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Immunoreactive calcitonin in the mother, neonate, child, and adult
Immunoreactive calcitonin in the mother, neonate, child, and adult NAGUIB
A.
GARLAND
SAMAAN, D.
MAYBELLE Houston,
M.D.,
ANDERSON, E.
PH.D. M.D.
ADAM-MAYNE,
PH.D.
Texas
Immunoreactive calcitonin (iCT) was measured in umbilical arterial and venous blood and in maternal peripheral blood in 32 normal deliveries. The results were comparedwith values found in nonpregnant adult females. The umbilical arterial blood contained significantly higher concentrations of iCT than umbilical venous blood (p < 0.001). The serum iCT in maternal peripheral blood was significantly higher than in normal nonpregnant subjects ranging
(p < 0.001). Serum iCT was also in age from 1 hour to 60 years. Serum
measured in 342 male and female subjects iCT was found to be high early in life and
to diminish with age. Our data suggest that calcitonin may be of physiologic bone formation during intrauterine life and childhood. High serum iCT responsible for the hypocalcemia seen in the neonatal period.
C ALC ITO N I N is a hormone secreted by the c-cells of the human thyroid.5 Its physiologic function, if any, is unknown, since its normal level in serum has until recently been unmeasurable.12‘14 We have recently reported development of a specific, sensitive radioimmunoassay of iCT in unextracted serum which is capable of measuring normal levels.x With this technique high serum calcitonin has been found in maternal and mixed arterial and venous cord blood at the time of
Supported by United States Public Health National Institutes of Health Grant No. CA-05831-13, and American Cancer Society No. CI-78B. Presented at the Twenty-first the Society for Gynecologic Angeles, California, March Receilved
for publication
Accepted
September
Service, Grant
Annual Meeting of Investigation, Los 27-29, 1974. May
12, 1974.
5, 1974.
Reprint requests: Naguib A. Samaan, M.D., Ph.D., Chief, Section of Endocrinology, M. D. Anderson Hospital, 6723 Bertner Ave., Houston, Texas
77025.
in
delivery. 6rr We now wish to report studies on serum calcium and calcitonin concentrations in umbilical arterial and venous blood collected at 32 normal deliveries. This work was undertaken to determine the site of origin of the high iCT level found in mixed cord blood. ICT was also measured in 342 subjects varying from 1 hour to 60 years of age to determine the normal levels of serum calcitonin at various ages. Material
From the Section of Endocrinology, Departments of Gynecology and Pathology, The Univesrity of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, Hermann Hospital, and the Texas Children Hospital.
significance
may also be
and
methcrds
Blood was collected at time of delivery selectively from the umbilical arteries and vein in each of 32 normal term babies. Serum iCT was measured in 342 male and female subjects (208 male and 134 female) ranging in age from 1 hour to 60 years: Blood was obtained by heel prick from babies and children at the same time that they were having blood collected for the study of metabolic or respiratory disturbances and by venous puncture from healthy adult volunteers. Serum was separated and stored at -20° C. until time of the assay. Serum was available for measurement of total serum calcium in someof thesesamples. The serum iCT concentrations were measured by a specific sensitive radioimmunoassay, which can
Volume Number
121 5
lmmunoreactive
Cord
Blood
Percent of subjects with Colcilonin
f32 Pis) Vem
calcitonin
623
level above normal range
Artery
T
1 Fig. 1. Serum iCT levels (mean + SD.) in nonpregnant subjects (32) compared to those found in the peripheral maternal serum and the umbilical venous and arterial serum at time of delivery in 32 patients studied.
Fig. 2. Serum iCT levels (mean k S.D.) in peripheral serum at different age groups. There was progressive fall of serum iCT level with age.
quantify as little as 0.032 ng. per milliliter of unextracted serum or 0.0032 ng. per incubating tube in terms of synthetic human calcitonin M MRC standard. The assay procedure, sensitivity, and reproducibility have been described in detail previously.” We also measured the effect of dilution on endogenous calcitonin in three umbilical cord serum samples from three patients. The serum samples were diluted with pooled serum from athyreotic patients in such a way as to have equal concentration of serum proteins in each diluted sample. Degree of recovery was measured by adding various concentrations of synthetic human calcitonin standard to four serum samples from the umbilical cord. Total serum calcium was measured by atomic absorption spectrophotometry.
