- Email: [email protected]
Relation of thyroid hormone secretion rate (TSR) in rats and fowl to hormone content of their thyroid glands
GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
25,25-30
(1975)
Relation of Thyroid Hormone Secretion Rate (TSR) in Rats and Fowl to Hormone Content of Their Thyroid Glands1-2 K. N. SINHA~, R. R. ANDERSON~, Department
of Dairy
AND C. W. TURNER
Husbandry, University of Missouri-Columbia, Columbia, Missouri 65201
Received July 28, 1974 By the determination of thyroid hormone secretion rate (TSR), the amount of L-thyroxine (L-Tq) secreted each 24 hr has been estimated. An attempt has been made in this study to determine by bioassay the relation of the daily TSR to the amount of stored hormone (thyroglobulin) in the thyroid of the rat and chicken in terms of l-T4 equivalents. The individual TSR of 28 rats and 36 chickens was determined. Two animals having the same TSR were grouped as the donor and recipient of the thyroid homogenate. To bioassay the L-T~ equivalents of donor thyroids, recipient rats and chickens were injected with graded levels of the thyroid homogenate until release of thyroidal 1311was blocked according to the technique used in the estimation of TSR. Mean L-T~ equivalents in the thyroids of donor rats and chickens were 13.9 and 154.4 pg with a daily secretion of 3.58 and 27.98 pg, respectively. The amount of L-T~ equivalent in the thyroid homogenate was 3.82 & 0.35 times the daily TSR in rats and 5.56 -t 0.23 times in chickens (P < 0.001). The data suggest that in rats and chickens there is a significant difference in the magnitude of bioassayable L-T, equivalents stored in the thyroid in terms of daily TSR.
hormone present in the thyroid glands of rats (Rattus norvegicus) and chickens (Callus domesricus) in relation to their daily TSR. In other words, the magnitude of the hormone storage of the thyroid glands to the daily TSR was determined. Since thyroglobulin stores the thyroid hormones, it was considered possible that graded amounts of the thyroid homogenate could be administered parenterally to block the secretion of the thyroidstimulating hormone (TSH) as is done in the case of TSR determination. This procedure would thus determine the bioassayable thyroid hormone in the thyroglobulin in terms of L-T~.
The thyroid stores its hormones within the lumen of the individual follicles as part of thyroglobulin. Prior to passage into the blood stream, the thyroglobulin is hydrolyzed and thyroxine (L-T~) and triiodothyronine (L-T~) flow into the blood as the biologically active hormones. By the determination of the daily thyroid hormone secretion rate (TSR) the amount of hormone secreted each day may be estimated in terms of L-T~ (Pipes et al., 1958). The object of this experiment was to determine the amount of biologically active 1 Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 5463. Approved by the Director. 2 Supported in part by a grant from the U. S. AEC, AT(1 l-l)COO-1758-6 and Contract AT(1 l-l)COO-301-137. 3 Present address: Department of Surgery, Surgical Research Unit, University of Cincinnati Medical Center, Cincinnati, Ohio 45267. 4 Address for reprint requests.
METHODS
Experiment Thyroids
Press, Inc. All rights of reproduction
Content
of Rat
The TSR of a group of 28 female Sprague-Dawley rats was determined individually by the method of 25
Copyright @I 197s by Academic Printed in the United States.
1. Thyroxine
in any form reserved.
26
SINHA.
ANDERSON
AND
Pipes et al. (1958). The method consisted of intraperitoneal (ip) injection of 10 @i of carrier-free 1311and 48 hr were allowed for thyroidal uptake of radioiodine. Graded amounts of L-T, were injected subcutaneously (SC) for 2 consecutive days until the release of thyroidal 13*1was blocked 95% or more. A goitrogen, Tapazole, was also injected daily SCduring the entire period of TSR determination at a level of 0.4 mg/lOO g body wt to reduce recycling of the metabolized 1311by the thyroid. Two rats having the same TSR were paired as the donor and recipient rat; thus, 14 rats served as donors of thyroid glands and 14 rats were recipients of thyroid homogenate. After the initial TSR determination, 3 wk were allowed for decay of remaining small amounts of 13’1 radioactivity in the thyroid, the donor rats were sacrificed. and the thyroid glands were collected on ice, weighed, and homogenized, each in 5 ml of cold 0.9% saline. To prevent thawing the whole homogenate for injection purposes, the thyroid homogenate from each rat was further subdivided into three glass vials containing 1.0, 1.5, and 2.0 ml of the homogenate and stored at -20°C until used. Recipient rats were injected ip with 10 &i la11 and the process of TSR estimation was repeated, but in this case thyroid homogenates were injected instead of L-T~. Graded levels of the homogenate (0.50, 0.75, and 1.00 ml) were injected ip into each recipient rat for 2 consecutive days until the release of thyroidal la11 was blocked 95% or more as compared to previous dose level, as in the method of TSR estimation. Tapazole was also injected into these rats to reduce the recycling of metabolized 1311by the thyroid.
