The Effect of Exercise on Circulating Immunoreactive Calcitonin in Men
Summary Moderate-duration exercise increases serum catecholamine and serum calcium levels and might as a result be also expected to increase the levels of circulating serum immunoreactive human calcitonin (HCT). To explore this possibility, HCT was studied during and after moderate duration symptom-limited dynamic exercise in 13 healthy males, mean age 28 ± 6.9 (SD) years. The mean duration of exercise using the Bruce treadmill protocol was 14.1 ± 2.2 (SD) minutes. The mean heart rate (HR) peaked at 185 + 6 (SD) bpm which was 96.1% of the predicted maximal HR for age. Values for HCT, uncorrected for changes in plasma volume, showed a minimal decrease in the recovery phase, whilst HCT corrected for changes in plasma volume did not alter during exercise or recovery. The serum parathyroid hormone (PTH) also did not change. At peak exercise, uncorrected but not corrected values for plasma noradrenaline, adrenaline and dopamine had increased significantly. Corrected plasma total calcium increased during recovery. In summary, dynamic weight-bearing moderate-duration exercise did not elevate HCT in healthy males. Key words Calcitonin — Exercise — Medullary Thyroid Carcinoma — Catecholamines — Parathyroid Hormone — Males
Introduction In order to use the measurement of circulating serum immunoreactive human calcitonin (HCT) as a reliable screening investigation for medullary carcinoma of the thyroid (MTC), it is important to have an understanding of the variables which might influence it. For example, eating {Wilson and Foster 1985) and alcohol ingestion (Weatherall, Ledingham and Warell 1984) are known to increase HCT.
Horm.metab.Res.22(1990)546-550 © Georg Thieme Verlag Stuttgart • New York
HCT secretion also increases in response to an acute increase in plasma ionized calcium (Hamburger, Crosnier and Grunfield 1979) and several studies have reported that moderate to high intensity exercise may increase plasma ionized calcium (Nielson, Christiansen, Hartling and Trap-Jansen 1977; Vora, Kukreja, York, Bowser, Hargis and Williams 1983; Ljunghall, Joborn, Benson, Fellstrom, Wide and Akerstrom 1984; Aloia, Rasulo, Deftos, Vaswani and Yeh 1985; Cunningham, Segre, Slatopolsky and Avioli 1985). Therefore, we hypothesized that exercise-related increases in plasma ionized calcium might increase HCT. Vora, Williams, Hargis, Bowser, Kawahara, Jackson, Henderson and Kukreja (1978) found that betaadrenergic stimulation with isoprenaline in man increased HCT but Epstein, Heath III and Bell (1983) did not, so that the question as to whether exercise-induced increases in circulating catecholamines might influence HCT was unresolved. The conflicting results found in previous studies that have explored the effect of exercise on HCT may be due to differences in the exercise intensities and durations tested, in the associated exercise-induced changes in calcium and in the sex of the subjects tested. Aloia et al. (1985) found that in males, there was an increase in HCT after 10 minutes of moderate intensity exercise at 40%—50% of maximal oxygen consumption, which was out of proportion to hemoconcentration changes and was associated with an increase in plasma total and ionized calcium levels. In contrast, Nishiyama, Tomoeda, Ohta, Higuchi and Matsuda (1988) also examined the HCT response in males to moderate intensity exercise (50% of maximum capacity), but found no change in HCT. They exercised their subjects for a more prolonged period of 30 minutes and it was associated with a decrease in ionized calcium levels. Cunningham et al. (1985) found that HCT was not influenced by maximal exercise of very short duration (60—130 seconds). Despite increasing plasma ionized calcium, this exercise duration may have been too brief to see an effect on HCT. Also, the inclusion of four females in their sample of 12 subjects may have minimized any HCT changes because females have lower calcium-stimulated HCT rises than males (Deftos, Weisman, Williams, Karpf Frumar, Davidson, Parthemore and Judd 1980).
Received: 24 July 1989
Accepted: 6 Apr. 1990
Downloaded by: University of Illinois. Copyrighted material.