Table I. The effect of dilution of serum from umbilical cord. The measured iCT multiplied the dilution factor remains constant Calcitonin
Umbilical cord serum
In
Dilutions
assay tube
undiluted serum 6.4 6.2 6.8 5.6
1:l 1:4 1:a
3.2 1.8 0.7 0.5
6.25 3.2 3.6 2.8 4.0
1:l 1:4 i:a
4.5 2.1 1.0 0.6
3.4 4.5 4.2 4.0 4.8
2
Mean
In
6.4 3.1 1.7 0.7
Mean
Mean 3
(ng./ml.)
1:l 1:4 1:a
1
Results In each of the 32 newborn infants, the serum calcitonin level in the umbilical artery was significantly higher than that in the vein (with the use of the pair test, p < 0.001). The mean level of serum iCT in the umbilical artery was 7.27 + S.D. 2.18 . .. ng. per mllhhter, whereas the umbilical vein concentration was 4.11 + SD. 1.19 ng. per milliliter (p < 0.001). The serum iCT concentrations both in the umbilical artery and vein were also significantly higher than that in the maternal peripheral blood, 1.39 k S.D. 0.47 ng. per milliliter (p < 0.001). This latter value, however, was in turn significantly higher (p < 0.001) than that found in normal
1 1
the by
4.4
nonpregnant subjects, 0.49 + S.D. 0.25 ng. per milliliter (Fig. 1 j . The serum samples from the umbilical cord which were diluted showed that the concentration of iCT in the serum obtained by reference to the standard curve fell linearly with the dilution and that the measured iCT multiplied by the dilution factor remained constant (Table I). This indicates that there is no immunologic difference between the endogenous hormone in serum found in the newborn infant and the synthetic calcitonin stan-
624
Samaan,
Anderson,
and
March 1, 1975 Am. J. Obstet. Gynecol.
Adam-Mayne
dard. There was 90 to 105 per cent recovery of synthetic calcitonin standard when it was added to serum from the cord. This is further evidence that there is no significant interference of plasma proteins in our calcitonin immunoassay system. In the group of patients studied at time of delivery the maternal serum calcium was 8.6 + SD. 0.65 mg. per 100 ml., the umbilical arterial serum calcium 9.4 k S.D. 0.42 mg. per 100 ml., and the umbilical venous serum calcium 10.6 + SD. 0.77 mg. per 100 ml. These differences are significant. For maternal serum calcium/umbilical vein serum calcium p < 0.001; and for umbilical arterial serum calcium/umbilical venous serum calcium p < 0.01. Among the 342 normal subjects studied serum iCT was greater than 1 ng. per milliliter (the upper limit for normal adults) in 96 per cent of 47 newborn infants 1 to 24 hours of age, 68 per cent of infants of 1 day to 1 month, 42 per cent of 77 infants of 1 month to 5 years, 24 per cent of 65 children 5 to 10 years of age, and 12 per cent of 70 subjects between 10 and 20 years of age (Fig.
2). In 20 newborn infants 1 to 24 hours of age the total serum calcium level was 6.6 + S.D. 1.3 mg. per 100 ml. In 20 neonates of 1 day to 1 month the serum calcium level was 7.2 f S.D. 1.3 mg. per 100 ml. In 18 children of 1 month to 5 years, the serum calcium was 10.1 + S.D. 1.3 mg. per 100 ml., in 23 children 6 to 10 years of age the serum calcium was 9.4 ? S.D. 1.4 mg. per 100 ml., and in 52 subjects 10 to 20 years of age the serum calcium was 9.5 t S.D. 0.86 mg. per 100 ml. In the 32 normal adult subjects the serum calcium was 9.7 + S.D. 0.24 mg. per 100 ml.