Experiment 2. Thyroxine Chicken Thyroids
Content of
At about 12 wk of age, the individual TSR of 36 white Leghorn cockerels was estimated by the method earlier described. To bioassay the L-T, equivalents in the chicken thyroids the same protocol
THECOMPARISONOF
TURNER
was followed as in the case of rats with the following modifications. Thirty microcuries of carrier-free 13’1 was injected SCper bird. Tapazole was injected SC at a level of 0.5 mg/lOO g body wt/day. The thyroid from each donor bird was homogenized in 10 ml of 0.9% saline. Graded levels of 1.25, 1SO, and 2.00 ml of the homogenate were injected SC into each recipient bird.
RESULTS
Experiment 1. Thyroxine of Rat Thyroids
The mean TSR of a group of 28 rats having a mean body weight of 300.8 g was 1.20 ,ug L-TJlOO g body wtlday with a range of 1.0 to 1.5 pg (Table 1). They were then divided into two groups based on equal TSR. To assay the 14 donor rat thyroids, graded amounts of the thyroid homogenate were injected ip into the 14 recipient rats. In seven recipient rats, the thyroid homogenate blocked thyroidal 1311 release (95% or more) at the l.O-ml level or less and in the remaining seven rats the extrapolation technique (Reineke and Singh, 1955) was used to compute the end points. The recipient rats with a mean body weight of 300.3 g required a mean of 3.58 /*g L-T, in their initial assay (Table 1). Since a mean of 1.48 ml of thyroid homogenate was equally effective, it was assumed that this volume of homogenate contained the same amount of L-T, equivalent. The mean total L-T, equivalent in 5
TABLE 1 TSR WITHTHYROIDGLANDCONTENTOFHORMONE
DOllOr
No. of animals 14 Rats 18 Chickens
Body weight” Cd 308 C_ 10 1554 k 24
Wet thyroid weight (mg)
TSR/IOO g body weight (PP)
18.88 i: I .OO 1.20 f_ 0.05 87.2 2 5.3 1.80~0.07
” Mean ? standard error. ’ Student’s I test I vs 2 P < 0.001.
Content
Recipient
Net daily secretion of L-T, k.4
Body weight (2)
TSR/i00 body weight w
3.61 f 0.20 27.98*1.07
300 r_ 9 1672+34
I.MC-0.04 1.80+0.07
g
Net dady secretion of L-T, (P&Y) 3.58+_0.18 30.21?1.37
BlOCkbIg dose of homogenate (ml) 1.48 f_ 0.17 2.00+0.10
L-T, content in donor thyroid gland (/a) 13.88r 1.48 154.4~7.3
Net content “s TSR CdaysY 3.82’ kO.35 5.56* 2 0.23
STORED
THYROXINE
IN RATS
ml of homogenate was calculated to be 13.88 pg. The average daily secretion of the L-T, of the donor rats with an average body weight of 308.2 g was 3.61 pg. Thus, the total thyroid hormone (present as thyroglobulin) was 3.82 + 0.35 times the estimated daily TSR, with a range of 2.09 to 6.22. Experiment 2. Thyroxine of Chicken Thyroid
Content
The mean TSR of a group of 36 white Leghorn cockerels at 12 wk of age was 1.8 lug/l00 g body wt with a range of 1.5 to 2.5 pg of L-T*. In all but three birds the thyroid homogenate of the donor bird blocked thyroidal 1311release at the 2.0-ml level or less. In these three birds, the extrapolation technique (Reineke and Singh, 1955) was used to compute the end point. The recipient birds with mean body weight of 1672 g required a mean of 30.2 1 pg of L-T~ in their initial assay. Since a mean of 2.0 ml of thyroid homogenate was equally effective, it was assumed that this volume of homogenate contained the same amount of L-T, equivalent. The mean total L-T, equivalent in 10 ml of homogenate was calculated to be 154.4 pg. The average daily secretion of L-T~ of the donor birds with an average body
COMPARISON
Number of animals 6 Rats 5 Rats 3 Rats 6 Chickens 6 Chickens 3 Chickens 2 Chickens 1 Chicken
OF TSR, Mean body weight (g)
THYROID
WEIGHT, TSR/100 g body weight w
291 -c 16” 318 f 13 326 + 32 1.556 1596 1483 1498 1544
a Mean f SE. * P:Student’s t test.
2 2 + f
49 32 66 91
1 vs 3 P < 0.01;
AND
27
FOWLS
weight of 1554 g was 27.98 pg. Thus, the total thyroid hormone (present in thyroglobulin) was 5.56 + 0.23 times the estimated daily TSR, with a range of 4.0 to 7.53 (Table l), which was significantly higher than 3.82 2 0.35 times the estimated daily TSR in the rats (P < 0.001). In Table 2, the mean wet thyroid weight and L-T~ content in the donor rat and chicken thyroids are shown. In the estimation of TSR, L-T, was injected SC in graded increments of 0.25 pg L-T,/lOO g body wtlday, then 0.50, 0.75, 1.00, 1.25, 1.50, and so on for 2 consecutive days until the release of thyroidal 1311 was blocked 95% or more as compared to the previous injected dose (Pipes et al., 1958). In the case of rats, the TSR ranged from 1.00 to 1.50 Fg/lOO g body wt. Rats with TSR of 1.OO, 1.25, and 1.50 pg/ 100 g body wt were grouped together to compare if there was any relationship between TSR, wet thyroid weight, and L-T, content. Likewise chickens with TSR of 1.50, 1.75, 2.00, 2.25, and 2.50 pg/lOO g body wt were grouped and mean wet thyroid weight and L-T, content in glands were calculated. Group means were compared by t test. In the case of chickens, net content versus TSR data indicated that there was a gradual decrease in stored hormone
TABLE 2 AND L-T, CONTENT
IN DONOR
Wet thyroid weight
RATS AND CHICKENS
@x4
f&Id
Net content vs TSR (days)
1.00 1.25 1.50
17.7 + 1.8 20.6 f 2.0 18.3 + 1.2
12.1 2 1.0 14.4 k 3.3 16.4 +- 4.2
4.26 +- 0.50 3.53 f 0.70 3.43 2 0.72
1.50 1.75 2.00 2.25 2.50
101.8 89.5 77.7 59.5 70.0
1 vs 4
P < 0.001; 2 vs 4 P < 0.001;
” * t f
11.2’ 4.62 11.73 0.5”
L-T, content in gland
146.6 153.4 144.3 162.6 221.4
5 vs 7
* f c +
13.3 10.7 17.8 16.1
6.20 5.50 4.83 4.81 5.73
f 0.415 f 0.416 k 0.417 AZ 0.18*
P < 0.05; 5 vs 8 P c 0.02.