M. E. O'Neill1, M. Wilkinson2, B. G. Robinson2, D. B. McDowall2, K. A. Cooper4(formerly1), A. S. Mihailidou , D. B. Frewin , P. Clifton-Bligh2 and S. N. Hunyor 'Cardiovascular Research Unit, Dept. of Cardiology, Royal North Shore Hospital, Sydney 2 Endocrinology Department, Royal North Shore Hospital, Sydney Department of Clinical and Experimental Pharmacology, University of Adelaide, South Australia 4 Catholic College of Education, North Sydney, Australia
Horm. metab. Res. 22 (1990)
Calcitonin Response to Exercise in Healthy Males
547
Table 1 Pre-, peak and post-exercise values for hormonal and biochemical variables for Group 1. Mean and pooled standard error (SE) determined from repeated measures analysis of variance (RMAV) are given. N
Pre
Peak
Post-Ex
RMAV
SE
Calcitonin (pg/ml) Corrected
11 10
36.4 35.6
39.7 35.1
31.3* 31.4
* NS
1.84 1.65
Total calcium (mmol/l) Corrected
12 10
Bicarbonate (mmol/l) Corrected
11 10
Phosphate (mmol/l) Corrected
11 10
1.14 1.14
1.43 + 1.31
1.13 1.23
NS
0.053 0.056
PTH (ng/ml) Corrected
12 10
0.21 0.21
0.22 0.20
0.23 0.27
NS NS
0.031 0.034
2.40 2.40 27.0 27.0
2.56* 2.35 20.4 + 18.8 +
2.37 2.52* 23.5 25.4
* ** ** ** **
0.022 0.033 1.2 1.3
Hence, with regard to interpreting HCT results, recent high-intensity short-duration exercise {Cunningham et al. 1985) or moderate-intensity prolonged exercise (Nishiyama et al. 1988) should not influence HCT. However, the study by Aloia et al. (1985) raises the possibility that moderate duration, moderate intensity exercise may influence HCT and the interpretation of HCT screening results. Their findings have not yet been confirmed by other studies. Therefore, the present study sought to confirm whether moderateduration, moderate to high intensity exercise in healthy males was associated with changes in HCT and whether exercise-related changes in catecholamines and/or plasma calcium occurred and could be linked with changes in HCT.
serum was not used to dilute calcitonin standards because of the difficulty in obtaining calcitonin-free serum. When calcitonin standards were prepared with sera from patients with severe untreated hypocalcaemia who had very low HCT values not significantly different from zero, and compared with standards prepared in buffer, identical curves were obtained for standards ranging between 0 and 50 pg per assay tube. Hence, we used standards prepared in buffer in the present study. Each serum sample was assayed in aliquots of 100 uL and 200 uL in triplicate. A non-specific binding blank was included in each sample. If proportional increases in the measured calcitonin value with increased aliquot volume did not occur, the values were discarded. Quality control sera at multiple dilutions were included with each assay. The interassay coefficient of variation for values of 200 pg/ml was 12%. The non-specific binding was 3%. The sensitivity of the assay was 20 pg/ml. The upper limit of the reference range was 200 pg/ml.
