377-400. 143 38. Chaney, A. L. Improvements in Determination of Iodine 1940 in Blood. Ind. and Eng. Chem., Anal. Ed., 12:179-181. 39. Chapman, A. The Relation of the Thyroid and the Pitui1941 tary Glands to Iodine Metabolism. Endocrinology, 29:680-685. 40. _______________ . Extrathyroidal Iodine Metabolism. 1941 crinology, 29:686-694. 41. Clarke, E. L., and Boyd, E. M. A Seasonal Study of the 1940 Iodine Content of the Blood of Birds. J. Biol. Chem., 135:691-695. 42. Cole, V. V., and Curtis, G. M. Cyclic Variations in 1933 Urinary Excretion of Iodine in Women. Proc. Soc. Exp. Biol, and Med., 31:29-30. 43. Connor, A. C., Swenson, R. E., Park, C. W., Gangloff, 1949 C. E., Lieberman, R., and Curtis, G. M. The Determination of the Blood Iodine. A Useful Method for the Clinical Laboratory. Surgery, 25:510-517. 44. Cooper, D., March, B., and Biely, J. The Effect of 1950 Feeding Thyroprotein and Thiouracil on the Vitamin A Requirement of the Chick. Endo­ crinology, 46:404-406. 45. Cortell, R., and Rawson, R. W. The Effect of Thyroxine I 9 4 4 on the Response of the Thyroid Gland to Thyro­ tropic Hormone. Endocrinology, 35:488-498. 46. Cosma, J. Utilization of Antithyroid Action Test for 1951 Bioassay of Thymus Hormone. Am. J. Physiol., 166:550-555. 47. C ourrier, R., and Aron, M. Sur le Passage de l'hormone 1929 Thyroidienne de la mere au foetus a trav ers le placenta. Compt. rend. Soc. de Biol., 100: 839-841. Endo- 144 48. Curtis, 1933 49. Danowski, T. S., Gow, R. C., Mateer, F. M., Everhart, 1950 W. C., Johnston, S. Y., and Greenman, J. H. Increases in Serum Thyroxine During Uncompli­ cated Pregnancy. Proc. Soc. Exp. Biol, and Med., 74:323-326. 50. Davis, C. B., Curtis, G. M., and Cole, V. V. Blood 1934 Iodine Studies. II. Normal Iodine Content of Human Blood. J. Lab. and Clin. Med., 19:818830. 51. Dempsey, E. W., and Astwood, E. B. Determination of 1943 the Rate of Thyroid Hormone Secretion at V ari­ ous Environmental Tem peratures. Endocrinology, 32:509-518. 52. ____________________. Fluorescent and Histochemical Reactions 1944 in the Rat Thyroid at Different Stages of Physi­ ological Activity. Endocrinology, 34:27-38. 53. De Robertis, E. The Intracellular Colloid of the Normal 1941 and Activated Thyroid of the Rat, Studied by the Free zing-Drying Method. Am. J. Anat., 68:317-337. 54. G. M., Davis, C. B., and Phillips, F. J. Significance of the Iodine Content of Human Blood. J. A. M. A., 101:901-905. . Proteolytic Enzyme Activity of Colloid Extracted from Single Follicles of the Rat Thyroid. Anat. Rec., 80:219-231. ________________________ 1941 55. 1942 56^ 1946 . Intracellular Colloid in the Initial Stages of Thyroid Activation. Anat. Rec., 84:125-135. , and Nowinski, W. W. The Proteolytic Activity of Normal and Pathological Human Thy­ roid Tissue. J. Clin. Indocrinol., 6:235-246. 145 57. __________________________________________ . The Mechanism of 1946 the Therapeutic Effect of Iodine on the Thyroid Gland. Science, 103:421-422. 58. ____________________• Cytological and Cytochemical Bases of 1949 Thyroid Function. Ann. N. Y. Acad. Sci., 50: 317-333. 59. Dougherty, J., Gross, J., and Leblond, C. P. Steady 1951 State of the Thyroidal Iodine. Endocrinology, 48:700-713. 60. Du Vigneaud, V., and Irish, O. J. The Role of the Acetyl 1937 Derivative as an Intermediary Stage in the Bio­ logical Synthesis of Amino Acids from Keto Acids. J. Biol. Chem., 122:349-370. 61. Eckles, 1943 62. Elm er, A. W., and Scheps, M. La Thyroxine est-elle 1934 Eliminee par les Reins Chez 1‘homme et chez les Basdowiens? Compt. rend. Soc. Biol., 115: 968-970. 63. Ershoff, B. H. An Antithyrotoxic Factor for the Rat 1949 Not Identical with Vitamin B j 2 - Proc, Soc. Exper. Biol, and Med., 71:209-211. 64. Fashena, Gladys J. A Study of the Blood Iodine in Child1938 hood. J. Clin. Invest., 17:179-188. 