'EEHE'E l I ! WE'VE“! E” “I” — E E" E E E“ w IE'E'E "4N 14>: , (mom a- “EYE-{E [NFLUENCE GEE LAE E‘" AEEQN QN 'E’EPEYEEOE [D SECRETEQR §t\?E AS 535% LEEAE'ED ESY A DE [EECE’ Cu WU? MWEJQD TE'EGSES Ea" E'Em Duane 3‘13 EVE. S. ALLClLGFJJ SE‘IEE‘EE " EECEEEESEE‘" Fritz. L. Lm‘scheicier E957 THEE- w“ LIBRARY Michigan Stan: Univcmty _ .- _—..-~...——- *wH-nm _._._ _.. Q THE INFLUENCE OF LACTATION ON THYROID SECRETION RATE AS EVALUATED BY A DIRECT OUTPUT METHOD BY Fritz L. Lorscheider A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Physiology 1967 ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. E. P. Reineke for his guidance and understanding in all phases of my laboratory and curricular endeavors while at Michigan State University. I am also indebted to Mrs. Linda Allison and Miss Barbara Brace for their assistance in the many tedious lab— oratory analyses required of this research. Special appreciation is due Drs. H. M. Klitgaard and R. C. Meade and my undergraduate professors at the University of Wisconsin for their tutelage, discipline and encouragement during my formative academic undertakings which helped me realize opportunities for graduate study. ,This work was supported by the National Institutes of Health grant No. AM-08515-02 and the Michigan State University Agricultural Experiment Station. ii TABLE OF CONTENTS Page ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . . . . . . . . iv LIST OF FIGURES. . . . . . . . . . . . . . . . . . . V INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW. . . . . . . . . . . . . . . . . . 2 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 6 I. General . . . . . . . . . . . . . . . . 6 II. Thyroxine Substitution Method . . . . . 9 III. Direct Output Method. . . . . . . . . . 9 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . 17 SUMMARY AND CONCLUSIONS. . . . . . . . . . . . . . . 52 REFERENCES CITED . . . . . . . . . . . . . . . . . . 54 APPENDICES . . . . . . . . . . . . . o . . . . . . . 40 iii TABLE 1. LIST OF TABLES Page Experimental design for comparison of thyroid secretion rates of lactating and control rats by T4 substitution and direct output methods at three levels of iodine intake. . . . . . . . 8 Computations for the total thyroxine activity equivalent of iodine. . . . . . . . . . . . . . 14 Influence of iodine supplementation on the thyroid secretion rate of non-lactating (control) rats as measured by the T4 substi- tution and direct output methods. . . . . . . . 18 Thyroid secretion rates of lactating and con— trol rats by T4 substitution and direct output methods at three levels of iodine intake. . . . 22 Mean cell heights of thyroid follicles in lac- tating (L) and control (C) rats at three levels of iodine intake. . . . . . . . . . . . . . . . 26 iv FIGURE 1-A. LIST OF FIGURES Page Thyroxine substitution method illustrating suppression of thyroidal iodine output with graded dosages of exogenous thyroxine . . . . 11 Thyroxine substitution method illustrating the extrapolation of the suppression end point of thyroidal 1131 output to thyroidal secretion rate. . . . . . . . . . . . . . . . 11 Direct output method illustrating the pro- cedure for determining the fractional daily output of total iodine from the thyroid . . . 11 Illustration of the static thyroid-pituitary state achieved by the T4 substitution method. 11 Bar graph illustration of thyroid secretion rates of lactating and control rats by thy- roxine substitution and direct output methods at three levels of iodine intake. . . . . . . 23 Proposed schema of thyroid—mammary competi— tion for iodine . . . . . . . . . . . . . . . 30 APPENDIX A. B. LIST OF APPENDICES Page Rat Feed Mixture. . . . . . . . . . . . . . 41 Quantitative Iodine Determination in the Thyroid . . . . . . . . . . . . . . . . . . 42 Comparative Activity of L—Thyroxine and L-Triiodothyronine. . . . . . . . . . . . . 44 Procedure and Calculations for Histological Evaluation. . . . . . . . . . . . . . . . . 