28
SINHA,
ANDERSON
in the thyroid as the TSR increased. Wet thyroid weight in birds with TSR of 1.50 pg/ 100 g body wt was significantly higher than that in birds with TSR of 2.00 (P < 0.01) and 2.25 (P < 0.001). However, in the case of rats no such difference was found. It might be suggested that wet thyroid weight may not differ significantly in rats with low TSR and high TSR in the euthyroid condition.
AND
TURNER
pounds and iodide, MIT, DIT, and L-T~ were identified in the plasma (Brown and Jackson, 1955, 1956). It has been shown that after the SC administration of a saline preparation of 1311-labeled thyroglobulin to guinea pigs, plasma and other tissues contained labeled iodide, MIT, DIT, and L-T, (Gilboa and Mitchell, 1964). The present study indicated that L-T, could be released after parenteral administration from its combination with a protein (thyroglobulin) DISCUSSION to enter the blood stream in order to block In the estimation of TSR, L-T~ was in- TSH secretion. It is not known whether jected SC in graded increments until the the iodotyrosines present in the thyroid are release of thyroidal 1311was blocked. The effective in inhibiting TSH secretion. The absorption of L-T~ into the blood thus observations presented are based upon the reached a concentration sufficient to block assumption that they are ineffective. the release of TSH from the pituitary. It might be suggested that the injection The question arises as to the rate of SC of thyroglobulin would stimulate the proabsorption of L-T, when it is injected in duction of antibodies which in turn might the form of thyroglobulin or iodinated ca- influence the biological activity of the sein (Protamone). Protamone is standardthyroglobulin. Since the homogenate was ized by the manufacturer to contain 1% injected for only 2-6 days, such an effect L-T+ In comparison with injected L-T~, on the assay of the hormone content of the was minimal. Protamone was found to be 112% effec- thyroglobulin tive in the rat (Bauman and Turner, 1966). When Tapazole is administered it preThe data indicated that L-T~ was absorbed vents the reutilization of metabolized iofrom a SC site when injected in the form of dide by the thyroid and blocks iodination the protein molecule of iodinated casein and coupling of iodotyrosines in the comparable to free L-T~. thyroid gland. However, it does not interfere with the release of preformed horThe method for estimating the daily TSR in terms of L-T, has been utilized in mones L-T, and L-T, from thyroglobulin. the present study to measure the total bioPrevious studies indicated that the biological activity of the thyroglobulin present logical activity (Shellabarger, 1955; Newin the thyroid glands of rats and chickens. comber, 1957) and half-lives (Heninger The TSR is measured in terms of daily in- and Newcomber, 1964; Singh et al., 1967) jections of L-T~, whereas the estimation of of L-T, and L-T, are equal in the chicken. the total thyroid hormone activity of the Srivastava and Turner (1967a, b) have rethyroglobulin is based on the injection of ported L-T~ to be slightly over twice as active biologically as L-T~ in fowl, similar to the thyroid homogenate. That thyroglobulin was hydrolyzed on injection and findings in rats (Pipes and Dale, 1963; Bauman et al., 1965) and in dairy cattle L-T, and L-T, were absorbed into the blood stream was indicated by the (Premachandra et al., 1961). Rosenberg et al. (1963) reported that blockage of the release of thyroidal 1311in L-T~ was not detected in digests of cockthe recipient rats. It has been reported that thyroglobulin given parenterally resulted in erel thyroid tissue. Vlijm (1958) reported the formation of several iodinated com- that L-T, in the digests of cockerels’
STORED THYROXINE
thyroid glands does not exceed 5% in stages of its synthesis after 1311injection. In the rat, hydrolysis of thyroid does not yield more than 3% of T, after 1311injection (Tong and Chaikoff, 1958). Since the amount of L-T~ in thyroglobulin is minimal, its presence should have little influence upon the results of this experiment even if the biological activity of L-T~ differs from L-T~. The bioassay of thyroid homogenate indicated that the amount of hormone stored as thyroglobulin in the thyroid was sufficient for normal secretion for periods of 4.0-7.5 days with a mean of 5.6 in the chickens and 2.1-6.2 days with a mean of 3.9 in the rats. It has been known that thyroid hormones are stored as thyroglobulin in the thyroid, but the magnitude of storage has not been investigated. The present study estimates the magnitude of stored thyroid hormones in L-T, equivalents in the gland by bioassay. When rats or chickens are subjected to cold environment, metabolism of thyroid hormones is increased and there is increased need of thyroid hormones for tissue. This leads to increased secretion of TSH which stimulates the thyroid for hormone synthesis and release. Under this condition preformed L-T, and L-T, in the thyroid may serve as an additional supply of hormones for tissue needs. The present study suggested that chicken thyroids contained significantly more bioassayable L-T, equivalents than those of rats in terms of daily TSR (P < 0.001). Perhaps chickens are better adapted than rats for short periods of sudden exposure to cold. Fowls placed in a cold environment showed a gradual increase in TSR (Stahl er al., 1961). As the TSR increased in chickens, there was a gradual decrease in stored hormone in the thyroid. Under these conditions, the thyroid weight, as well as the reserve supply of hormone, decreased.