65. Folley, S. J., and White, P. The Effect of Thyroxine on 1936 Milk Secretion and on the Phosphatase of the Blood and Milk of the Lactating Cow. Proc. Roy. Soc. Lond. Ser. B, 120:346-365. 66. F o ster, G. L», and Smith, P. E. Hypophysectomy and 1926 Replacement Therapy in Relation to Basal Me­ tabolism and Specific Dynamic Action in the Rat. J. A. M. A., 87:2151-2153. C. H., Combs, W. B., and Macy, H. Milk and Milk Products. 3rd. Ed. 7th Impression. McGraw-Hill Book Co., New York, page 49. 146 ^7. 1929 68. .The Isolation, of 3,5-diiodotyrosine from the Thyroid. J. Biol. Chem., 83:345-346. Freedm an, N. H., and Gordon, A. S. Effects of Thyroid1950 ectomy and of Thiouracil on Adrenal Weight and Ascorbic Acid. Proc. Soc. Exp. Biol, and Med., 75:729-732. 6 9 . Galli-Mainini, G. 1941 70. 71. Effect of Thyroid and Thyrotropic Hormones upon Oxygen Consumption of the Thyroid of the Guinea Pig. Endocrinology, 29: 674-679. Gardner, J. H. Effects of Inunction of Alpha-Estradiol 1949 on Testes and Thyroids of Albino Rats. Proc. Soc. Exp. Biol, and Med., 72:306-309. Gersh, I., and Caspersson, T. Total Protein and Organic 194:0 Iodine in the Colloid of Cells of Single Follicles of the Thyroid Gland. Anat. Rec., 78:303-319. 72. Ghosh, B. N., Woodbury, D. M., and Sayers, G. Quanti1951 tative Effects of Thyrotrophic Hormone on 1^31 accumulation in Thyroid and Plasm a Proteins of Hypophysectomized Rats. Endocrinology, 48: 631-642. 73. Glimm, E., and Isenbruch, J. Uber die Bestimmung 1929 Kleinster Jodmengen. Biochem. Ztschr., 207: 368-376. 74. Goldberg, R. C., and Chaikoff, I. L. The Cytological 1950 Changes that Occur in the Anterior Pituitary Glands of Rats Injected with Various Doses of jl31 an(j their Significance in the Estimation of Thyroid Function. Endocrinology, 46:91-104. 75. Goldsmith, E. D. Phylogeny of the Thyroid: Descriptive 1949 and Experimental. Ann. N. Y. Acad. Sci., 50: 283-313. 147 76. Gorbman, A. Reactions of Thyroid Glands to Juxtathy1950 roidal Implants of Thyrotropic Agents. Endo­ crinology, 46:397-402. 77. G reer, M. A. Nutrition and Goiter. Physiol. 1950 513-548. 78. Griesbach, W. E., and Purves, H. D. The Significance 1945 of the Basophil Changes in the Pituitary Ac­ companying Various forms of Thyroxine De­ ficiency. Brit. J. Exp, Path., 26:13-17. 79. Gross, J., and Leblond, C. P. Distribution of a Large 1947 Dose of Thyroxine Labeled with Radioiodine in theOrgans and Tissues of the Rat. J. Biol. Chem., 171:309-320. 80. 1950 81. 1950 82. 1951 ro 00 1951 84. Rev., 30: , Franklin, A. E., and Quastel J. H. Presence of Iodinated Amino Acids in Unhydrolyzed Thyroid and Plasm a. Science, 111:605-608. Metabolism of the Thyroid Hormone in the Rat as Shown by Physiological Doses of Labeled Thyroxine. J. Biol. Chem., 184:489-500. Metabolites of Thyroxine. Proc. Soc. Exp. Biol, and Med., 76:686-689. The Presence of Free Iodinated Compounds in the Thyroid and Their Passage into the Circulation. J. Biol. Chem., 48:714-725. Gudernatsch, F. Ann. N. Y. Acad. Sci., 50:313-316. 1949 85. Gutman, A. B., Benedict, E. N., Baxter, B., and Palm er, 1932 W. W. The Effect of Administration of Iodine on the Total Iodine, Inorganic Iodine, and Thy­ roxine Content of the Pathological Thyroid Gland. J. Biol. Chem., 97:303-324. 148 86. Halmi, N. S., and Bogdonove, E. M. Effect of Thyroid1951 ectomy of ACTH Content of Rat Adenohypophysis. P ro c. Soc. Exp. Biol, and Med., 77:518-520. 87. Hansborough, L. A., and Seay, H. Accumulation of Radio1951 iodine (I131) in Thyroid Gland of the Hamster Embryo. P roc. Soc. Exp. Biol, and Med., 78:481-483. 88. Harington, C. R., and Barger, G. Chemistry of Thyroxine. 1927 III. Constitution and Synthesis of Thyroxine. Biochem. J., 21:169-181. 89. 1935. Biochemical Bases of Thyroid Function Lancet, 228:1199-1204. 1935 Biochemical Bases of Thyroid Function. II. Lancet, 228:1261-1266. 90. 91. 1944 Thyroxine: Its biosynthesis and its Imnunochemistry. Proc. Roy. Soc. London, Series B, 132:223-238. 