46 vi INTRODUCTION Studies over the past seventeen years have shown sig- nificant secretion of iodide in the milk of many species, including man. More recently it has been suggested that lactation may tend to become self-limiting due to reduced thyroid function resulting from a thyroid—mammary competition for limited available iodine. Despite efforts to substantiate this theory conclusively, little evidence has been obtained in the lactating animal which is amenable to statistical analysis. In studies reported to date, both a thyroxine substitu- tion and a direct output method have been used as indices of thyroid function. Employment of the substitution method in rats has indicated an elevated thyroid secretion rate during lactation. But when evaluated by the direct output method, preliminary studies of nonlactating rats indicated a corre- lation between iodine supply and thyroid secretion rate. Thus it was apparent that application of the latter method might actually prove a decreased thyroid secretion rate dur- ing lactation. Therefore, in view of these findings, the work reported herein attempts to compare the two methods for assessment of thyroid function in lactating rats maintained on various levels of dietary iodine. L I TE RATURE REV IEW Several studies, beginning with work by Courrier gt _l. (1949), have revealed that a considerable amount of labeled iodide is secreted in the milk. Subsequent research has shown this to occur in many species including man (Honour §£__l,, 1952; Noble and Rowlands, 1953), cows (Lengeman §£_§l,, 1955; Lengeman and Swanson, 1957), goats (Wright_gg.al., 1955; Flamboe and Reineke, 1959; Reineke, 1961), dogs (Van Middles- worth g£_§l,, 1953; Van Middlesworth gt al., 1954), rabbits (Brown-Grant, 1956; Brown-Grant, 1957; Brown-Grant, 1958), rats (Potter and Chaikoff, 1956; Potter gt al,, 1959: Grosvenor, 1960), and mice (Rugh, 1951). Protein bound iodide in milk was reported to be 50-60% in the dog (Van Middlesworth, 1955; Van Middlesworth, 1954) and 85% in the rat (Potter and 1.. 1959), Where up to 50% of 1131 Chaikoff, 1956; Potter gt was recovered in total mammary tissue plus milk twenty-four hours post administration (Potter gt al., 1959). Secretion of 1131 by the mammae is of such magnitude that thyroidal accumulation of administered 1131 is markedly decreased (Brown-Grant, 1956; Potter and Chaikoff, 1956; Rugh, 1951). That this is an active secretion is evidenced by the high in_yiyg_milk to serum ratios of 1131 (Honour 2; al., 1952; Flamboe and Reineke, 1959; Brown-Grant, 1957; Potter and Chaikoff, 1956; Grosvenor, 1960), and the same active concentration of I131 by lactating rat mammae has also been demonstrated in_yi£;g (Freinkel and Ingbar, 1956; Maqsood and Reineke, 1960a). The similarity of mammae to the thy— roid in response to goitrogens has also been shown both in_vivo (Brown-Grant, 1957; Potter gt al., 1959) and in vitro (Maqsood and Reineke, 1960a). Neither concentration of I131 nor goitrogen response was noted in other tissues to the degree shown by the mammae (Maqsood and Reineke, 1960b). In vivo secretion of iodine into milk occurs both as free iodide and as monoiodotyrosine (MIT) in peptide linkage in milk protein (Potter and Chaikoff, 1956). It was indicated that this iodide concentrating ability of the mammae is prob- ably associated with an oxidative enzyme system (Maqsood and Reineke, 1960a). This and an additional enzymatic system for the formation of MIT were further characterized as being associated with the lactation process and requiring cofactors of Cu++ and Mn++. Both 1131 concentration and MIT formation decreased with mammary involution (Reineke, 1965). Obviously iodine combined with milk protein is not available for hor— mone synthesis and thus measurements of thyroxine output might be expected to be altered. Over the past twenty-five years various methods have been developed to estimate thyroxine secretion rate (TSR); these have included the goitrogen technique (Dempsey and Astwood, 1945) and the thyroxine degradation procedure (Sterling 2: al., 1954; Ingbar and Freinkel, 1955). A thy- roxine (T4) substitution method for determining thyroid hormone secretion rate was first described in individual animals (Henneman _§_§l., 1952) and subsequently applied in several other species (Reineke, 1959)° This method has yielded consistent values under ordinary conditions