Methods and Design Group 1 consisted of 13 healthy males who exercised to a symptom-limited maximal level using the standard Bruce treadmill protocol. Venous blood samples (25 to 30 mls) were taken in the standing position prior to exercise, at peak exercise and 30 minutes into recovery for the following estimations: HCT, parathyroid hormone (PTH), biochemical profile (for plasma total calcium, bicarbonate and phosphate), hematocrit and hemoglobin. Group 2 comprised a subset of 7 of these males who had extra blood withdrawn at 3 and 9 minutes of exercise for the above variables, in addition to venous samples before exercise, and at 3 and 9 minutes and at peak exercise for noradrenaline (NA), adrenaline (AD) and dopamine (DA). Hemolyzed samples were excluded. During exercise, heart rate (HR) was monitored continuously and systolic blood pressure (SBP) was measured every three minutes. A wide range of fitness levels was represented among the subjects, all of whom were free of any medications. The study was approved by the institutional Ethics Review Committee and all subjects gave informed consent. They abstained from food, alcohol and smoking, for at least 4 hours prior to their test. HCT was measured by a modification of the method of Heynen and Franchimont (1974). Anti-human calcitonin antibody raised in a goat, which bound selectively to the 17—32 amino acid sequence of the calcitonin molecule was used. Synthetic human calcitonin (Organon) was used to prepare standards in a buffer described by Heynen and Franchimont (1974) and for labelling with I125 by the procedure of Marx, Woodward and Aurbach (1972), which is known to preserve the biological activity of the labelled calcitonin. Nondescript
The radio-immunoassay method previously described by Kleerekoper, Ingham, McCarthy and Posen (1974) was used to measure PTH (1—84). It has inter- and intra-assay variabilities of 9.2% and 8.1 % respectively at the upper limit of the normal range, which was 0.4 ng/ml. Venous samples for NA, AD and DA were collected on ice in heparinized tubes (lithium heparin 125 U/10 ml) containing glutathione (5 mmol/l), spun immediately in a refrigerated centrifuge, separated and kept at - 20 °C until assayed. A modification of the radioenzymatic technique of Da Prada and Zurcher (1976), as described by Cummings, Russell, Frewin and Miller (1984) was used. In our laboratory, the recovery rate was 60—62% and the coefficient of variation of the assay was 5 % at the concentrations found in this study. The lower limit of detection was 0.05 pmol/ml. Corrected results, which take into account plasma volume changes with exercise and recovery, were calculated using equations developed by van Beaumont, Underkofler and van Beaumont (1981). Due to clotting of some of the blood samples for hemoglobin and hematocrit, the number of subjects included in the corrected data is less than for the uncorrected data. This accounts for the slight difference in the group 1 uncorrected and corrected baseline values. Data analysis was performed using the Minitab Statistics Package. The values given in the text are means (± standard deviation [SD ]). Repeated measures analysis of variance (RMAV) was used as the test of statistical significance. The significance level was set at P < 0.05. The number of subjects (N) with complete sets of data
Downloaded by: University of Illinois. Copyrighted material.
Values corrected for changes in plasma volume with exercise and recovery are indicated by "Corrected", NS/not significant, *P < 0.05, P
Summary Moderate-duration exercise increases serum catecholamine and serum calcium levels and might as a result be also expected to increase the levels of circulating serum immunoreactive human calcitonin (HCT). To explore this possibility, HCT was studied during and after moderate duration symptom-limited dynamic exercise in 13 healthy males, mean age 28 ± 6.9 (SD) years. The mean duration of exercise using the Bruce treadmill protocol was 14.1 ± 2.2 (SD) minutes. The mean heart rate (HR) peaked at 185 + 6 (SD) bpm which was 96.1% of the predicted maximal HR for age. Values for HCT, uncorrected for changes in plasma volume, showed a minimal decrease in the recovery phase, whilst HCT corrected for changes in plasma volume did not alter during exercise or recovery. The serum parathyroid hormone (PTH) also did not change. At peak exercise, uncorrected but not corrected values for plasma noradrenaline, adrenaline and dopamine had increased significantly. Corrected plasma total calcium increased during recovery. In summary, dynamic weight-bearing moderate-duration exercise did not elevate HCT in healthy males. Key words Calcitonin — Exercise — Medullary Thyroid Carcinoma — Catecholamines — Parathyroid Hormone — Males
Introduction In order to use the measurement of circulating serum immunoreactive human calcitonin (HCT) as a reliable screening investigation for medullary carcinoma of the thyroid (MTC), it is important to have an understanding of the variables which might influence it. For example, eating {Wilson and Foster 1985) and alcohol ingestion (Weatherall, Ledingham and Warell 1984) are known to increase HCT.