29
IN RATS AND FOWLS
REFERENCES BAUMAN,
T. R., PIPES, G. W., AND TURNER,
C. W.
(1965). Relative potency of several analogues when substituted for L-thyroxine in the estimation of thyroid secretion rate in rats. Endocrinology
76, 537-538.
C. W. (1966). Comparison of biological activity of orally administered and injected L-thyroxine, L-triiodothyronine and thyroprotein in rats. Proc. Sot. Exp. Biol. Med. 123, 9-14. BROWN, F., AND JACKSON, H. (1955). Metabolism of thyroglobulin labelled with iodine131. Nature 175, BAUMAN,
T. R., AND TURNER,
472-413. BROWN, F.,
AND JACKSON, H. (1956). The fate of 1311-labelled homologous and heterologous thyroglobulins in the rat, dog, monkey and rabbit. Biochem. J. 62, 295-301. GILBOA, Y., AND MITCHELL, M. L. (1964). Metabolism of thyroglobulin-1131 in the guinea pig in vivo and in vitro. Metabolism 13, 661-668. HENINGER, R. W., AND NEWCOMER, W. S. (1964). Plasma protein binding, half-life and erythrocyte uptake of thyroxine and triiodothyronine in chickens. Proc. Sot. Exp. Biol. Med. 116, 624-628. NEWCOMBER,
W. S. (1957). Relative potencies of thyroxine and triiodothyronine based on various criteria in thiouracil-treated chickens. Amer. J.
Physiol. 190, 413-418. PIPES, G. W., AND DALE,
H. E. (1963). A comparison of the relative potencies of L-thyroxine, Dthyroxine and L-triiodothyronine in the rat. Fed. Proc. 22, 62 1. PIPES, G. W., PREMACHANDRA, B. N., AND TURNER, C. W. (1958). Measurement of the thyroid hormone secretion rate of individual fowls. Poultry Sci.
37,
36-4
1.
B. N., PIPES, G. W., AND TURNER, C. W. (1961). Comparative abilities of thyroxine and triiodothyronine as inhibitors of pituitary thyrotropin secretion in cattle. Amer. ./. Physiol.
PREMACHANDRA,
201, 77-80. REINEKE, E. P., AND
SINGH, 0. N. (1955). Estimation of thyroid hormone secretion rate of intact rat. Proc. Sot. Exp. Biol. Med. 88, 203-207.
ROSENBERG,
L. L., DIMICK,
M.
K., AND
LAROCHE,
G. (1963). Thyroid function in chickens and rats: Effect of iodine content of the diet and hypophysectomy on iodine metabolism in white Leghorn cockerels and Long-Evans rats. Endocrinology 72, 749-758. SHELLABARGER,
C. J. (1955). A comparison of triiodothyronine and thyroxine in the chick goiterprevention test. Poultry Sci. 34, 1437-1440.
30
SINHA,
ANDERSON
SINGH, A.. REINEKE, E. P., AND RINGER, R. K. ( 1967). Thyroxine and triiodothyronine turnover in the chicken and the bobwhite and Japanese quail. Gen. Comp. Endocrinol. 9, 353-361. SRIVASTAVA, L. S., AND TURNER, C. W. (1967a). Comparison of thyroxine secretion rate of docile and flighty lines of fowls. Proc. Sot. Exp. Biol. Med. 124, 325-327. SRIVASTAVA, L. S., AND TURNER, C. W. (1967b). Comparison of biological activity of injected and orally administered L-thyroxine, t-triiodothyronine and thyroprotein in fowls. Proc. Sot. Exp.
AND
TURNER
Biol. Med. 126, 157-161. STAHL, P. R., PIPES, G. W., AND TURNER, C. W. (1961). Time required for low temperature to influence thyroxine secretion rate in fowls. Poultry Sri. 40, 646-650. TONG, W., AND CHAIKOFF, 1. L. (1958). Hydrolysis of r3*I-thyroprotein by pancreatic enzymes. J. Biol. Chem. 232, 939-950. VLIJM, L. (1958). On the production of hormones in the thyroid glands of birds. A quantitative study with the help of radioiodine and antithyroid drugs. Arch. Neerland. Zool. 12, 467-53 1.