of management. As further application of the thyroxine degradation method has pointed out, a quantitative assessment of thyroid function necessitates a definition of the proportional meta- bolic effect of triiodothyronine (T3) (Gregerman, 1965). And in addition to necessary corrections for T3 activity, experiments have demonstrated that iodine intake may also influence thyroid secretion rate. In a preliminary report a more direct measurement of thyroxine secretion (fractional output rate times thyroidal iodine content), which included corrections for the more biologically active T3 component, revealed a significant correlation between dietary iodine supply and thyroxine secretion rate. But the thyroxine sub- stitution method exhibited no such correlation (Reineke, 1964). Using the substitution method it was first proposed by Flamboe and Reineke (1959) that lactation might become a self-limiting process due to reduced thyroid function result- ing from a thyroid-mammary—kidney competition for limited available iodine. However, Grosvenor and Turner (1958) had reported significantly elevated thyroxine secretion rates in lactating rats when employing the thyroxine substitution method of Reineke and Singh (1955). Since Reineke (1964) had demonstrated with the direct output method that a corre- lation exists between iodine supply and thyroxine secretion, it was hypothesized that under conditions of limited avail- able iodine, such as lactation, this direct method might actually reveal a decreased thyroid secretion rate. In view of these findings a comparison of the thyroxine substitution and direct output methods for evaluating thyroid function was made in lactating rats on various levels of iodine intake. With the latter method corrections were made for T3 release. It was attempted to substantiate the theory that lactation is a self-limiting process and to further define the interrelationships of thyroid-mammary function during lactation. MATERIALS AND METHODS I. General Thyroid secretion rates (ug T4/100 gm. B.W./day) of lactating and control rats maintained on three levels of dietary iodine were determined by the thyroxine substitution and direct output methods. Three groups of female rats of the Carworth CFN strain weighing approximately 200 grams each were fed a corn-soybean diet for a thirty day period beginning two weeks prior to isotope administration until sixteen days post partum. Laboratory temperature was maintained at 24.50C..i 10 and lights were on 14 hours per day. The feed (Appendix A) con- sisted of a finely ground corn meal, soybean oil meal, vitamin supplements, and a special mineral salt premix pre- pared at the Michigan State University Department of Animal Husbandry feed mixing plant. The diet of group number I was low in iodine. A 0.2% stock solution of potassium iodide was added to the diet at levels of 0.65 ug iodine and 1.50 ug iodine/gram of feed for groups II and III respectively. Subcutaneous injections of 5 no and 8 no of carrier free I131 were given each control and lactating rat, respectively. External thyroid and body background counts (epigastric region) were taken under Nembutal anesthesia (5 mg/100 gm of body weight), for all subjects on alternate days starting with the third day post 1131 administration for a series of six counts. Counting was achieved with a radiation analyzer-scaler connected to a well—type collimated NaI scintillation detector monitored with a count rate meter for optimal geometry. The 2” NaI crystal was mounted beneath a 2.5 cm aperture of a 2.5 x 25 x 55 cm. lead shield platform. A standard prepared at one-tenth of the injected dose was counted with each group. Counts were corrected for body and room background by the method of Wolff (1951). Thyroidal 1131 concentration was expressed as a per cent of the injected dose to correct for physical isotopic decay using the formula: Thyroidal 1131,as per cent of injected dose = thyroid count _ (body backgroundgroom background) x 100 (1) (standard count-room background) The thyroid secretion rate (TSR) of rats on three levels of iodine intake was evaluated by a comparison of the thy- roxine substitution method (Reineke