Horm.metab.Res.22(1990)546-550 © Georg Thieme Verlag Stuttgart • New York
HCT secretion also increases in response to an acute increase in plasma ionized calcium (Hamburger, Crosnier and Grunfield 1979) and several studies have reported that moderate to high intensity exercise may increase plasma ionized calcium (Nielson, Christiansen, Hartling and Trap-Jansen 1977; Vora, Kukreja, York, Bowser, Hargis and Williams 1983; Ljunghall, Joborn, Benson, Fellstrom, Wide and Akerstrom 1984; Aloia, Rasulo, Deftos, Vaswani and Yeh 1985; Cunningham, Segre, Slatopolsky and Avioli 1985). Therefore, we hypothesized that exercise-related increases in plasma ionized calcium might increase HCT. Vora, Williams, Hargis, Bowser, Kawahara, Jackson, Henderson and Kukreja (1978) found that betaadrenergic stimulation with isoprenaline in man increased HCT but Epstein, Heath III and Bell (1983) did not, so that the question as to whether exercise-induced increases in circulating catecholamines might influence HCT was unresolved. The conflicting results found in previous studies that have explored the effect of exercise on HCT may be due to differences in the exercise intensities and durations tested, in the associated exercise-induced changes in calcium and in the sex of the subjects tested. Aloia et al. (1985) found that in males, there was an increase in HCT after 10 minutes of moderate intensity exercise at 40%—50% of maximal oxygen consumption, which was out of proportion to hemoconcentration changes and was associated with an increase in plasma total and ionized calcium levels. In contrast, Nishiyama, Tomoeda, Ohta, Higuchi and Matsuda (1988) also examined the HCT response in males to moderate intensity exercise (50% of maximum capacity), but found no change in HCT. They exercised their subjects for a more prolonged period of 30 minutes and it was associated with a decrease in ionized calcium levels. Cunningham et al. (1985) found that HCT was not influenced by maximal exercise of very short duration (60—130 seconds). Despite increasing plasma ionized calcium, this exercise duration may have been too brief to see an effect on HCT. Also, the inclusion of four females in their sample of 12 subjects may have minimized any HCT changes because females have lower calcium-stimulated HCT rises than males (Deftos, Weisman, Williams, Karpf Frumar, Davidson, Parthemore and Judd 1980).
Received: 24 July 1989
Accepted: 6 Apr. 1990
Downloaded by: University of Illinois. Copyrighted material.
M. E. O'Neill1, M. Wilkinson2, B. G. Robinson2, D. B. McDowall2, K. A. Cooper4(formerly1), A. S. Mihailidou , D. B. Frewin , P. Clifton-Bligh2 and S. N. Hunyor 'Cardiovascular Research Unit, Dept. of Cardiology, Royal North Shore Hospital, Sydney 2 Endocrinology Department, Royal North Shore Hospital, Sydney Department of Clinical and Experimental Pharmacology, University of Adelaide, South Australia 4 Catholic College of Education, North Sydney, Australia
Horm. metab. Res. 22 (1990)
Calcitonin Response to Exercise in Healthy Males
547
Table 1 Pre-, peak and post-exercise values for hormonal and biochemical variables for Group 1. Mean and pooled standard error (SE) determined from repeated measures analysis of variance (RMAV) are given. N
Pre
Peak
Post-Ex
RMAV
SE
Calcitonin (pg/ml) Corrected
11 10
36.4 35.6
39.7 35.1
31.3* 31.4
* NS
1.84 1.65
Total calcium (mmol/l) Corrected
12 10
Bicarbonate (mmol/l) Corrected
11 10
Phosphate (mmol/l) Corrected
11 10
1.14 1.14
1.43 + 1.31
1.13 1.23
NS
0.053 0.056
PTH (ng/ml) Corrected
12 10
0.21 0.21
0.22 0.20
0.23 0.27
NS NS
0.031 0.034
2.40 2.40 27.0 27.0
2.56* 2.35 20.4 + 18.8 +
2.37 2.52* 23.5 25.4
* ** ** ** **
0.022 0.033 1.2 1.3
Hence, with regard to interpreting HCT results, recent high-intensity short-duration exercise {Cunningham et al. 1985) or moderate-intensity prolonged exercise (Nishiyama et al. 1988) should not influence HCT. However, the study by Aloia et al. (1985) raises the possibility that moderate duration, moderate intensity exercise may influence HCT and the interpretation of HCT screening results. Their findings have not yet been confirmed by other studies. Therefore, the present study sought to confirm whether moderateduration, moderate to high intensity exercise in healthy males was associated with changes in HCT and whether exercise-related changes in catecholamines and/or plasma calcium occurred and could be linked with changes in HCT.
