Correlation of Serum Triiodothyronine (T,) and Thyroxine (T,) With Biologic Effects of Thyroid Hormone Replacement in Propylthiouracil-treated Rats Roz D. Frumess and P. Reed Larsen To study the role of T, to Ts conversion in the biologic action of T,, thyroidectomized, hypothyroid rats were given subcutaneous T4 (0.8 or 1.6 rg/l OOg/day) with or without intraperitoneal propylthiouracil (PTU) (1 mg/lOOg/day). Rats were killed after 5, 10, 12, or 15 days of treatment and serum Ts and T4 levels were correlated with serum TSH, liver mitochondrial aGPD activity and weight gain. In rats killed at 5 days, PTU treatment resulted in higher serum T,, lower serum Ts, and a markedly elevated serum Tl:Ts ratio, demonstrating that PTU inhibits peripheral conversion of T, to Ts in the rat. Despite higher T4 levels, mean serum TSH was higher in the two groups receiving PTU as well as T4. In rats receiving 0.8 Ag T,, growth rate was also slower with concomitant PTU administration. In other groups of rats treated with 0.8 pg T4 for 10 and 15 days, PTU treat-
ment resulted in similar differences in Ts, T,, and T4:Ts ratios and serum TSH. At 15 days, rats treated with 0.8 /,rg T, + PTU had signiticantly lower crGPD activity than rats receiving 0.8 pg T, alone. PTU treatment had no effect on aGPD activity in rats maintained on 0.1 pg Ts/lOOg/day indicating that there was no inhibition of this biologic response to Ts by this agent. PTU without T, had no significant effect on TSH, weight gain, or aGPD activity. In addition, the dialyzable fraction of Ts and T4 in serum was not altered by this agent. These data show that in animals treated with T,, with and without PTU, TSH sup pression, aGPD activity and growth correlate better with serum Ts concentrations than with serum T,. This suggests that for maximum biologic activity, T, must be converted to Ts.
N
UMEROUS STUDIES have demonstrated that 6-n-propylthiouracil (PTU) decreases the biologic response to a given dose of L-thyroxine (T4) in the rat. Concomitant administration of PTU leads to decreased effects of Tq on liver, heart, and kidney mitochondrial alpha glycerophosphate dehydrogenase ((uGPD) activity,lm3 and impairs T4-induced suppression of TSH release from the pituitary.4-6 It has been speculated that this effect of PTU is associated with its inhibition of the peripheral deiodination of T4.‘-” Recently, Oppenheimer, Schwartz, and Surks demonstrated that extrathyroidal conversion of tracer T4 to 3,5,3’-triiodo-L-thyronine (T3) occurs in the rat,13 and that this is inhibited by PTU. I4 Recent studies have suggested that about two thirds of the TJ utilized per day by iodine-sufficient rats arises from T4.‘* As postulated by the above authors, PTU-induced inhibition of this important pathway for T3 production provides an attractive explanation for the
From
the Division
of Endocrinology
Pittsburgh School of Medicine. Receivedforpublication Supported by National a grant from
Department
of Medicine,
University
of
Pa. 15261.
June 27, 1974. Institute
of Arthritis,
the University of Pittsburgh
Presented in part at the Ftfty-sixth 0 1974 by Grune
and Metabolism.
Pittsburgh,
& Stratton,
Metabolism
School of Medicine
Annual Meeting
and Digestive
Diseases Grant 14283 and
Alumni Association.
of The Endocrine Society, June, 1974.
Inc.
Metabolism, Vol. 24, No. 4 (April), 1975
547
548
FRUMESS
AND LARSEN
observed antagonism to the biologic effectiveness of Tq.14It would be compatible with the original speculation of Gross and Pitt-Rivers that, in order to be metabolically active, T4 must be deiodinated to T3.16 To substantiate this hypothesis, it is necessary to demonstrate that the administration of PTU to thyroidectomized rats maintained on physiologic replacement doses of T4 is associated with low serum T3 concentrations as well as decreased hormonal activity when compared to rats given T4 alone. The thyroid status of rats receiving PTU + T4 should then correlate better with circulating T3 than T4 concentrations. MATERIALS
AND METHODS
Surgically thyroidectomized (with parathyroid reimplantation) or sham-operated SpragueDawley male rats (Zivic Miller) were maintained on Purina rat chow and tap water ad-lib. at a constant temperature of 75 + I’ F. They were left untreated for 4-8 wk to allow the thyroidectomized animals to become hypothyroid. Three sets of experiments were then performed to study the effect of PTU on the biologic action of T4. In each of these experiments, half of the thyroidectomized animals were given daily intraperitoneal injections of PTU in 2.0 ml of physiologic saline alkalinized with NaOH (pH 8.5) at a dose of I mg/ 100 g body wt/day. The remaining animals received control injections of the vehicle. These injections were started I day prior to and continued throughout the course of the hormone injections. Injections were used to avoid variations in PTU intake that might occur with administration of the drug in the diet.