and Singh, 1955) and di- rect output method (Reineke and Lorscheider, in publication) under both lactating and nonlactating conditions (Table 1). Litters were equally distributed to establish a similar degree of nursing for all lactating subjects. Table 1. Experimental design for comparison of thyroid secretion rates (TSR) of lactating (L) and control (C) rats by T4 substitution and direct output methods at three levels of iodine intake. —{ h Dietary iodine level TSR TSR (Hg I added/gm. feed) TA substitution Direct output Group L C L C I 0.00 II 0.65 III 1.50 II. Thyroxine Substitution Method A 0.1% stock solution of L—thyroxine was prepared by first dissolving the T4 pentahydrate crystals in NaOH and then neutralizing the alkaline solution to a pH of approxi- mately 8.6 with HCl. At this point the T4 precipitates as a fine opalescent suspension. With the thyroxine substitution method graded dosages of L-thyroxine (Merck) are injected subcutaneously daily and increased every third day (0.75, 1.50, 2.25, 5.00, 5.75 ug T4/100 gm. body weight) until thyroidal 1131 output is maximally supressed (Figure 1-A). The per cent of the previous per cent of the injected dose vs. ug T4/1OO gm. body weight is then replotted linearly. The end point of maximal suppression (97.5%) yields the amount of T4 needed to establish a static thyroid—pituitary state (Figures 1-B and 1—D). Thus TSR by the T4 substitution method is that level of exogenous thyroid hormone that reduces further out- put of previously collected thyroidal I131 to 97.5% of the prior count. III. Direct Output Method By the direct output method TSR was estimated as the product of daily fractional output rate times the thyroxine equivalent of total thyroidal iodine content (Figure 1—C). The calculations used to compute fractional turnover of iodine per day are the following: 10 .UonumE COHusuHquSm we mnu >9 Um>ma£om mumum SMMUAsuHmnpflouwfiu oaumww ms“ mo coflumuumDHHH .uHoussu was Eoum mCHUOH Hmuou mo usmpso SHflmp HMCOHpomuw m£p mEHcHE Inmuwp how muspmuoum mnu mafipmuuwsaafl pozumfi usmuso uUmMHQ .mumu coaumnomm Hmoflouwnu ou usmuso amHH Hmofloumnu mo ucflom Usm COHmmmHQQSm msu mo soaumHommnuxm mzu msflumuumsaafl Uonpme coausuflquSm ocflxouwse .mcflxouhsu msosmmoxm mo mmmmmop UmpmumESuHB usmuso H64H Hmpfiouwzu mo GOflmmmummsm msflumuumsaafl Cozumfi sowusufluwflsm mcflxouhzfi H mudmfim .mé .¢-a 11 INJECTED DOSE X Ola musmflm .ooxm E. .50.". m> .50) on the 1.50 ug iodine diet. This seems to indicate similarity in the two methods of 17 18 Table 5. Influence of iodine supplementation on the thyroid secretion rate of non-lactating (control) rats as measured by the thyroxine substitution and direct output methods. The number of rats in each trial is shown in parentheses. Diet TSR TSR Group (ug I added/ T4 Substitution Direct Output gm. feed) Method Method (ug T4/100 gm.) (ug T4/100 gm.) I none 2.05 0.65 (7) (5) .x. vsP > .50 P < .15 II 0.65 2.05 0.80 (9) (10) P > .25 P < .05 III 1.50 2.55 2.29 (7) (7) ‘X' P - Mann4Whitney U test (Siegel, 1956). 19 evaluating TSR when iodine supply is sufficient. The 1.50 ug iodine diet is somewhat in excess of previously reported 1., 1951) but compares favorably with values (Parker g£_ Wayne and Purina normal diets of 1.00-1.50 ug iodine per gram of feed. These data presented in Table 5 agree favorably with data previously reported by Reineke (1964): first, that no difference is exhibited in TSR by the T4 substitution method employed in rats on various levels of dietary iodine, and second, that TSR by the direct output method is significantly decreased when iodine supply is limited and that a corre- lation can be shown between TSR and iodine availability. From the foregoing results it may be concluded that the T4 substitution method measures the apparent thyroid hormone demand, but can represent the actual TSR only if iodine supply is adequate. Thus, under conditions of limited iodine, such as lactation, the direct output method would appear more applicable. Because of the lengthy laboratory procedure and large numbers of animals needed for adequate statistical evaluation, the goitrogen technique of Dempsey and Astwood (1945) for measuring TSR