92. Heidelberger, M., and Pedersen, K. O. The Molecular 1935 Weight and Isolectric Point of Thyroglobulin. J; Gen. Physiol., 19:95-108. 93. Hektoen, L., Carlson, A. J., and Schulhof, K. The P re 1923 cipitin Reaction of Thyroglobulin. Presence of Thyroglobulin in the Thyroid Lymph of Goitrous Dogs. J. A. M. A., 81:86-88. 94 # 1927 . Further Attempts to Increase Experimentally the Hormone Output by the Thyroid Gland. Am. J. Physiol., 81:661-664. 95. Herring, P. T. The Action of Thyroid Upon the Growth 1917 of the Body and Organs of the White Rat. Quart. J. Exp. Physiol., 11:231-253. 149 96. Hertz, R., Allen, M. J., and Tullner, W. W. Effects of 1950 Amphenone u Btl on Thyroid, Adrenals, and Genital T ract of the Female Rat. Proc. Soc. Exp. Biol. Med., 75:627-630. 97. Hibbs, J. 1946 9 S. 99. W., and Krauss, W. E. The Effect of Thyroprotein (Protamone) on Milk Production and on Some of the Constituents of the Milk and Blood of Dairy Cattle. J. Anim. Sci., 5:401. (Abs.) ___________________________________________ . Effect of Thyroprotein 1947 (Protamone) on Milk Production and On Some of the Constituents of the Milk and Blood of Dairy Cows. J. Anim. Sci., 6:161-173. Hilditch, 1936 T. P ., and Paul, H. The Occurrence and Possible Significance of Some of the Minor Com­ ponent Acids of Cow Milk Fat. Biochem. J., 30:1905-1914. 100. Hoffman, E. Thyroxine Secretion Rate and Growth in the 1950 White Pekin Duck. Poultry Sci., 29:109-114. 101. Hoskins, R. G. Congenital Thyroidism: An Experimental 1910 Study of the Thyroid in Relation to Other Organs of Internal Secretion. Am. J. Physiol., 26:426438. 102. ______________ . Thyroid Secretion as a Factor in Adrenal 1910 Activity. J. A. M. A., 55:1724-1725. 103. Hudson, 1931 G. E. The Permeability of the Placenta to Inorganic Iodides. J. A. M. A., 97:1513-1517. 104. Hum, R. F., Goldberg, R. C., and Chaikoff, I. L. Effect 1951 of Excess Iodide Upon Anterior Pituitary Cytol­ ogy of the Completely Thyroidectomized Rat and its Bearing on the Question of Extra thy­ roidal Thyroxine Synthesis. Endocrinology, 49: 21-24. 150 105. Hutt, F. B., and Gowe, R. S. On the Supposed Effect 1948 of Iodocasein Upon Egg Production. Poultry Sci., 27:286-293. 106. Karp, L., and Kostkiewicz, B. Goitre Colloidal Experi193 3 mental Provogue par le Folliculine. Comp, rend. Soc. de Biol., 114:1339-1342. 107. Kellaway, P. E., Hoff, H. E., and Leblond, C. P. The 1945 Response to Thyroxine after Subtotal Hep ate ctomy. Endocrinology, 36:272-279. 108. Kemmerer, A. R., Bolomey, R. A., Vavich, M. G., and 1946 Davis, R. N. Effect of Thyroprotein on the Vitamin Content of Milk. Proc. Soc. Exp. Biol, and Med., 63:309-310. 109. Kendall, E. C., and Richardson, F. S. Determination of 1920 Iodine in Blood and Animal Tissues. J. Biol. Chem., 43:161-170. 110. Keston, A. S. The Shardinger Enzyme in Biological 1944 Iodinations. J. Biol. Chem., 153:335-336. 111. Koneff, A. A., Nichols, C. W., J r., Wolff, H., and Chai1949 koff, I. L., The Fetal Bovine Thyroid: Morpho­ genesis as Related to Iodine Accumulation. Endocrinology, 45:242-249. 112. Laidlaw, J. Nature of the Circulating Thyroid Hormone. 1949 Nature, 164:927-928. 113. Leblond, C. P ., and Gross, P. Thyroglobulin Formation 1948 in the Thyroid Follicle Visualized by the "Coated Autograph" Technique. Endocrinology, 43:306324. 114. Lein, A. 1943 Studies on the Fixation of Radioactive Iodine by the Rabbit Thyroid. Endocrinology, 32:429432. 151 115. Leland, J. P ., and F o ster, G. L. A Method for the De1932 termination of Thyroxine in the Thyroid. J. Biol. Chem., 95:165-179. 116. Lerman, J. Iodine Components of the Blood. Circulat1940 ing Thyro-Globulin in Normal Persons and in P ersons with Thyroid Disease. J. Clin. Invest., 19:555-560. 117. Loeb, L., and Bassett, R. B. Effect of Hormones of 1929 Anterior Pituitary on Thyroid Gland in the Guinea Pig. Proc. Soc. Exp. Biol, and Med., 26:860- 862. 