AND
COMPARATIVE
ENDOCRINOLOGY
25,25-30
(1975)
Relation of Thyroid Hormone Secretion Rate (TSR) in Rats and Fowl to Hormone Content of Their Thyroid Glands1-2 K. N. SINHA~, R. R. ANDERSON~, Department
of Dairy
AND C. W. TURNER
Husbandry, University of Missouri-Columbia, Columbia, Missouri 65201
Received July 28, 1974 By the determination of thyroid hormone secretion rate (TSR), the amount of L-thyroxine (L-Tq) secreted each 24 hr has been estimated. An attempt has been made in this study to determine by bioassay the relation of the daily TSR to the amount of stored hormone (thyroglobulin) in the thyroid of the rat and chicken in terms of l-T4 equivalents. The individual TSR of 28 rats and 36 chickens was determined. Two animals having the same TSR were grouped as the donor and recipient of the thyroid homogenate. To bioassay the L-T~ equivalents of donor thyroids, recipient rats and chickens were injected with graded levels of the thyroid homogenate until release of thyroidal 1311was blocked according to the technique used in the estimation of TSR. Mean L-T~ equivalents in the thyroids of donor rats and chickens were 13.9 and 154.4 pg with a daily secretion of 3.58 and 27.98 pg, respectively. The amount of L-T~ equivalent in the thyroid homogenate was 3.82 & 0.35 times the daily TSR in rats and 5.56 -t 0.23 times in chickens (P < 0.001). The data suggest that in rats and chickens there is a significant difference in the magnitude of bioassayable L-T, equivalents stored in the thyroid in terms of daily TSR.
hormone present in the thyroid glands of rats (Rattus norvegicus) and chickens (Callus domesricus) in relation to their daily TSR. In other words, the magnitude of the hormone storage of the thyroid glands to the daily TSR was determined. Since thyroglobulin stores the thyroid hormones, it was considered possible that graded amounts of the thyroid homogenate could be administered parenterally to block the secretion of the thyroidstimulating hormone (TSH) as is done in the case of TSR determination. This procedure would thus determine the bioassayable thyroid hormone in the thyroglobulin in terms of L-T~.
The thyroid stores its hormones within the lumen of the individual follicles as part of thyroglobulin. Prior to passage into the blood stream, the thyroglobulin is hydrolyzed and thyroxine (L-T~) and triiodothyronine (L-T~) flow into the blood as the biologically active hormones. By the determination of the daily thyroid hormone secretion rate (TSR) the amount of hormone secreted each day may be estimated in terms of L-T~ (Pipes et al., 1958). The object of this experiment was to determine the amount of biologically active 1 Contribution from the Missouri Agricultural Experiment Station. Journal Series No. 5463. Approved by the Director. 2 Supported in part by a grant from the U. S. AEC, AT(1 l-l)COO-1758-6 and Contract AT(1 l-l)COO-301-137. 3 Present address: Department of Surgery, Surgical Research Unit, University of Cincinnati Medical Center, Cincinnati, Ohio 45267. 4 Address for reprint requests.
METHODS
Experiment Thyroids
Press, Inc. All rights of reproduction
Content
of Rat
The TSR of a group of 28 female Sprague-Dawley rats was determined individually by the method of 25
Copyright @I 197s by Academic Printed in the United States.
1. Thyroxine
in any form reserved.
26
SINHA.
ANDERSON
AND
Pipes et al. (1958). The method consisted of intraperitoneal (ip) injection of 10 @i of carrier-free 1311and 48 hr were allowed for thyroidal uptake of radioiodine. Graded amounts of L-T, were injected subcutaneously (SC) for 2 consecutive days until the release of thyroidal 13*1was blocked 95% or more. A goitrogen, Tapazole, was also injected daily SCduring the entire period of TSR determination at a level of 0.4 mg/lOO g body wt to reduce recycling of the metabolized 1311by the thyroid. Two rats having the same TSR were paired as the donor and recipient rat; thus, 14 rats served as donors of thyroid glands and 14 rats were recipients of thyroid homogenate. After the initial TSR determination, 3 wk were allowed for decay of remaining small amounts of 13’1 radioactivity in the thyroid, the donor rats were sacrificed. and the thyroid glands were collected on ice, weighed, and homogenized, each in 5 ml of cold 0.9% saline. To prevent thawing the whole homogenate for injection purposes, the thyroid homogenate from each rat was further subdivided into three glass vials containing 1.0, 1.5, and 2.0 ml of the homogenate and stored at -20°C until used. Recipient rats were injected ip with 10 &i la11 and the process of TSR estimation was repeated, but in this case thyroid homogenates were injected instead of L-T~. Graded levels of the homogenate (0.50, 0.75, and 1.00 ml) were injected ip into each recipient rat for 2 consecutive days until the release of thyroidal la11 was blocked 95% or more as compared to previous dose level, as in the method of TSR estimation. Tapazole was also injected into these rats to reduce the recycling of metabolized 1311by the thyroid.