has proved relatively impractical. By this indirect method TSR was reported to be 5.20 ug T4/100 gm. at 250C. The thyroxine degradation procedure involves protein bound iodine (PBI) determinations which iodine intake may influence markedly (Sterling et al., 1954; Ingbar and 20 Freinkel, 1955) and by this indirect method TSR for the female rat has been estimated at 1.22 ug T4/100 gm. but, this method does not correct for the T3 component. Previous TSR values by the T4 substitution method ranged from 1.70 to 2.21 ug T4/100 gm. in the adult female rat (Reineke, 1964; Reineke and Singh, 1955), and this agrees favorably with data of the present study. The slight elevation of the present TSR may represent the goitrogenic effects of trace amounts of thiocyanate (SCN) in the corn-soybean protein. It is recognized that fluctuations of TSR in the rat may indicate not only differences in methodology, but also variances due to age, sex, strain and environmental temperature. Though most attempts to evaluate TSR have used rats receiving normal levels of iodine, the present studies which varied the dietary levels of iodine reveal no differences in the TSR as a function of diet when using the T4 substitution method. But this method bypasses the thyroid and assesses only indirect parameters of thyroid function. Thus, the present study employs the direct output method which yields significantly lower TSR on reduced iodine intake, and this apparently substantiates the theory first proposed by Flamboe and Reineke (1959) that if inadequate amounts of iodine are taken up for normal hormone synthesis, thyroid secretion rate may decrease. Table 4 presents the TSR of lactating and control rats by T4 substitution and direct output at three levels of 21 iodine intake. Figure 2 is a graphical illustration of the TSR data contained in Table 4. It should be noted that the non-lactating control data of Table 4 is that previously discussed in Table 5. It, therefore, becomes appropriate to discuss these data in context since lactating and control data for both TSR evaluation methods were run simultaneously for a given dietary level of iodine. Table 4 indicates and Figure 2 illustrates that, with the T4 substitution method at 0.00 and 0.65 ug iodine added/ gm. diet, TSR of lactating rats is significantly elevated above control TSR values. This finding is in accord with previous results obtained by this method (Grosvenor and Turner, 1958) that showed elevated TSR during lactation. This method appears to reflect elevated TSH output caused by a hyperactive pituitary as a result of pre—existing iodine deficiency instead of an increase in TSR. Based on a normal Wayne diet of 1.00 ug I/gm. feed, the slightly increased 1.50 ug showed no difference between lactating and control TSR values. Furthermore, T4 may actually enhance lactation so that more hormone is required to maintain the increased lac- tation state. This would necessitate additional T4 for TSH suppression and thereby result in a positive feedback cycle. Using the direct output method (Table 4) it is evident that with inadequate iodine intake (Group I) thyroidal iodine is markedly reduced. This reflects the mammary drain on limited iodine which would otherwise be available for thyroid 22 .UMSOHmEm ma ummulu Umuflmmcs cm mmHBHmnuo “Umaaflmasm uog ma huflmcmmofion mUCMHHm> mo coaumESmmm map ma mflmhamcm oauumfimummgog m .Awmma .Hmmmfimv ummu D wmcuflnzwscmz n m * mo. v om. A om. A m Ase AoHv Aev Lose 15L LAC HHH «m.o mm.m m>.o mm.am N©.md mm. mm.m m¢.m om.H mo. v om. A $00. v m Lose Ame AOfiv Amv Amy Acav HH mw.o om.o mm.o m¢.m mm.m >m.a mo.m m¢.m no.0 moo. v ,NooJ NS. 0 *3 Amy Ame Ame Ame Ase Amv H mm.o mm.o mfi.o mm.m HH.H Hm.fi mo.m am.m msos o\q o q o q o\q o A msouo A.Em ooa\«e miv mcHUoH A.Em OOH\.B w1¢ Apmmm .Em mme Hmufloumse Hmuoe m: mms \umuum H mac usmuso uomuflo soflusufluuhsm «s umflo e .mmp\.u3 moon .Em ooa\ua 51 mm memmumxw ma Mme .mmmmnugmumm :fl czonm we amauu 30mm CH wumn mo Hmnfizc mna .mxmusa wGH00fl mo mHm>mH mmusu um moonumfi usmuso uomuflp 0cm Goausu IHquSm EB >3 mumu AOL Houucoo Usm ARV mcflumuomH mo mwumn coaumuumm UHOHNSB .w magma : lulu-ll can a 1....