Experiment A At the time of thyroidectomy, rats in this study weighed 150-200 g. After 8 wk, by which time growth had plateaued for 2 wk, the thyroidectomized rats were divided into six groups of 9-10 animals of equal mean weights (256 g). The experimental groups were given subcutaneous T4 (0.8 or 1.6 rg/lOO g/day) with or without PTU. The dose of 0.8 rg T4/100 g/day was chosen because it approximated the calculated daily T4 production rate based on our previous studies.15 To control for nonspecific toxic effects of PTU as well as for any direct effect of PTU on TSH levels, one group of thyroidectomized rats received PTU alone. The other two control groups consisted of untreated thyroidectomized and sham-operated rats. Injections were continued for 5 days and each rat was identified and weighed every other day. Animals were killed by cardiac puncture after chloroform anesthesia and the necks were examined for any thyroid remnants. Serum T4 was measured in duplicate at two dilutions by methods previously described.” The method was modified to use 10 and 5 ~1 aliquots of rat serum in place of human serum. Serum Ts was measured by methods and modifications described previously using duplicate aliquots of 100 and 50 ~1.‘~ Sera containing small quantities of T3 ( < 20 ng/lOO ml) were reassayed using 200 and 100 ~1 samples. Rat serum TSH was measured in duplicate at two dilutions by the procedure described in the protocol supplied with the rat TSH immunoassay materials kindly donated by the National Institute of Arthritis, Metabolism and Digestive Diseases. Equal numbers of control and experimental samples were included in all assays to minimize interassay variation. Mean normal values & SD for Zivic Miller male Sprague-Dawley rats weighing 200 g or more are: 3.7 + 0.89 pg T4/100ml; 38 f I7 ng Ts/lOOml; I91 + 249 rtJ TSH/ml. The dialyzable fractions of Ts and T4 (DFTs and DFT4) were determined in duplicate by the method of Oppenheimer, Squef, Surks, and Haver with minor modifications.” Absolute free Ts and T4 concentrations
were the product
of the dialyzable
fractions
and individual
total Ts and T4 concentrations.
Experiment B To study the effect of PTU on liver mitochondrial aGPD, rats of a mean weight of 400-450 g at the time of surgery were treated as above, except that only one dose of T4 (0.8 pg/loO g/day) was used and rats were treated for 10 and I5 days. Liver mitochondrial aGPD activity was determined by the method of Lee and Lardy.20 Smce only five mitochondrial samples, performed in duplicate, could be assayed at one time, one representative from each of the differently
SERUM
TRIIODOTHYRONINE
AND THYROXINE
549
treated groups was run in each assay to minimize interassay variation. The same control sample was also determined with each assay and no significant variation in this sample was noted. Rats were killed and assays performed as described above.
Experiment C Rats weighing 150-200 g at the start were used in this experiment. Starting 4 wk after thyroidectomy, these rats received two equal subcutaneous doses of Ts each day at 8 a.m. and 5 p.m. (total dose 0.1 pegTs/lOO g/day) with and without PTU. This dose was estimated to be about two thirds of the daily T3 production rate.‘* A third group was untreated. After 12 days of treatment, assays of liver aGPD, Ts, T4. and TSH were carried out as above. All statistical analyses were performed using Student’s t test for unpaired samples.*’ RESULTS
The data in Table 1 show that in thyroidectomized rats maintained on 0.8 pg T,/lOO g/day and 1 mg PTU/lOO g/day for 5 days (experiment A), serum T4 was higher and serum T3 was lower than when T4 was given alone. This resulted in markedly higher T4:T3 ratios in the PTU-treated group. Despite the higher T4 levels, TSH was higher and growth rate was slower in the group receiving PTU + T4. In the two sets of animals receiving 1.6 pg T,/lOO g/day, similar differences were seen in T4, T3, and TSH values and the serum T4:T3 ratio was again markedly increased in the PTU-treated group. Weight gain was not different in these two groups, and both were similar to that observed in the animals receiving 0.8 pg T4/100 g/day, suggesting that maximum growth rate was attained at that dosage. PTU without T4 had no effect on T4 and TSH levels or on weight gain in otherwise untreated thyroidectomized rats. T3 levels were lower in rats receiving PTU alone in this experiment, suggesting some residual thyroid hormone production in these animals. The TSH levels in sham-operated animals were not different from those in the groups receiving 0.8 pg T,/day also supporting the validity of the theoretical estimates of daily T4 production. No meaningful growth data could be obtained for the sham-operated rats, since their weights were approximately twice that of the thyroidectomized rats at the time of the experiment. Using previously published kinetic data for normal and PTU-treated rats,14 it is possible to calculate Table 1. Effect of PTU on Serum T4, T3, and TSH levels and Weight Thyroidactomized
Rats Maintained
T4 Treatment*
n
(us/100
ml)
T4
10
4.9 f 0.2
0.8 pg T4 + PTU
10
5.7 f 0.2
0.8
pg
< 0.025
P 1.6m T4 1.6pgT4+
PTU
P Control THX PTU alone P Sham-operated
lT4
9
7.3 f 0.2
10
9.6 f 0.5
ment resulted in similar differences in Ts, T,, and T4:Ts ratios and serum TSH. At 15 days, rats treated with 0.8 /,rg T, + PTU had signiticantly lower crGPD activity than rats receiving 0.8 pg T, alone. PTU treatment had no effect on aGPD activity in rats maintained on 0.1 pg Ts/lOOg/day indicating that there was no inhibition of this biologic response to Ts by this agent. PTU without T, had no significant effect on TSH, weight gain, or aGPD activity. In addition, the dialyzable fraction of Ts and T4 in serum was not altered by this agent. These data show that in animals treated with T,, with and without PTU, TSH sup pression, aGPD activity and growth correlate better with serum Ts concentrations than with serum T,. This suggests that for maximum biologic activity, T, must be converted to Ts.