Experiment 2. Thyroxine Chicken Thyroids
Content of
At about 12 wk of age, the individual TSR of 36 white Leghorn cockerels was estimated by the method earlier described. To bioassay the L-T, equivalents in the chicken thyroids the same protocol
THECOMPARISONOF
TURNER
was followed as in the case of rats with the following modifications. Thirty microcuries of carrier-free 13’1 was injected SCper bird. Tapazole was injected SC at a level of 0.5 mg/lOO g body wt/day. The thyroid from each donor bird was homogenized in 10 ml of 0.9% saline. Graded levels of 1.25, 1SO, and 2.00 ml of the homogenate were injected SC into each recipient bird.
RESULTS
Experiment 1. Thyroxine of Rat Thyroids
The mean TSR of a group of 28 rats having a mean body weight of 300.8 g was 1.20 ,ug L-TJlOO g body wtlday with a range of 1.0 to 1.5 pg (Table 1). They were then divided into two groups based on equal TSR. To assay the 14 donor rat thyroids, graded amounts of the thyroid homogenate were injected ip into the 14 recipient rats. In seven recipient rats, the thyroid homogenate blocked thyroidal 1311 release (95% or more) at the l.O-ml level or less and in the remaining seven rats the extrapolation technique (Reineke and Singh, 1955) was used to compute the end points. The recipient rats with a mean body weight of 300.3 g required a mean of 3.58 /*g L-T, in their initial assay (Table 1). Since a mean of 1.48 ml of thyroid homogenate was equally effective, it was assumed that this volume of homogenate contained the same amount of L-T, equivalent. The mean total L-T, equivalent in 5
TABLE 1 TSR WITHTHYROIDGLANDCONTENTOFHORMONE
DOllOr
No. of animals 14 Rats 18 Chickens
Body weight” Cd 308 C_ 10 1554 k 24
Wet thyroid weight (mg)
TSR/IOO g body weight (PP)
18.88 i: I .OO 1.20 f_ 0.05 87.2 2 5.3 1.80~0.07
” Mean ? standard error. ’ Student’s I test I vs 2 P < 0.001.
Content
Recipient
Net daily secretion of L-T, k.4
Body weight (2)
TSR/i00 body weight w
3.61 f 0.20 27.98*1.07
300 r_ 9 1672+34
I.MC-0.04 1.80+0.07
g
Net dady secretion of L-T, (P&Y) 3.58+_0.18 30.21?1.37
BlOCkbIg dose of homogenate (ml) 1.48 f_ 0.17 2.00+0.10
L-T, content in donor thyroid gland (/a) 13.88r 1.48 154.4~7.3
Net content “s TSR CdaysY 3.82’ kO.35 5.56* 2 0.23
STORED
THYROXINE
IN RATS
ml of homogenate was calculated to be 13.88 pg. The average daily secretion of the L-T, of the donor rats with an average body weight of 308.2 g was 3.61 pg. Thus, the total thyroid hormone (present as thyroglobulin) was 3.82 + 0.35 times the estimated daily TSR, with a range of 2.09 to 6.22. Experiment 2. Thyroxine of Chicken Thyroid
Content
The mean TSR of a group of 36 white Leghorn cockerels at 12 wk of age was 1.8 lug/l00 g body wt with a range of 1.5 to 2.5 pg of L-T*. In all but three birds the thyroid homogenate of the donor bird blocked thyroidal 1311release at the 2.0-ml level or less. In these three birds, the extrapolation technique (Reineke and Singh, 1955) was used to compute the end point. The recipient birds with mean body weight of 1672 g required a mean of 30.2 1 pg of L-T~ in their initial assay. Since a mean of 2.0 ml of thyroid homogenate was equally effective, it was assumed that this volume of homogenate contained the same amount of L-T, equivalent. The mean total L-T, equivalent in 10 ml of homogenate was calculated to be 154.4 pg. The average daily secretion of L-T~ of the donor birds with an average body
COMPARISON
Number of animals 6 Rats 5 Rats 3 Rats 6 Chickens 6 Chickens 3 Chickens 2 Chickens 1 Chicken
OF TSR, Mean body weight (g)
THYROID
WEIGHT, TSR/100 g body weight w
291 -c 16” 318 f 13 326 + 32 1.556 1596 1483 1498 1544
a Mean f SE. * P:Student’s t test.
2 2 + f
49 32 66 91
1 vs 3 P < 0.01;
AND
27
FOWLS
weight of 1554 g was 27.98 pg. Thus, the total thyroid hormone (present in thyroglobulin) was 5.56 + 0.23 times the estimated daily TSR, with a range of 4.0 to 7.53 (Table l), which was significantly higher than 3.82 2 0.35 times the estimated daily TSR in the rats (P < 0.001). In Table 2, the mean wet thyroid weight and L-T~ content in the donor rat and chicken thyroids are shown. In the estimation of TSR, L-T, was injected SC in graded increments of 0.25 pg L-T,/lOO g body wtlday, then 0.50, 0.75, 1.00, 1.25, 1.50, and so on for 2 consecutive days until the release of thyroidal 1311 was blocked 95% or more as compared to the previous injected dose (Pipes et al., 1958). In the case of rats, the TSR ranged from 1.00 to 1.50 Fg/lOO g body wt. Rats with TSR of 1.OO, 1.25, and 1.50 pg/ 100 g body wt were grouped together to compare if there was any relationship between TSR, wet thyroid weight, and L-T, content. Likewise chickens with TSR of 1.50, 1.75, 2.00, 2.25, and 2.50 pg/lOO g body wt were grouped and mean wet thyroid weight and L-T, content in glands were calculated. Group means were compared by t test. In the case of chickens, net content versus TSR data indicated that there was a gradual decrease in stored hormone
TABLE 2 AND L-T, CONTENT
IN DONOR
Wet thyroid weight
RATS AND CHICKENS
@x4
f&Id
Net content vs TSR (days)
1.00 1.25 1.50
17.7 + 1.8 20.6 f 2.0 18.3 + 1.2
12.1 2 1.0 14.4 k 3.3 16.4 +- 4.2
4.26 +- 0.50 3.53 f 0.70 3.43 2 0.72
1.50 1.75 2.00 2.25 2.50
101.8 89.5 77.7 59.5 70.0
1 vs 4
P < 0.001; 2 vs 4 P < 0.001;
” * t f
11.2’ 4.62 11.73 0.5”
L-T, content in gland
146.6 153.4 144.3 162.6 221.4
5 vs 7
* f c +
13.3 10.7 17.8 16.1
6.20 5.50 4.83 4.81 5.73
f 0.415 f 0.416 k 0.417 AZ 0.18*
P < 0.05; 5 vs 8 P c 0.02.