- a Gilli d u . ,... .. .u - flutllLL I E 4 E II. El I - II: .II . n . I - o in.~[~\ 0 E03 \XUuUflJJ I...~H¢ AJAJIH\.‘.HN ma. 'dL. mvvfivllllfl'hflfllflxmluv ”VHS. V,Nm..fi Min» H mimic-dd uU> . Unfi .lul-a.l.\/I.I- llrIA-‘nnu uo\ tied-vou- 00000 Ujl,In-l1v yi.vndnuui pin-n» .q...‘,‘-. in u‘fi‘—-.uh 25 0w>._d0.v.u .Illl .Illl 0w>4.00..na .IIIIIIIIII “3.x a . \~\\. - 10; 0, N (m6 oon/vi EN!) 391 I Q [0 5&8 Dues 0 fl 2550 Emma u U 295:5me E 0 fl zetfizmmnm fl ._ U I cum... E3326 H a: 00. 0mm“. onuovuo H o: no.0 _ cum... 63330 H on 00.0 E. .>MU\.u3 MUOQ .Em 00a\¢a 01 mm memmumxw mum mma How mmsHm> .mxmucH weapofl Ho mHm>mH mmn£u um mpozumfi usmpso uomufip 0cm cofiusuflumnsm «9 HQ mum“ HOV Houucoo paw HHV mcflumuomH mo mmumu coaumuomm UHou>£B .N ousmfim 24 incorporation. Even in cases where lactating and control levels of iodine are similar (Group II) K'4 values (Equation 2) tended to be lower in lactating rats. This is contrary to an earlier report where a simultaneous decrease in thy- roidal uptake was observed with an increased thyroidal re- lease rate of isotope (Brown—Grant, 1956)° When K'4 values were corrected for recycling of iodine to K4 values (Equation 5), release rates were still not necessarily greater during lactation. In applying the Brownell equation to the direct output method it may be generally stated that the greater the dietary deficiency in iodine for non-lactating (control) rats, the greater the attempt to conserve iodine as indicated by larger U uptake values (Equation 5). As U becomes larger in control rats on reduced iodine intake, K4 becomes correspond— ingly larger. This correction by a mathematical thyroid analog in lactating animals is apparently not as significant. This may possibly be the result of mammary iodine partition- ing acting as an effective trap to prevent recycling of iodine. The TSR as measured by the direct output method (Table 4 and Figure 2) shows a significant reduction during lac— tation when compared to controls on all three levels of iodine intake. However, with increased levels of iodine, the lactating TSR values are significantly increased to approach control values. In addition, histological evaluation, as 25 described in Appendix D, indicated that endogenous TSH stimulation had effected a significant hyperplasia of thy- roid follicular cells (Table 5) of lactating rats on 0.00 ug of added iodine, with a similar tendency for lactating rats on 0.65 ug of added iodine. This hyperplasia was pre- sumed to indicate a functional hypothyroidism. Long term PBI and histology studies of Grosvenor (1962) tend to agree with these observations. It should be noted that the direct output lactating TSR of 0.78 ug T4/100 gm° may actually be larger than that calculated. Recent studies indicate that 1131 output rates may be masked by recycling of 113:- back to the mother by way of the litter urine. Capek and Jelinek (1956) demonstrated that micturition in neonate rats is dependent on perineal stimulation by the mother for up to 16 days post partum. In current research this phenomenon has been observed to occur in rabbits as well. Present studies and those reported by l., 1965; Samel and Caputa, 1965) have n“— others (Samel e; demonstrated a significant amount of 1131 from tagged rat and rabbit neonates to be present in the untagged mother 24 1131 injection. Since milk contains a high iodine hours post titer, it was proposed from this observation that the recycl- ing of iodine, via the litter urine injested by the mother, acted as a conserving mechanism for iodine. The direct output method is based on three assumptions as applied to this study. First, that all of the iodine 26 .ummulb xmcuflnzncsmz * who. moo. V m * N>.HH m¢.fid mm.afi NN.MH om.Na mm.