118. 1930 119. , and Friedman, H. Further Investigations Concerning the Stimulating Effect of Anterior Pituitary Gland Preparation on the Thyroid Gland. Proc. Soc. Exp. Biol, and Med., 28:209-213. Loeser, A., and Thompson, K. W. Hypophysenvorderlappen, 1934 Jod und Schilddruse. Der Mechanismus der Schilddrusenwirkung des Jods. Endokronologie, 14:144-150. 120. Long, J. 1951 121. Mann, E. B., Smirnow, A. E., Gildea, E. F., and P eters, 1942 J. P. Serum Iodine Fractions in Hyperthy­ roidism. J. Clin. Invest., 21:773-780. 122. 123. F., Gilmore, L. O., Curtis, G. M., and Rife, D. C. The Bovine Protein-Bound Iodine as Related to Age, Sex and Breed. J. Amin. Sci., 10:1027-1028. (Abs.) Mann, W., Leblond, C. P., and Warren, S. L. Iodine 1942 Metabolism of the Thyroid Gland. J. Biol. Chem., 142:905-912. Maqsood, M. Effects of Thyroxine on Oxygen Con sump1950 tion of Mammalian Spermotozoa. Abst. of Comm. 18th Inter. Physiol. Congr., Copenhagen. 152 124. Marine, D., and. Lenhart, G. H. Effects of the Adminis1909 tration or the Withholding of Iodine - Containing Compounds in Normal, Colloid or Actively Hy­ perplastic (Perenchymatous) Thyroids of Dogs. Some Experiments on (Congenital) Prenatal Thyroid Hyperplasia in dogs: Remarks on the Clinical Manifestations Associated with Marked Thyroid Hyperplasia. Arch. Int. Med., 4:253270. 125. _____________, Manley, O. T., and Baumann, E. The Influence 1924 of Thyroidectomy, Gonadectomy, Suprarenolectomy, and Splenectomy on the Thymus Gland of Rabbits. J. Exp. Med., 40:429-443. 126. _____________, and Rosen, S. H. The Effect of the Thyro1934 tropic Hormone on Auto- and Homeotransplants of the Thyroid, and its Bearing on the Question of Secretory Nerves. Am. J. Physiol., 107: 677-680. 127. Matthews, N. L., Curtis, G. M., and Mayer, J. H. The 1939 Effect of Increased Iodine Feeding Upon the Iodine Content of Cow’s Milk. J. Dairy Res., 10:395-402. 128. McClendon, J. F., and Foster, W. C. Protein-Bound 1944 Iodine in Erythrocytes and Plasma. J. Biol. Chem., 154:619-622. 129. McCullagh, E. P., and McCullagh, D. R. Clinical Ex1936 periences in the Use of Determinations of Blood Iodine. Arch. Int. Med., 57:1061-1066. 130. McDonald, M. R., Riddle, O., and Smith, G. C. Action 1945 of Thyroxine on Estrogen-Induced Changes in Blood Chemistry and Endosteal Bone. Endo­ crinology, 37:23-28. 131. McKenzie, F. F., and Berliner, V. The Reproductive 1937 Capacity of Rams. Mo. Agr. Exp. Sta. Res. Bui., 338. 153 132. Meites, 1948 133. Money, W. L., Kraintz, L., Fager, J., Kirschner, J., 1951 and Rawson, R. W. The Effects of Various Steroids on the Collection of Radioactive Iodine by the Thyroid Gland of the Rat. Endocrinology, 48:682-690. 134. Monroe, R. A., and Turner, C. W. The Metabolism of 1949 Iodine. Mo. Agr. Exp. Sta. Res. Bui. 446. 135. Morton, 1941 136. ____________________________________, Anderson, E., and Chaikoff, 1942 I. L. Radioactive Iodine as an Indicator of the Metabolism of Iodine. V. The Effects of Hypopyphectomy on the Distribution of Labeled Thy­ roxine and Diiodotyrosine in the Thyroid Gland and in the Plasma. Endocrinology, 30:495-501. 137. 1943 138. J., and Chandrashaker, B. Effect of the Thyroid Status on Response of the Gonads to Pregnant M ares’ Serum in Two Different Species. J. Anim. Sci., 7:542. (Abs.) M. E., Perlman, I., and Chaikoff, I. L. Radio active Iodine as an Indicator of theMetabolism of Iodine. III. The Effect of Thyrotropic Hor­ mone on the Turnover of Thyroxine and Diiodotyrosine in the Thyroid Gland and Plasma. J. Biol. Chem., 140:603-611. , and Chaikoff, I. L. The Formation In Vitro of Thyroxine and Diiodotyrosine by Thy­ roid Tissue with Radioactive Iodine as Indi­ cator. J. Biol. Chem., 147:1-9. _____________________________Reinhardt, W. O., and Ander1943 son, E. Radioactive Iodine as an Indicator of the Metabolism of Iodine. VI. The Formation of Thyroxine and Diiodotyrosine by the Completely Thyroidectomized Animal. J. Biol. Chem., 147:757-769. 