28
SINHA,
ANDERSON
in the thyroid as the TSR increased. Wet thyroid weight in birds with TSR of 1.50 pg/ 100 g body wt was significantly higher than that in birds with TSR of 2.00 (P < 0.01) and 2.25 (P < 0.001). However, in the case of rats no such difference was found. It might be suggested that wet thyroid weight may not differ significantly in rats with low TSR and high TSR in the euthyroid condition.
AND
TURNER
pounds and iodide, MIT, DIT, and L-T~ were identified in the plasma (Brown and Jackson, 1955, 1956). It has been shown that after the SC administration of a saline preparation of 1311-labeled thyroglobulin to guinea pigs, plasma and other tissues contained labeled iodide, MIT, DIT, and L-T, (Gilboa and Mitchell, 1964). The present study indicated that L-T, could be released after parenteral administration from its combination with a protein (thyroglobulin) DISCUSSION to enter the blood stream in order to block In the estimation of TSR, L-T~ was in- TSH secretion. It is not known whether jected SC in graded increments until the the iodotyrosines present in the thyroid are release of thyroidal 1311was blocked. The effective in inhibiting TSH secretion. The absorption of L-T~ into the blood thus observations presented are based upon the reached a concentration sufficient to block assumption that they are ineffective. the release of TSH from the pituitary. It might be suggested that the injection The question arises as to the rate of SC of thyroglobulin would stimulate the proabsorption of L-T, when it is injected in duction of antibodies which in turn might the form of thyroglobulin or iodinated ca- influence the biological activity of the sein (Protamone). Protamone is standardthyroglobulin. Since the homogenate was ized by the manufacturer to contain 1% injected for only 2-6 days, such an effect L-T+ In comparison with injected L-T~, on the assay of the hormone content of the was minimal. Protamone was found to be 112% effec- thyroglobulin tive in the rat (Bauman and Turner, 1966). When Tapazole is administered it preThe data indicated that L-T~ was absorbed vents the reutilization of metabolized iofrom a SC site when injected in the form of dide by the thyroid and blocks iodination the protein molecule of iodinated casein and coupling of iodotyrosines in the comparable to free L-T~. thyroid gland. However, it does not interfere with the release of preformed horThe method for estimating the daily TSR in terms of L-T, has been utilized in mones L-T, and L-T, from thyroglobulin. the present study to measure the total bioPrevious studies indicated that the biological activity of the thyroglobulin present logical activity (Shellabarger, 1955; Newin the thyroid glands of rats and chickens. comber, 1957) and half-lives (Heninger The TSR is measured in terms of daily in- and Newcomber, 1964; Singh et al., 1967) jections of L-T~, whereas the estimation of of L-T, and L-T, are equal in the chicken. the total thyroid hormone activity of the Srivastava and Turner (1967a, b) have rethyroglobulin is based on the injection of ported L-T~ to be slightly over twice as active biologically as L-T~ in fowl, similar to the thyroid homogenate. That thyroglobulin was hydrolyzed on injection and findings in rats (Pipes and Dale, 1963; Bauman et al., 1965) and in dairy cattle L-T, and L-T, were absorbed into the blood stream was indicated by the (Premachandra et al., 1961). Rosenberg et al. (1963) reported that blockage of the release of thyroidal 1311in L-T~ was not detected in digests of cockthe recipient rats. It has been reported that thyroglobulin given parenterally resulted in erel thyroid tissue. Vlijm (1958) reported the formation of several iodinated com- that L-T, in the digests of cockerels’
STORED THYROXINE
thyroid glands does not exceed 5% in stages of its synthesis after 1311injection. In the rat, hydrolysis of thyroid does not yield more than 3% of T, after 1311injection (Tong and Chaikoff, 1958). Since the amount of L-T~ in thyroglobulin is minimal, its presence should have little influence upon the results of this experiment even if the biological activity of L-T~ differs from L-T~. The bioassay of thyroid homogenate indicated that the amount of hormone stored as thyroglobulin in the thyroid was sufficient for normal secretion for periods of 4.0-7.5 days with a mean of 5.6 in the chickens and 2.1-6.2 days with a mean of 3.9 in the rats. It has been known that thyroid hormones are stored as thyroglobulin in the thyroid, but the magnitude of storage has not been investigated. The present study estimates the magnitude of stored thyroid hormones in L-T, equivalents in the gland by bioassay. When rats or chickens are subjected to cold environment, metabolism of thyroid hormones is increased and there is increased need of thyroid hormones for tissue. This leads to increased secretion of TSH which stimulates the thyroid for hormone synthesis and release. Under this condition preformed L-T, and L-T, in the thyroid may serve as an additional supply of hormones for tissue needs. The present study suggested that chicken thyroids contained significantly more bioassayable L-T, equivalents than those of rats in terms of daily TSR (P < 0.001). Perhaps chickens are better adapted than rats for short periods of sudden exposure to cold. Fowls placed in a cold environment showed a gradual increase in TSR (Stahl er al., 1961). As the TSR increased in chickens, there was a gradual decrease in stored hormone in the thyroid. Under these conditions, the thyroid weight, as well as the reserve supply of hormone, decreased.
