Thyroidal Response to Graded Doses of Bovine Thyrotropin R. P. ULLER, A. J. VAN HERLE, AND I. J. CHOPRA Department of Medicine, UCLA School of Medicine, Los Angeles, California ABSTRACT. Nine normal euthyroid human subjects (5 males, 4 females) were given varying doses of bovine thyrotropin (bTSH) (6 subjects, 2 IU and 3 subjects, 0.4 IU) im, and their sera were tested at timed intervals for thyroglobulin (HTg), triiodothyronine (T3), thyroxine (T4) and bTSH concentrations. Data from a previous study on 5 normal euthyroid subjects (3 males, 2 females) given 10 IU were compared, and it was found from both studies that regardless of the dose of bTSH employed, the same qualitative response pattern was observed. T3 and T4 peaked close together and HTg peaked much later. Furthermore, it was shown that when the dose of bTSH was decreased there was no change in the sequence of response of the various thyroidal products. This suggests that the difference between the pattern of HTg response to bTSH and to thyrotropin releasing hormone (TRH) noted previously
N
ORMAL euthyroid subjects demonstrate a delayed response in serum thyroglobulin (HTg) to bovine thyrotropin (bTSH) as compared with the response in thyroxine (T4) and triiodothyronine (T3) (1). Following thyrotropin releasing hormone (TRH), these subjects showed essentially a simultaneous peak response in the same parameters. One possible explanation for these findings is that the discrepancy between the effects of exogenous bTSH and endogenous human TSH (hTSH) was related to the degree of thyroid stimulation attained with 10 IU bTSH as compared with that attained with TRH; previous studies have shown that maximal serum bTSH concentrations reached about 150 /xU/ml after 10 IU of bTSH (2) and about 13 fiV/ml of hTSH after TRH (1). Available evidence indicates
probably is not due to the variation in degree of thyroidal stimulation but to a qualitative difference in human thyroidal response to bTSH and endogenous human TSH (hTSH). In addition, the characteristics of the dose-response curves of the human thyroidal response to bTSH were examined. These data indicated that the mean maximal increase in the various thyroidal products were logarithmically proportional to the dose of bTSH administered. Also, the net secretory responses of various thyroidal products, as measured by the integrated area under the curve, were related essentially linearly to the logarithm of the dose of bTSH injected. Peak serum concentration of bTSH occurred uniformly at about 2 h after the injection and the values were related essentially linearly to the logarithm of the dose of bTSH employed. (J Clin Endocrinol Metab 45:312, 1977)
that modest degrees of thyroid stimulation may result in a relatively lesser degree of proteolysis than of endocytosis (3,4), which could then result in an earlier release of thyroglobulin into the circulation. An alternative explanation for these results could be that the human thyroid gland responds qualitatively differently to bTSH than to endogenously released hTSH. The present study was designed to test whether or not variations in the dose of bTSH would influence the qualitative pattern of the response of the normal human thyroid to exogenous bTSH. Materials and Methods
Nine healthy euthyroid subjects, 5 males and 4 females, ranging in age from 19 to 27 years, were given bTSH (Thytropar®) im. Six subjects (3 males and 3 females) were each given 2 IU and 3 subjects (2 males and 1 female) were each Received June 24, 1975. given 0.4 IU. Blood samples were obtained Supported by grants from NIH Research Cancer before injection of bTSH and then at 2, 4, 6, 8, CA 13447, USPHS AM-16155, CRC Grant RR 00865, and NIH Research Career Development Award, 10, 12, 24, 36, 48, 72 and 96 h after injection. Serum concentrations of T4, T3, and HTg were KO4 AM-70225 (Dr. Chopra). Reprint requests to: Andre J. Van Herle, M.D., Dept. determined by radioimmunoassay methods (5of Medicine, UCLA School of Medicine, Los Angeles, 7). A bTSH assay was developed in order to determine the levels of bTSH attained in the Ca. 90024.