154 139. Nichols, C. W., J r ., Chaikoff, I. L., and Wolf, J. The 1949 Relative Growth of the Thyroid Gland in the Bovine Fetus. Endocrinology, 44:502-509. 140. Palm er, W. W., Leland, J. P., and Gutman, A. B. The 193 8 Microdetermination of Thyroxine in the Thyroid Gland of the New Born. J. Biol. Chem., 125: 615-623. 141. Paschkis, K. E., Cantarow, A., Eberhard, T., and Boyle, 1950 D. Thyroid Function in the Alarm Reaction. Proc. Soc. Exp. Biol, and Med., 73:116-118. 142. Perkin, H. J., Brown, B. R., and Lang, J. The Blood 1934 Iodine Content of Normal and Thyrotoxic Indi­ viduals: An Iodine Tolerance Test. Canad. M. A. J., 31:365-368. 143. 144. __________________________________. The Influence of the Thy1938 roid Gland and of the Ovary on the Metabolism of Iodine. Endocrinology, 22:538-542. Perlm an, I., Morton, M. E., and Chaikoff, I. L. Radio1941 active Iodine as an Indicator of the Metabolism of Iodine. 1. The Turnover of Iodine in the Tissues of the Normal Animal, with P articular Reference to the Thyroid. J. Biol. Chem., 139: 433-447. 145. 1941 ________________________________________ . Radio active Iodine as an Indicator of the Metabolism of Iodine. II. The Rates of Formation of Thy­ roxine and Diiodotyrosine by the Intact Normal Thyroid Gland. J. Biol. Chem., 139:449-456. 146. P e rry , W. F. The Action of Cortisone and ACTH on 1951 Thyroid Function. Endocrinology, 49:284-288. 147. P etersen, W. E., Spielman, A., Pomeroy, B. S., and Boyd, 1941 W. L. Effect of Thyroidectomy upon Sexual Behavior of the Male Bovine. Proc. Soc. Exp. Biol, and Med., 46:16-17. 155 148. Puntriano, G., and Meites, J. The Effects of Continuous 1951 Light or Darkness on Thyroid Function in Mice. Endocrinology, 48:217-224. 149. Purves, H. D., and Griesbach, W. E. The Site of Thyro1951 trophin and Gonadotrophin Production in the Rat Pituitary Studied by McManus-Hotchkiss Staining for Glycoprotein. Endocrinology, 49:244-264. 150. Rail, J. E., Power, M. H., and Albert, A. Distribution 1950 of Radioiodine in Erythrocytes and Plasm a of Man. Proc. Soc. Exp. Biol, and Med., 74:460461. 151. Ralston, N. P., Cowsert, W. C., Ragsdale, A. C., Her1940 man, H. A., and Turner, C. W. The Yield and Composition of the Milk of Dairy Cows and Goats as Influenced by Thyroxine. Mo. Agr. Exp. Sta. Res. Bui., 317. 152. Randall, R. V., and Albert, A. The Effect of Hypophy1951 sectomy on the Uptake of Radioactive Iodine by the Thyroid of the Rat. Endocrinology, 48: 327-332. 153. Rawson, R. W., Sterne, G. O., and Aub, J. C. Physio1942 logical Reactions of Thyroid Stimulating Hor­ mone of Pituitary. I. Its Inactivation by Ex­ posure to Thyroid Tissue in Vitro. Endocrin­ ology, 30:240-245. 154. 1943 155. 1949 , Graham, R. M., and Riddell, C. B. Physiological Reactions of the Thyroid Stimu­ lating Hormone of Pituitary; Effect of Normal and Pathological Human Thyroid Tissues on Activity of Thyroid Stimulating Hormone. Ann. Int. Med., 19:405-414. .PhysiologicalReactionsofthe ThyroidStimulating Hormone. Ann. N. Y. Acad. Sci., 50:491-507. 156 156. Reece, R. P., and Man, E. B. Serum Precipitable and 1952 Butanol Extractable Iodine of Bovine Sera. Proc. Soc. Exp. Biol, and Med., 79:208-210. 157. Reed, O. E. Report of the Chief of the Bureau of Dairy 1951 Industry, Agricultural Research Administration. 158. Reineke, E. P ., Bergman, A. J., and Turner, C. W. Ef­ 1941 fect of Thyroidectomy of Young Goats upon Certain Anterior Pituitary Hormones. Endo­ crinology, 29:306-312. 159. 1942 160. 1944 161. 1945 162. Non-Permeability of the Mammary Gland to Thyroid Hormone. J. Dairy Sci., 27:793-805. , Mixner, J. P., and Turner, C. W. Ef­ fect of Graded Doses of Thyroxine on Metabo­ lism and Thyroid Weight of Rats Treated with Thiouracil. Endocrinology, 36:64-67. 