N
UMEROUS STUDIES have demonstrated that 6-n-propylthiouracil (PTU) decreases the biologic response to a given dose of L-thyroxine (T4) in the rat. Concomitant administration of PTU leads to decreased effects of Tq on liver, heart, and kidney mitochondrial alpha glycerophosphate dehydrogenase ((uGPD) activity,lm3 and impairs T4-induced suppression of TSH release from the pituitary.4-6 It has been speculated that this effect of PTU is associated with its inhibition of the peripheral deiodination of T4.‘-” Recently, Oppenheimer, Schwartz, and Surks demonstrated that extrathyroidal conversion of tracer T4 to 3,5,3’-triiodo-L-thyronine (T3) occurs in the rat,13 and that this is inhibited by PTU. I4 Recent studies have suggested that about two thirds of the TJ utilized per day by iodine-sufficient rats arises from T4.‘* As postulated by the above authors, PTU-induced inhibition of this important pathway for T3 production provides an attractive explanation for the
From
the Division
of Endocrinology
Pittsburgh School of Medicine. Receivedforpublication Supported by National a grant from
Department
of Medicine,
University
of
Pa. 15261.
June 27, 1974. Institute
of Arthritis,
the University of Pittsburgh
Presented in part at the Ftfty-sixth 0 1974 by Grune
and Metabolism.
Pittsburgh,
& Stratton,
Metabolism
School of Medicine
Annual Meeting
and Digestive
Diseases Grant 14283 and
Alumni Association.
of The Endocrine Society, June, 1974.
Inc.
Metabolism, Vol. 24, No. 4 (April), 1975
547
548
FRUMESS
AND LARSEN
observed antagonism to the biologic effectiveness of Tq.14It would be compatible with the original speculation of Gross and Pitt-Rivers that, in order to be metabolically active, T4 must be deiodinated to T3.16 To substantiate this hypothesis, it is necessary to demonstrate that the administration of PTU to thyroidectomized rats maintained on physiologic replacement doses of T4 is associated with low serum T3 concentrations as well as decreased hormonal activity when compared to rats given T4 alone. The thyroid status of rats receiving PTU + T4 should then correlate better with circulating T3 than T4 concentrations. MATERIALS
AND METHODS
Surgically thyroidectomized (with parathyroid reimplantation) or sham-operated SpragueDawley male rats (Zivic Miller) were maintained on Purina rat chow and tap water ad-lib. at a constant temperature of 75 + I’ F. They were left untreated for 4-8 wk to allow the thyroidectomized animals to become hypothyroid. Three sets of experiments were then performed to study the effect of PTU on the biologic action of T4. In each of these experiments, half of the thyroidectomized animals were given daily intraperitoneal injections of PTU in 2.0 ml of physiologic saline alkalinized with NaOH (pH 8.5) at a dose of I mg/ 100 g body wt/day. The remaining animals received control injections of the vehicle. These injections were started I day prior to and continued throughout the course of the hormone injections. Injections were used to avoid variations in PTU intake that might occur with administration of the drug in the diet.