312
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313
THYROIDAL RESPONSE TO bTSH IN MAN
TABLE 1. Serum HTg, T3, T4 and bTSH levels following im administration of 10 IU of bTSH in normal subjects Time of bleeding post bTSH (h) Subjects
0
2
4
6
8
12
24
36
48
72
96
120
1 HTg' T3" T4t bTSH'
2.7 120 9.0 0.9
4.0 158 7.0 3.5
5.1 280 10.8 3.6
3.9 280 9.5 2.3
5.4 305 13.0 1.5
7.4 253
HTg Ta T4 bTSH
4.2 130 6.6 0.6
5.6 100 5.0 2.2
7.7 115 5.3 1.8
8.0 145 7.5 1.9
HTg Ta T4 bTSH
5.1 138 0.4
4.6 250 5.6 7.8
6.6 330 8.5 5.4
HTg T, T4 bTSH
2.4 120 8.2 0.6
2.6 150 7.3 6.0
2.9 210 8.3 5.2
HTg T, T4 bTSH
6.0 150 9.5 0.5
— — —
_ _ —
_ —
4.If ±0.7
4.2t* ±0.6
5.6ff* ± 1.0
5.7f* ± 1.4
16.5 270
17.0 290 13.2 0.9
16.0
7.6 125
210
13.2
_
_
1.0
—
—
10.0
9.4 130 8.4 0.6
11.3
10.7
1.1
1.0
8.7 130 6.0 2.0
16.5 145 7.0 0.8
145 8.0 1.4
21.0 135 8.0 1.1
8.2 370 9.3 1.9.
9.2 340 11.5 1.6
11.5 350 9.0 0.8
16.5 310 10.8 0.7
26.0 — — —
250 9.8 0.6
2.6 215 10.3 4.2
2.4 280 13.3 4.1
2.0 285 16.0 2.5
2.6 240 13.5 1.3
7.2 _
5.2 _
—
0.8
6.0 150 9.2 0.7
26.5 215 12.8 0.7
14.0
13.3 2.7
16.5 270 15.3 0.7
20.0
280 13.0 1.5
7.1t* ± 1.4
9.4ft ±3.1
13.8 ft ±2.8
19.5ft ±3.5
10.0 1.0
2 17.0
23.0
140 _ 0.9
13.8
35.0
22.0 225
1.0
5.6 105 6.4 0.6
3
4.4
8.1 —
16.5 185 6.5 0.5
— — —
4 5.0 9.6 0.8
2.8 105 8.8 0.6
5
mean ± SEM HTg
132H* ±6
T3
7.5$ ±0.9
T4 bTSH
9.8
164$ ±31 6.2$ ±0.6
234 H ±47
8.2$ ± 1.1
0.6
4.9
4.0
±0.1
± 1.2
±0.8
252 ft ± 48
264ft ±46
262 ff ±33
9.2ft ±0.6
11.4tt ± 1.4
2.6 ±0.5
2.2
1.1
± 0.4
±0.1
' ng/ml. •• ng/dl.
11.3ft ± 1.6
241ff ±35
213tt ± 45
225
—
5.8 155
15.3
12.5
11.3
0.7
0.3
0.4
13.0H ±3.4
8.0$ ±2.8
19.8 ±4.8 228ft ± 12
11.7H ± 1.2
11.311 ± 1.7
1.0 ±0.1
0.9
0.8
± 0.1
±0.1
12.8ft ± 1.6
172tf ± 27
157$ ± 16
6.6 170 9.8 0.6
5.6t* ± 1.0 126f* ± 15
9.0$ ± 1.0
8.8$ ± 0.8
0.7
0.6
±0.2
±0.1
0.7 ±0.1
10.9ft ± 1.3
$ P < 0.05 when compared to mean peak value. f*P < 0.01 when compared to mean peak value, ft*/' < 0.005 when compared to mean peak value.
11 NS compared to mean peak value.
serum following its im administration. All samples from each patient were analyzed in duplicate in the same assay. Purified bTSH was used to prepare tracer and standards.1 Bovine TSH was labeled with 125I by the method of Hunter et al. (8). The standard curve obtained was linear from 0.5 ng/ml (15 /x.U/ml) to 50 ng/ml (1500 /nU/ml). Net secretory response for each parameter studied was computed, as described by Foley et al. (9). The base line 1 Highly purified bTSH (30 U/ml) and rabbit antibTSH antibody were generously provided by Dr. John G. Pierce, (Department of Biochemistry, UCLA School of Medicine).
value was subtracted from the values obtained at each of the subsequent time intervals. The values between each time interval were then averaged and multiplied by the time lapse between each point. The sum total of the values was calculated to reflect net secretory response and was expressed as ng/ml h. In order to examine the relative degree of response in the various parameters studied, all data were converted to ng/ml before computation of the net secretory response. The dose-response (d-r) characteristic of the net secretory responses to bTSH was examined by the least mean square method (10). The d-r curve was computed using the formula y = a + b x; where a = y intercept,
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JCE & VI • 1977 Vol 45 • No 2
ULLER, VAN HERLE AND CHOPRA
314
b = slope, x = log dose bTSH. Significance of the slope was calculated by the t test. All subjects had undetectable antithyroglobulin antibodies in their serum as measured by the tanned red cell hemagglutination technique (11).