>fi H10 .us Hamo m 0H 0H m mumu mo HmQEDZt U A U A U A mmm.Em\Um00m H 01 0m.H ummu .sm\umuum H m: mm.o vowm .Em\pmppm H 01 00.0 .mxmucfl mCHUOH mo mHm>mH mmunu um mums HOV Houucoo 02m Adv moduMHomH CH mmHoHHHow Uflouhnu mo munmflwn HHmo Gmmz .m mHQMB 27 normally released from the gland is in hormonal form; second, that T4 and T3 are released in the same proportions in which they are found in the thyroid; and third, that under conditions of iodine deficiency and/or during lacta- tion the ratio of T3 txb T4 released is not significantly altered. If the first assumption is not met it would result in an overestimation of hormone output. Pitt-Rivers and Rall (1961) have suggested evidence in thyroid-blood equilibrium studies to support the second assumption. Rosenberg §£_al. (1966) have shown that despite early heterogeneous labelling and turnover of iodine, after a few days homogeneous label— ling is attained. In regard to the third assumption, the i vivo studies of Matsuda and Greer (1965) which measured thyroid venous effluent in the rat, have suggested that under elevated TSH stimulation there is a substantial proportional increase in the secretion of iodotyrosines, chiefly DIT, and in the amount of iodothyronines, mainly T3. Further, Shimoda and Greer (1966) have suggested that rats under iodine de— ficient conditions, propylthiouracil, or TSH all evidenced increased in_vitro labelling of iodothyronines, mainly T3. However, Heninger and Albright (1966) measured total thyroidal quantities of T4 and T3 in iodine deficient rats to be 0.17 ug and 0.10 ug respectively per gland. Using these values the T4 plus T3 equivalent <1f iodine in terms of T4 is calculated to be 2.25 from Table 2 and a final correction 28 factor of 5.42 is obtained (Reineke and Lorscheider, in publication). Even if this factor is applied to the present data the increase in TSR is still markedly reduced in lac— tating subjects. Thus, the apparently more efficient shift to T3 production under iodine deficiency fails to compensate adequately in elevating TSR to normal. In view of these findings the T4 substitution method appears to reflect the physiological demand for thyroxine and/or may represent a latent TSH suppression by exogenous T4 as a result of iodine deficiency. However, this estimate represents the true hormone output only if the thyroid is receiving sufficient iodine to meet the hormone demand. The direct output method appears to be a more valid measure- ment of actual thyroxine released under conditions such as lactation which limit iodine available for thyroid hormone synthesis. With an adequate supplement of iodine, estimates of thyroid secretion rates are similar, employing either method of evaluation, under non-lactating conditions. During lactation there is sufficient loss of iodine in the milk that secretion of thyroxine is reduced below normal, resulting in a functional hypothyroidism, as evi- denced by histology, unless adequate iodine is supplied in the diet. By increasing the intake of iodine, the deficiency in hormone production is significantly alleviated. However, the exact extent of alleviation could not be assessed at this time since recycling of 1131 by way of the litter urine 29 back to the mother masks, to some degree, the true output rate of hormone. Once this is clarified, it might still prove necessary to determine whether this disparity between lactating and control animals can be overcome by additional iodine supplementation. The proposed thyroid-mammary-kidney distribution of iodine is illustrated in Figure 5. The size of the arrow is indicative of the approximate proportional distribution for several species. The chart shows that lactating females do not receive an adequate supply of thyroidal iodine even when their dietary intake is doubled to normal. Thyroid hormone enhances milk production. However, increased mammary secretion results in increased competition with the thyroid for available iodine, thus tending to make lactation a self— limiting process. The chart also shows that the thyroid does not take up more iodine than it requires, in non- lactating females, so that the amount going to the thyroid remains constant. As indicated, some species recycle iodine via the litter urine. This phenomenon tends to complicate the evaluation of certain thyroid-mammary parameters. In support of the data presented herein, it is interest- ing to note that clinical studies have indicated low serum butanol extractable iodine values in women during the latter part of the third trimester (Man 23 al., 1964), near the on- set of lactation. 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