1945 , and Turner, C. W. The Relative Thy­ roidal Potency of 1- and d,l- Thyroxine. Endo­ crinology, 36:200-206. 1945 Seasonal Rhythm in the Thyroid Hormone Secretion of the Chick. Poultry Sci., 24:499-504. 1946 The Effect of Synthetic Thyroprotein on Sterility in Bulls. The Problem of Fertility. Princeton University P re ss , Princeton, N. Y. 163. 164. 165. , and Turner, C. W. Formation in Vitro of Highly Active Thyroproteins, Their Biologic Assay, and P ractical Use. Mo. Agr. Exp. Sta. Res. Bui. 355. Remington, R. E., and Supplee, G. C. Studies on the 1934 Iodine Content of Milk. II. Variations in the Mixed Milk of Herds. J. Dairy Sci., 17:19-28. 157 16 6 . ______________________> Coulson, E. J., and Levine, H. Studies 1936 on the Relation of Diet to Goiter. IV. The Antigoitrogenic Value of Some Foods. J. Nut., 12:27-37. 167. Riggs, D. S., Lavictes, P. J., and Man, E. B. Investi1942 gations on the Nature of Blood Iodine. J. Biol. Chem., 143:363-372. 168. Salter, W. T., and Lerman, J. The Genesis of Thyroid 1936 Protein: Clinical Assays of Artificial Thyroid Protein in Human Myxedema. Endocrinology, 20:801-808. 169. ________________. The Endocrine Function of Iodine, Cam1940 bridge University P re ss . 170. ________________, Bassett, A. M., and Sappington, T. S. 1941 Protein-Bound Iodine in Blood: Its Relation to Thyroid Function in 100 Clinical Cases. Am. J. Med. Sci., 202:527-542. 171. Schultze, A. B., and Turner, C. W. The Rate of Thy1944 roxine Secretion by the Thyroid Glands of White Leghorn Cockerels. Yale J. Biol. Med., 17: 269. (abst. Annotated Bibliography of the Bio­ logical Literature on Iodinated Protein and Thy­ roxine, Cerophyl Laboratories, 1944, page 1 [1951]). 172. ____________________________________________ . The Determination 1945 of the Rate of Thyroxine Secretion by Certain Domestic Animals. Mo. Agr. Exp. Sta. Res. Bui. 392. 173. ____________________f and Davis, H. P. The Influence of Feed1946 ing Synthetic Thyroprotein on Fertility of Bulls. J. Dairy Sci., 28:534-535. . 174. 1947 Some Effects of Adding Thyroxine to Bovine Semen. 543-544. J. Dairy Sci., 30: 158 175. ______________________ :_________________________. Effect of Thyroxine 1948 on Oxygen Consumption of Bovine Spermatozoa and Semen. J. Dairy Sci., 31:946-950. 176. Scott, K. G., Reaves, J. C., Saunders, W. W., White, 1951 W. E. The Use of 1 ^ 1 Red Cell Plasm a Ratio as a Measure of Thyroid Function. Proc. Soc. Exp. Biol, and Med., 76:592-595. 177. Seidell, A., and Fenger, F. Seasonal Variation in the 1913 Iodine Content of the Thyroid Gland. J. Biol. Chem., 13:517-526. 178. Severinghaus, A. E., Smelser, G. K., and Clark, H. M. 1934 Ant. Pituitary Changes in Adult Male Rats Fol­ lowing Thyroxin Injections or Thyroid Feeding. Proc. Soc. Exp. Biol, and Med., 31:1125-1127. 179. 1934 Ant. Pituitary Changes in the Adult Male Rate Following Thyroidectomy. Proc. Soc. Exp. Biol, and Med., 31:1127-1129. 180. Sharpless, G. R., Pearsons, J., and Prato, G. S. P ro 1939 due tion of Goiter in Rats With Raw and With Treated Soybean Flour. J. Nut., 17:545-555. 181. Sherwood, T. C., and Luckner, W. G. Further Studies 1935 on the Effect of Cod Liver Oil on the Thyroid Gland. J. Nut., 9:123-129. 182. Silver, S. Nature of Blood Iodine and its Determination. 1942 J. Lab. and Clin. Med., 28:329-335. 183. Smogyi, M. A Method for the Preparation of Blood 1930 F iltrates for the Determination of Sugar. J. Biol. Chem., 86:655-663. 184. Spielman, A. A., Peterson, W. E., and Fitch, J. B. The 1944 Effect of Thyroidectomy on Lactation in the Bovine. J. Dairy Sci., 27:441-448. 159 185. _____________________________ _ ___________ 1945 Pomeroy, B. S. General Appearance, Growth and Reproduction of Thyroidectomized Bovine. J. Dairy Sci., 28:329-337. 