Results The individual and mean serum concentrations of HTg, T3 T4, and bTSH at each depicted time interval following administration of 10, 2, and 0.4 IU bTSH are shown in Tables 1-3. The data showing mean HTg, T3, and T4 responses to 10 IU bTSH have been reported previously (1); they are noted here only to compare with the data obtained using the smaller doses of bTSH (Table 1). Serum HTg levels rose in each instance and the mean maximal increase above basal was 376% for 10 IU, 109% for 2 IU, and 35.5% for 0.4 IU of bTSH. Mean maximal increases above the base line were 108%, 74% and 48.5% for T3 and 76.8%, 42% and 15% for T4 following 10 IU, 2 IU and 0.4 IU of bTSH, respectively. Quantitatively larger doses of bTSH produced a greater increment above basal for each thyroidal substance measured. However, the qualitative pattern observed remained essentially the same regardless of the dose of bTSH administered (Fig. 1). A sequential pattern was observed with T3 and T4 peaking first followed by HTg. No significant differences were found between peak T3 values at 8 h when compared with values reached from 2 to 24 h following 10 IU bTSH. Likewise, the peak serum T3 value at 6 h following 2 IU bTSH was not significantly different than T3 levels from 2 to 4 h (Fig. 1, Table 2). As for T4, a peak value reached at 24 h following 10 IU bTSH was not significantly different when compared with values at 6 to 72 h. Peak values at 6 h were not significantly different from those at 4 to 48 h after 2 IU bTSH. HTg values peak at 48 h and 24 h after administration of 10 and 2 IU bTSH, respectively. Likewise, there was no signifi-
cant difference when these were compared with values from 12 to 72 h (10 IU) and from 12 to 48 h (2 IU). The pattern noted in T3 and HTg response differed from that of T4; the former displayed a peak whereas the latter showed a plateau. Circulating bTSH levels were related to the doses administered (Fig. 1 and Table 1). 400-i 300HTg
UJ LLJ
to < < 03 UJ
>
o
CO < UJ CO
CE
•
4
8
10 l.U. b-TSH 2 I U b-TSH 0.4 l.U. b-TSH
12 24 48 72 96 120 HOURS
FIG. 1. Mean per cent increment above basal concentrations for HTg, T3, T4 and bTSH following im administration of varying doses of bTSH.
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315
THYROIDAL RESPONSE TO bTSH IN MAN
TABLE 2. Serum HTg, T3, T4 and bTSH levels following im administration of 2 IU of bTSH in normal subjects Time of bleeding post bTSH (h) Subjects
0
2
4
6
8
12
24
36
48
13.1
14.2
10.7
— _
160 5.7
—
173 6.3 0.2
140 5.1 0.5
255 9.0
N
ORMAL euthyroid subjects demonstrate a delayed response in serum thyroglobulin (HTg) to bovine thyrotropin (bTSH) as compared with the response in thyroxine (T4) and triiodothyronine (T3) (1). Following thyrotropin releasing hormone (TRH), these subjects showed essentially a simultaneous peak response in the same parameters. One possible explanation for these findings is that the discrepancy between the effects of exogenous bTSH and endogenous human TSH (hTSH) was related to the degree of thyroid stimulation attained with 10 IU bTSH as compared with that attained with TRH; previous studies have shown that maximal serum bTSH concentrations reached about 150 /xU/ml after 10 IU of bTSH (2) and about 13 fiV/ml of hTSH after TRH (1). Available evidence indicates
probably is not due to the variation in degree of thyroidal stimulation but to a qualitative difference in human thyroidal response to bTSH and endogenous human TSH (hTSH). In addition, the characteristics of the dose-response curves of the human thyroidal response to bTSH were examined. These data indicated that the mean maximal increase in the various thyroidal products were logarithmically proportional to the dose of bTSH administered. Also, the net secretory responses of various thyroidal products, as measured by the integrated area under the curve, were related essentially linearly to the logarithm of the dose of bTSH injected. Peak serum concentration of bTSH occurred uniformly at about 2 h after the injection and the values were related essentially linearly to the logarithm of the dose of bTSH employed. (J Clin Endocrinol Metab 45:312, 1977)
that modest degrees of thyroid stimulation may result in a relatively lesser degree of proteolysis than of endocytosis (3,4), which could then result in an earlier release of thyroglobulin into the circulation. An alternative explanation for these results could be that the human thyroid gland responds qualitatively differently to bTSH than to endogenously released hTSH. The present study was designed to test whether or not variations in the dose of bTSH would influence the qualitative pattern of the response of the normal human thyroid to exogenous bTSH. Materials and Methods
Nine healthy euthyroid subjects, 5 males and 4 females, ranging in age from 19 to 27 years, were given bTSH (Thytropar®) im. Six subjects (3 males and 3 females) were each given 2 IU and 3 subjects (2 males and 1 female) were each Received June 24, 1975. given 0.4 IU. Blood samples were obtained Supported by grants from NIH Research Cancer before injection of bTSH and then at 2, 4, 6, 8, CA 13447, USPHS AM-16155, CRC Grant RR 00865, and NIH Research Career Development Award, 10, 12, 24, 36, 48, 72 and 96 h after injection. Serum concentrations of T4, T3, and HTg were KO4 AM-70225 (Dr. Chopra). Reprint requests to: Andre J. Van Herle, M.D., Dept. determined by radioimmunoassay methods (5of Medicine, UCLA School of Medicine, Los Angeles, 7). A bTSH assay was developed in order to determine the levels of bTSH attained in the Ca. 90024.