serum was not used to dilute calcitonin standards because of the difficulty in obtaining calcitonin-free serum. When calcitonin standards were prepared with sera from patients with severe untreated hypocalcaemia who had very low HCT values not significantly different from zero, and compared with standards prepared in buffer, identical curves were obtained for standards ranging between 0 and 50 pg per assay tube. Hence, we used standards prepared in buffer in the present study. Each serum sample was assayed in aliquots of 100 uL and 200 uL in triplicate. A non-specific binding blank was included in each sample. If proportional increases in the measured calcitonin value with increased aliquot volume did not occur, the values were discarded. Quality control sera at multiple dilutions were included with each assay. The interassay coefficient of variation for values of 200 pg/ml was 12%. The non-specific binding was 3%. The sensitivity of the assay was 20 pg/ml. The upper limit of the reference range was 200 pg/ml.
Methods and Design Group 1 consisted of 13 healthy males who exercised to a symptom-limited maximal level using the standard Bruce treadmill protocol. Venous blood samples (25 to 30 mls) were taken in the standing position prior to exercise, at peak exercise and 30 minutes into recovery for the following estimations: HCT, parathyroid hormone (PTH), biochemical profile (for plasma total calcium, bicarbonate and phosphate), hematocrit and hemoglobin. Group 2 comprised a subset of 7 of these males who had extra blood withdrawn at 3 and 9 minutes of exercise for the above variables, in addition to venous samples before exercise, and at 3 and 9 minutes and at peak exercise for noradrenaline (NA), adrenaline (AD) and dopamine (DA). Hemolyzed samples were excluded. During exercise, heart rate (HR) was monitored continuously and systolic blood pressure (SBP) was measured every three minutes. A wide range of fitness levels was represented among the subjects, all of whom were free of any medications. The study was approved by the institutional Ethics Review Committee and all subjects gave informed consent. They abstained from food, alcohol and smoking, for at least 4 hours prior to their test. HCT was measured by a modification of the method of Heynen and Franchimont (1974). Anti-human calcitonin antibody raised in a goat, which bound selectively to the 17—32 amino acid sequence of the calcitonin molecule was used. Synthetic human calcitonin (Organon) was used to prepare standards in a buffer described by Heynen and Franchimont (1974) and for labelling with I125 by the procedure of Marx, Woodward and Aurbach (1972), which is known to preserve the biological activity of the labelled calcitonin. Nondescript
The radio-immunoassay method previously described by Kleerekoper, Ingham, McCarthy and Posen (1974) was used to measure PTH (1—84). It has inter- and intra-assay variabilities of 9.2% and 8.1 % respectively at the upper limit of the normal range, which was 0.4 ng/ml. Venous samples for NA, AD and DA were collected on ice in heparinized tubes (lithium heparin 125 U/10 ml) containing glutathione (5 mmol/l), spun immediately in a refrigerated centrifuge, separated and kept at - 20 °C until assayed. A modification of the radioenzymatic technique of Da Prada and Zurcher (1976), as described by Cummings, Russell, Frewin and Miller (1984) was used. In our laboratory, the recovery rate was 60—62% and the coefficient of variation of the assay was 5 % at the concentrations found in this study. The lower limit of detection was 0.05 pmol/ml. Corrected results, which take into account plasma volume changes with exercise and recovery, were calculated using equations developed by van Beaumont, Underkofler and van Beaumont (1981). Due to clotting of some of the blood samples for hemoglobin and hematocrit, the number of subjects included in the corrected data is less than for the uncorrected data. This accounts for the slight difference in the group 1 uncorrected and corrected baseline values. Data analysis was performed using the Minitab Statistics Package. The values given in the text are means (± standard deviation [SD ]). Repeated measures analysis of variance (RMAV) was used as the test of statistical significance. The significance level was set at P < 0.05. The number of subjects (N) with complete sets of data
Downloaded by: University of Illinois. Copyrighted material.
Values corrected for changes in plasma volume with exercise and recovery are indicated by "Corrected", NS/not significant, *P < 0.05, P