Experiment A At the time of thyroidectomy, rats in this study weighed 150-200 g. After 8 wk, by which time growth had plateaued for 2 wk, the thyroidectomized rats were divided into six groups of 9-10 animals of equal mean weights (256 g). The experimental groups were given subcutaneous T4 (0.8 or 1.6 rg/lOO g/day) with or without PTU. The dose of 0.8 rg T4/100 g/day was chosen because it approximated the calculated daily T4 production rate based on our previous studies.15 To control for nonspecific toxic effects of PTU as well as for any direct effect of PTU on TSH levels, one group of thyroidectomized rats received PTU alone. The other two control groups consisted of untreated thyroidectomized and sham-operated rats. Injections were continued for 5 days and each rat was identified and weighed every other day. Animals were killed by cardiac puncture after chloroform anesthesia and the necks were examined for any thyroid remnants. Serum T4 was measured in duplicate at two dilutions by methods previously described.” The method was modified to use 10 and 5 ~1 aliquots of rat serum in place of human serum. Serum Ts was measured by methods and modifications described previously using duplicate aliquots of 100 and 50 ~1.‘~ Sera containing small quantities of T3 ( < 20 ng/lOO ml) were reassayed using 200 and 100 ~1 samples. Rat serum TSH was measured in duplicate at two dilutions by the procedure described in the protocol supplied with the rat TSH immunoassay materials kindly donated by the National Institute of Arthritis, Metabolism and Digestive Diseases. Equal numbers of control and experimental samples were included in all assays to minimize interassay variation. Mean normal values & SD for Zivic Miller male Sprague-Dawley rats weighing 200 g or more are: 3.7 + 0.89 pg T4/100ml; 38 f I7 ng Ts/lOOml; I91 + 249 rtJ TSH/ml. The dialyzable fractions of Ts and T4 (DFTs and DFT4) were determined in duplicate by the method of Oppenheimer, Squef, Surks, and Haver with minor modifications.” Absolute free Ts and T4 concentrations
were the product
of the dialyzable
fractions
and individual
total Ts and T4 concentrations.
Experiment B To study the effect of PTU on liver mitochondrial aGPD, rats of a mean weight of 400-450 g at the time of surgery were treated as above, except that only one dose of T4 (0.8 pg/loO g/day) was used and rats were treated for 10 and I5 days. Liver mitochondrial aGPD activity was determined by the method of Lee and Lardy.20 Smce only five mitochondrial samples, performed in duplicate, could be assayed at one time, one representative from each of the differently
SERUM
TRIIODOTHYRONINE
AND THYROXINE
549
treated groups was run in each assay to minimize interassay variation. The same control sample was also determined with each assay and no significant variation in this sample was noted. Rats were killed and assays performed as described above.
Experiment C Rats weighing 150-200 g at the start were used in this experiment. Starting 4 wk after thyroidectomy, these rats received two equal subcutaneous doses of Ts each day at 8 a.m. and 5 p.m. (total dose 0.1 pegTs/lOO g/day) with and without PTU. This dose was estimated to be about two thirds of the daily T3 production rate.‘* A third group was untreated. After 12 days of treatment, assays of liver aGPD, Ts, T4. and TSH were carried out as above. All statistical analyses were performed using Student’s t test for unpaired samples.*’ RESULTS
The data in Table 1 show that in thyroidectomized rats maintained on 0.8 pg T,/lOO g/day and 1 mg PTU/lOO g/day for 5 days (experiment A), serum T4 was higher and serum T3 was lower than when T4 was given alone. This resulted in markedly higher T4:T3 ratios in the PTU-treated group. Despite the higher T4 levels, TSH was higher and growth rate was slower in the group receiving PTU + T4. In the two sets of animals receiving 1.6 pg T,/lOO g/day, similar differences were seen in T4, T3, and TSH values and the serum T4:T3 ratio was again markedly increased in the PTU-treated group. Weight gain was not different in these two groups, and both were similar to that observed in the animals receiving 0.8 pg T4/100 g/day, suggesting that maximum growth rate was attained at that dosage. PTU without T4 had no effect on T4 and TSH levels or on weight gain in otherwise untreated thyroidectomized rats. T3 levels were lower in rats receiving PTU alone in this experiment, suggesting some residual thyroid hormone production in these animals. The TSH levels in sham-operated animals were not different from those in the groups receiving 0.8 pg T,/day also supporting the validity of the theoretical estimates of daily T4 production. No meaningful growth data could be obtained for the sham-operated rats, since their weights were approximately twice that of the thyroidectomized rats at the time of the experiment. Using previously published kinetic data for normal and PTU-treated rats,14 it is possible to calculate Table 1. Effect of PTU on Serum T4, T3, and TSH levels and Weight Thyroidactomized
Rats Maintained
T4 Treatment*
n
(us/100
ml)
T4
10
4.9 f 0.2
0.8 pg T4 + PTU
10
5.7 f 0.2
0.8
pg
< 0.025
P 1.6m T4 1.6pgT4+
PTU
P Control THX PTU alone P Sham-operated
lT4
9
7.3 f 0.2
10
9.6 f 0.5