312
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313
THYROIDAL RESPONSE TO bTSH IN MAN
TABLE 1. Serum HTg, T3, T4 and bTSH levels following im administration of 10 IU of bTSH in normal subjects Time of bleeding post bTSH (h) Subjects
0
2
4
6
8
12
24
36
48
72
96
120
1 HTg' T3" T4t bTSH'
2.7 120 9.0 0.9
4.0 158 7.0 3.5
5.1 280 10.8 3.6
3.9 280 9.5 2.3
5.4 305 13.0 1.5
7.4 253
HTg Ta T4 bTSH
4.2 130 6.6 0.6
5.6 100 5.0 2.2
7.7 115 5.3 1.8
8.0 145 7.5 1.9
HTg Ta T4 bTSH
5.1 138 0.4
4.6 250 5.6 7.8
6.6 330 8.5 5.4
HTg T, T4 bTSH
2.4 120 8.2 0.6
2.6 150 7.3 6.0
2.9 210 8.3 5.2
HTg T, T4 bTSH
6.0 150 9.5 0.5
— — —
_ _ —
_ —
4.If ±0.7
4.2t* ±0.6
5.6ff* ± 1.0
5.7f* ± 1.4
16.5 270
17.0 290 13.2 0.9
16.0
7.6 125
210
13.2
_
_
1.0
—
—
10.0
9.4 130 8.4 0.6
11.3
10.7
1.1
1.0
8.7 130 6.0 2.0
16.5 145 7.0 0.8
145 8.0 1.4
21.0 135 8.0 1.1
8.2 370 9.3 1.9.
9.2 340 11.5 1.6
11.5 350 9.0 0.8
16.5 310 10.8 0.7
26.0 — — —
250 9.8 0.6
2.6 215 10.3 4.2
2.4 280 13.3 4.1
2.0 285 16.0 2.5
2.6 240 13.5 1.3
7.2 _
5.2 _
—
0.8
6.0 150 9.2 0.7
26.5 215 12.8 0.7
14.0
13.3 2.7
16.5 270 15.3 0.7
20.0
280 13.0 1.5
7.1t* ± 1.4
9.4ft ±3.1
13.8 ft ±2.8
19.5ft ±3.5
10.0 1.0
2 17.0
23.0
140 _ 0.9
13.8
35.0
22.0 225
1.0
5.6 105 6.4 0.6
3
4.4
8.1 —
16.5 185 6.5 0.5
— — —
4 5.0 9.6 0.8
2.8 105 8.8 0.6
5
mean ± SEM HTg
132H* ±6
T3
7.5$ ±0.9
T4 bTSH
9.8
164$ ±31 6.2$ ±0.6
234 H ±47
8.2$ ± 1.1
0.6
4.9
4.0
±0.1
± 1.2
±0.8
252 ft ± 48
264ft ±46
262 ff ±33
9.2ft ±0.6
11.4tt ± 1.4
2.6 ±0.5
2.2
1.1
± 0.4
±0.1
' ng/ml. •• ng/dl.
11.3ft ± 1.6
241ff ±35
213tt ± 45
225
—
5.8 155
15.3
12.5
11.3
0.7
0.3
0.4
13.0H ±3.4
8.0$ ±2.8
19.8 ±4.8 228ft ± 12
11.7H ± 1.2
11.311 ± 1.7
1.0 ±0.1
0.9
0.8
± 0.1
±0.1
12.8ft ± 1.6
172tf ± 27
157$ ± 16
6.6 170 9.8 0.6
5.6t* ± 1.0 126f* ± 15
9.0$ ± 1.0
8.8$ ± 0.8
0.7
0.6
±0.2
±0.1
0.7 ±0.1
10.9ft ± 1.3
$ P < 0.05 when compared to mean peak value. f*P < 0.01 when compared to mean peak value, ft*/' < 0.005 when compared to mean peak value.