186. S tarr, P ., and Patton, H. The Effect of Pregnancy Urine 1934 Extract and Ovarian Eollicular Hormone on Hy­ perthyroidism. Endocrinology, 18:113-116. 187. Sure, B., and Buchanan, K. S. Antithyrogenic Action of 1937 Crystalline Vitamin B. J. Nut., 5:513-519. 188. __________, and Esterling, L. The Protective Action of 1950 Vitamin B 12 Against the Toxicity of d,l-thyroxine. J. Nut., 42:221-226. 189. Sweet, J. E., and Ellis, J. W. The Influence Upon the 1915 Spleen and the Thyroid of the Complete Removal of the External Function of the Pancreas. J. Exp. Med., 22:732-738. 190. Swett, W. W., Matthews, C. A., Miller, F. W., and Graves, 1937 R. R. Variations Recorded in the Study of the Conformation and Anatomy of 593 Dairy Cows Having Records of Production. B. D. I., U. S. D. A. M. 589 (Revised). 191. Talbot, N. B., Butler, A. A., Saltzman, A. H., and Rod1944 riquez, P. M. The Colorimetric Estimation of Protein-Bound Serum Iodine. J. Biol. Chem., 153:479-488. 192. Taurog, A., and Chaikoff, I. L. On the Determination of 1946 Plasm a Iodine. J. Biol. Chem., 163:313-322. 193. 1946 .TheDeterminationof Thyroxine in the Thyroid Gland of the Rat. J. Biol. Chem., 163:323-328. ,and 160 194. 1946 195. 1947 196. 1947 • The Relation of the Thyroxine Content of the Thyroid Gland and of the Level of Protein-Bound Iodine of Plasm a to Iodine Intake. J. Biol. Chem., 165:217-222. , and Entenman, C. The Rate of Turnover of Protein-Bound Iodine in the Plasm a of the Dog as Measured with Radio­ active Iodine. Endocrinology, 40:86-91. The Metabolic In te rrela ­ tions of Thyroxine and Diiodotyrosine in the Thyroid Gland as Shown by a Study of Their Specific Activity-Time Relations in Rats In­ jected With Radioactive Iodine. J. Biol. Chem., 169:49-56. 1948 The Nature of the C ir­ culating Thyroid Hormone. J. Biol. Chem., 176:. 639-656. 1949 , and Tong, W. On the Occurrence of Monoiodotyrosine in the Thyroid Gland. J. Biol. Chem., 178:997-998. 197. 198. 199. 1950 200. 1951 The Nature of Plasm a Iodine as Revealed by Filter Paper Partition Chromatography. J. Biol. Chem., 184: 99-104. Briggs, E. N., and Chaikoff, I. L. I*^1-Labeled 1-Thyroxine. 1. An Inidentified Excretion Product in Bile. J. Biol. Chem., 191:28-34. 201. ___________ , Tong, W., and Chaikoff, I. L. Non-Thyroglob1951 ulin Iodine of the Thyroid Gland. II. Inorganic Iodide. J. Biol. Chem., 191:677-682. 202. Thompson, W. O., Thompson, P . K.., Taylor, S. G., Nadler, 1935 S. B., and Dickie, L. F. The Pharmacology of the Thyroid in Man. J. A. M. A., 104:972-980. 161 203. Tong, W., Taurog, A., and Chaikoff, I. L. Non-Thyro1951 globulin Iodine of the Thyroid Gland. 1. Free Thyroxine and Diiodotyrosine. J. Biol. Chem., 191:665-675. 204. Trevorrow, V. Studies on the Nature of the Iodine of 1939 Blood. J. Biol. Chem., 127:737-750. 205. Trues dell, C. The Effect of Feeding Thyroid Extract 1926 on Gastric Secretion. Am. J. Physiol., 76: 20-27. 206. Turner, C. W., and Cupps, P. T. The Thyrotropic Hormone in the Pituitary of the Albino Rat During 1939 Growth, Pregnancy and Lactation. Endocrinology, 24:650-655. 207. 1945 , Irwin, M. P., and Reineke, E. P. Effect of the Thyroid Hormone Egg Production of White Leghorn Hens. Poultry Sci., 24:171-180. 1945 , Kempster, H. L., Hall, N. M., and Reineke, E. P. The Effect of Thyroprotein on Egg P r o ­ duction. Poultry Sci., 24:522-533. 1948 Effect of Age and Season on the Thy­ roxine Secretion Rate of White Leghorn Hens. Poultry Sci., 27:146-160. 208. 209. 210. Turner, K. B., DeLamater, A., and Province, W. D. Observations on the Blood Iodine. 1. The Blood 1940 Iodine in Health, in Thyroid and Cardiorenal Disease, and in Leukemia. J. Clin. Invest., 19:515-524. 211. The Iodine Content Turner, R. G., and Matthews, C. W. of Blood in Certain Pathological Conditions. 1931 Abst. J. Biol. Chem., 92:lxxxviii. 212. 1934 Iodine and Thyroid Hyperplasia. 1. The Iodine Content of Human Skimmed Milk from Goitrous and Non-Goitrous Regions. Am. J. Dis. Child., 48:1209-1227. 