29
IN RATS AND FOWLS
REFERENCES BAUMAN,
T. R., PIPES, G. W., AND TURNER,
C. W.
(1965). Relative potency of several analogues when substituted for L-thyroxine in the estimation of thyroid secretion rate in rats. Endocrinology
76, 537-538.
C. W. (1966). Comparison of biological activity of orally administered and injected L-thyroxine, L-triiodothyronine and thyroprotein in rats. Proc. Sot. Exp. Biol. Med. 123, 9-14. BROWN, F., AND JACKSON, H. (1955). Metabolism of thyroglobulin labelled with iodine131. Nature 175, BAUMAN,
T. R., AND TURNER,
472-413. BROWN, F.,
AND JACKSON, H. (1956). The fate of 1311-labelled homologous and heterologous thyroglobulins in the rat, dog, monkey and rabbit. Biochem. J. 62, 295-301. GILBOA, Y., AND MITCHELL, M. L. (1964). Metabolism of thyroglobulin-1131 in the guinea pig in vivo and in vitro. Metabolism 13, 661-668. HENINGER, R. W., AND NEWCOMER, W. S. (1964). Plasma protein binding, half-life and erythrocyte uptake of thyroxine and triiodothyronine in chickens. Proc. Sot. Exp. Biol. Med. 116, 624-628. NEWCOMBER,
W. S. (1957). Relative potencies of thyroxine and triiodothyronine based on various criteria in thiouracil-treated chickens. Amer. J.
Physiol. 190, 413-418. PIPES, G. W., AND DALE,
H. E. (1963). A comparison of the relative potencies of L-thyroxine, Dthyroxine and L-triiodothyronine in the rat. Fed. Proc. 22, 62 1. PIPES, G. W., PREMACHANDRA, B. N., AND TURNER, C. W. (1958). Measurement of the thyroid hormone secretion rate of individual fowls. Poultry Sci.
37,
36-4
1.
B. N., PIPES, G. W., AND TURNER, C. W. (1961). Comparative abilities of thyroxine and triiodothyronine as inhibitors of pituitary thyrotropin secretion in cattle. Amer. ./. Physiol.
PREMACHANDRA,
201, 77-80. REINEKE, E. P., AND
SINGH, 0. N. (1955). Estimation of thyroid hormone secretion rate of intact rat. Proc. Sot. Exp. Biol. Med. 88, 203-207.
ROSENBERG,
L. L., DIMICK,
M.
K., AND
LAROCHE,
G. (1963). Thyroid function in chickens and rats: Effect of iodine content of the diet and hypophysectomy on iodine metabolism in white Leghorn cockerels and Long-Evans rats. Endocrinology 72, 749-758. SHELLABARGER,
C. J. (1955). A comparison of triiodothyronine and thyroxine in the chick goiterprevention test. Poultry Sci. 34, 1437-1440.
30
SINHA,
ANDERSON
SINGH, A.. REINEKE, E. P., AND RINGER, R. K. ( 1967). Thyroxine and triiodothyronine turnover in the chicken and the bobwhite and Japanese quail. Gen. Comp. Endocrinol. 9, 353-361. SRIVASTAVA, L. S., AND TURNER, C. W. (1967a). Comparison of thyroxine secretion rate of docile and flighty lines of fowls. Proc. Sot. Exp. Biol. Med. 124, 325-327. SRIVASTAVA, L. S., AND TURNER, C. W. (1967b). Comparison of biological activity of injected and orally administered L-thyroxine, t-triiodothyronine and thyroprotein in fowls. Proc. Sot. Exp.
AND
TURNER
Biol. Med. 126, 157-161. STAHL, P. R., PIPES, G. W., AND TURNER, C. W. (1961). Time required for low temperature to influence thyroxine secretion rate in fowls. Poultry Sri. 40, 646-650. TONG, W., AND CHAIKOFF, 1. L. (1958). Hydrolysis of r3*I-thyroprotein by pancreatic enzymes. J. Biol. Chem. 232, 939-950. VLIJM, L. (1958). On the production of hormones in the thyroid glands of birds. A quantitative study with the help of radioiodine and antithyroid drugs. Arch. Neerland. Zool. 12, 467-53 1.