11 NS compared to mean peak value.
serum following its im administration. All samples from each patient were analyzed in duplicate in the same assay. Purified bTSH was used to prepare tracer and standards.1 Bovine TSH was labeled with 125I by the method of Hunter et al. (8). The standard curve obtained was linear from 0.5 ng/ml (15 /x.U/ml) to 50 ng/ml (1500 /nU/ml). Net secretory response for each parameter studied was computed, as described by Foley et al. (9). The base line 1 Highly purified bTSH (30 U/ml) and rabbit antibTSH antibody were generously provided by Dr. John G. Pierce, (Department of Biochemistry, UCLA School of Medicine).
value was subtracted from the values obtained at each of the subsequent time intervals. The values between each time interval were then averaged and multiplied by the time lapse between each point. The sum total of the values was calculated to reflect net secretory response and was expressed as ng/ml h. In order to examine the relative degree of response in the various parameters studied, all data were converted to ng/ml before computation of the net secretory response. The dose-response (d-r) characteristic of the net secretory responses to bTSH was examined by the least mean square method (10). The d-r curve was computed using the formula y = a + b x; where a = y intercept,
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JCE & VI • 1977 Vol 45 • No 2
ULLER, VAN HERLE AND CHOPRA
314
b = slope, x = log dose bTSH. Significance of the slope was calculated by the t test. All subjects had undetectable antithyroglobulin antibodies in their serum as measured by the tanned red cell hemagglutination technique (11).
Results The individual and mean serum concentrations of HTg, T3 T4, and bTSH at each depicted time interval following administration of 10, 2, and 0.4 IU bTSH are shown in Tables 1-3. The data showing mean HTg, T3, and T4 responses to 10 IU bTSH have been reported previously (1); they are noted here only to compare with the data obtained using the smaller doses of bTSH (Table 1). Serum HTg levels rose in each instance and the mean maximal increase above basal was 376% for 10 IU, 109% for 2 IU, and 35.5% for 0.4 IU of bTSH. Mean maximal increases above the base line were 108%, 74% and 48.5% for T3 and 76.8%, 42% and 15% for T4 following 10 IU, 2 IU and 0.4 IU of bTSH, respectively. Quantitatively larger doses of bTSH produced a greater increment above basal for each thyroidal substance measured. However, the qualitative pattern observed remained essentially the same regardless of the dose of bTSH administered (Fig. 1). A sequential pattern was observed with T3 and T4 peaking first followed by HTg. No significant differences were found between peak T3 values at 8 h when compared with values reached from 2 to 24 h following 10 IU bTSH. Likewise, the peak serum T3 value at 6 h following 2 IU bTSH was not significantly different than T3 levels from 2 to 4 h (Fig. 1, Table 2). As for T4, a peak value reached at 24 h following 10 IU bTSH was not significantly different when compared with values at 6 to 72 h. Peak values at 6 h were not significantly different from those at 4 to 48 h after 2 IU bTSH. HTg values peak at 48 h and 24 h after administration of 10 and 2 IU bTSH, respectively. Likewise, there was no signifi-
cant difference when these were compared with values from 12 to 72 h (10 IU) and from 12 to 48 h (2 IU). The pattern noted in T3 and HTg response differed from that of T4; the former displayed a peak whereas the latter showed a plateau. Circulating bTSH levels were related to the doses administered (Fig. 1 and Table 1). 400-i 300HTg
UJ LLJ
to < < 03 UJ
>
o
CO < UJ CO
CE
•
4
8
10 l.U. b-TSH 2 I U b-TSH 0.4 l.U. b-TSH
12 24 48 72 96 120 HOURS
FIG. 1. Mean per cent increment above basal concentrations for HTg, T3, T4 and bTSH following im administration of varying doses of bTSH.
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315
THYROIDAL RESPONSE TO bTSH IN MAN
TABLE 2. Serum HTg, T3, T4 and bTSH levels following im administration of 2 IU of bTSH in normal subjects Time of bleeding post bTSH (h) Subjects
0
2
4
6
8
12
24
36
48
13.1
14.2
10.7
— _
160 5.7
—
173 6.3 0.2
140 5.1 0.5
255 9.0
![[Pancreatic response to graded doses of synthetic secretion: comparison between intravenous and pernasal administration in man].](/assets/img/hold.png)