162 213. Tyndale, H. H., and Levin, L. Ovarian Weight Responses 1937 to Menopause Urine Injections in Normal, Hypo physectomized and Hypophysectomized Thyroxine Treated Immature Rats. Am. J. Physiol., 120: 486-493. 214. Vanderlaan, J. E., Vanderlaan, W. P., and Logan, M. A. 1941 Effect of Administering Thyrotropic Hormone with and without Iodine on Thyroid Tissue Metabolism. Endocrinology, 29:93-95. 215. 1947 . The Iodide Concentration Mechanism of the Rat Thyroid and its Inhibition by Thiocyanate. Endocrinology, 40:403-416. 216. Vanderlaan, W. P ., and Greer, M. A. Some Effects of 1950 the Hypophysis on Iodine Metabolism by the Thyroid Gland of the Rat. Endocrinology, 47: 36-47. 217. Van Landingham, A. H., Henderson, H. O., and Weakley, 1944 C. E. The Effect of Iodinated Casein on Milk and Butterfat Production and on the Ascorbic Acid Content of the Milk. J. Dairy Sci., 27: 385-396. 218. 1946 219. 220. , Hyatt, J r., G., and Weakley, J r., C. A. Effect of Feeding Iodinated Casein to Dairy Cows on the Protein Composition and Con­ tent of Milk. J. Dairy Sci., 29:533-534. ________________________________________________________________________ 1947 , and Henderson, H. O. Further Observa­ tions on the Effects, of Feeding Thyroprotein to Dairy Cows. J. Dairy Sci., 30:576-577. Watman, R. N., and Nasset, E. S. Thyroid Activity and 1949 Resistance to Histamine-Induced Peptic Ulcer and to Acute Histamine Poisoning. Am. J. Physiol., 157:216-220. 163 221. __________________________________ * Evidence for a Non1951 thyroxine Thyroid Factor which Affects Gastric Function. Am. J. Physiol., 166:131-136. 222. Webster, B., and Chesney, A. M. Endemic Goiter in 192 8 Rabbits. III. Effect of Administration of Iodine. Bull. Johns Hopkins Hosp., 43:291. 223. Welch, H. Cause and Prevention of Hairless Pigs in the 1917 United States. Mont. Agr. Exp. Sta. Circ. 71: 37-47. 224. Westerfeld, W. W., and Lowe, C. The Oxidation of p1942 cresol by Peroxidase. J. Biol. Chem. 145:463470. 225. Williams, R. H. Relation of Obesity to the Function of 1948 the Thyroid Gland, Especially as Indicated by the Protein-Bound Iodine Concentration in the Plasm a. J. Clin. Endocrinology, 8:257-261. 226. Wilmanns, H. Z. Ges-Exp. Med., 102-269. (Quoted by 193 3 Taurog, A., and Chaikoff, I. L., The Nature of the Circulating Thyroid Hormone. J. Biol. Chem., 176:639-656. [1948].) 227. Winchester, C. F ., Comar, C. L., and Davis, G. K. ThyI r) 1 1949 roid Destruction by I , and Replacement Ther­ apy. Science, 110:302-304. 228. Wolff, J., Chaikoff, I. L., Taurog, A., and Rubin, L. The 1946 Disturbance of Iodine Metabolism Produced by Thiocyanate: The Mechanism of its Goitrogenic Action with Radioactive Iodine as Indicator. Endocrinology, 39:140-148. _________________ 229. 1947 . The Relation of the Thyroxine to Total Iodine in the Thyroid Gland. crinology, 41:295-298. Endo­ 164 230. ________________________________. The Inhibiting Action of Exces1948 sive Iodide Upon the Synthesis of Diiodotyrosine and of Thyroxine in the Thyroid Gland of the Normal Rat. Endocrinology, 43:174-179. 231. ________________________________, and Nichols, J r., C. W. The 1949 Accumulation of Thyroxine-like and other Iodine Compounds in the Fetal Bovine Thyroid. Endo­ crinology, 44:510-519. 232. Wolterink, L. F., and Lee, C. C. Relationships Between 1950 Thyroid Activity as Assayed by the ThiouracilThyroxine Method and by the Thyroid Turnover of Radioiodine in P air-fed Rats. Fed. Proc. 9:138. (Abs.) 233. 234. _________________________ *Olsen, K., and Murray, 1950 M. Effect of Estrogen on Iodine Turnover in Thyroids of Rats and Mice. Fed. Proc. 9:13 8. (Abs.) Zilversmit, D. B., Entemman, C., and F ishier, M. C. 1943 On the Calculation of "Turnover Time" and "Turnover Rate" from Experiments Involving the Use of Labeling Agents. J. Gen. Physiol., 26:325-331.