That serum calcium in the umbilical vein is higher than that in the maternal blood indicates that there is active transport of calcium through the placenta to the fetus.“, 9 That serum calcium in the umbilical artery was lower than in the umbilical vein shows that calcium is metabolized by the fetus. It has been previously shown by Pearse”! 5 that the human fetal thyroid gland has a greater number of calcitonin-producing cells than does the adult gland. The levels of circulating calcitonin at various ages have not been reported previously. Our data show that serum calcitonin is elevated not only during neonatal life but also during childhood. There is no significant sex difference. Low serum calcium has been reported to occur in term infants during the first few days in life.‘” Premature, low-birth-weight infants and infants with respiratory difficulties have also been found to have lower serum calciums than term neonates.l”t I1 The low serum calcium found in the newborn infants in the present series may be attributed to the fact that most of them were premature with low birth weights or had respiratory difficulties. A sluggish response of the parathyroid glands to hypocalcemia has been proposed as the mechanism of hypocalcemia commonly seen in the first few days of life. I6 The possible significance of calcitonin has not been studied previously. The results of this study suggest that the high level of serum calcitonin in the newborn infant may be important in producing the hypocalcemia commonly seen in the first days of life. Furthermore, the high level of serum calcitonin in the newborn period and early childhood may play an important role in bone formation and calcium metabolism during the growth years.
Comment Serum iCT concentrations in the maternal and cord serum have previously been shown to be significantly higher than those found in normal adult nonpregnant human subjects.G-8 The high calcitonin levels found in the arterial cord blood as compared with the umbilical venous blood indicate that calcitonin is produced by the fetus and not the placenta. Milhaud and associates3 have shown that calcitonin does not cross the placental barrier in rats.
REFERENCES
1.
Fairney, A., Jackson, D., Dis. Child. 48: 419, 1973.
The authors wish to express their sincere gratitude to Dr. Robert Neher, Ciba Limited, Basle, Switzerland, for the synthetic human calcitonin M and the British Medical Research Council for the supply of calcitonin standard. We also wish to thank Drs. W. A. McRoberts, ,Jr., B. Held, and H. S. Gallager for their most helpful suggestions. We are also indebted to Mrs. Socorro Castillo and Mrs. Pamela Schultz for their excellent technical assistance.
2. and
Clayton,
B. E.: Arch. 3.
Hallman, 1953. Milhaud,
N.,
and
Salmi,
G., Maukhtar,
I.: M.
Acta
Pediatr.
S., Perault-Staub,
42:
126,
A. M.,
Volume Number
121 5
and Coutris, G.: In Taylor, S., and Foster, G. V., editors: Calcitonin, Proceedings Second International Symposium, London, 1969, William Heinemann Med. Books, p. 182. 4. Pearse, A. G. E.: In Taylor, S., and Foster, G. V., editors: Calcitonin, Proceedings Second International Symposium, London, 1969, William Heinemann Med. Books, p. 125. 5. Pearse, A. G. E.: Proc. R. Sot. Biol. 164: 478, 1966. 6. Samaan, N. A., Hill, C. S., Jr., Beciero, J. R., and Schultz, P. N.: .J. Lab. Clin. Med. 81: 671, 1973. 7. Samaan, N. A., Wigoda, C., and Castillo, S.: In Tavlor. S.. and Foster. G. V.. editor: Calcitonin, Proceebings Fourth International Symposium, Len: don, 1974, William Heinemann Med. Books. 8. Samaan, N. A., Castillo, S., and Wigoda, C.: Abst. No. 256, Endocrine Society, Chicago, Ill., June 20-22, 1973.
lmmunoreactive
9. 10. 11. 12. 13. 14. 15. 16.
calcitonin
625
Widdowson, E. M., and McCance, R. A.: Pediatr. Clin. North Am. 12: 595, 1965. Acharya, P. T., and Payne, W. W.: Arch. Dis. Child. 40: 430, 1965. Rosh, A., and Fanconi, A.: Helv. Pediatr. Acta 28: 443, 1973. Clark, M. B., Boyd, G. W., Byfield, P. G. H., and Foster, G. V.: Lancet 2: 74, 1969. Tashjian, A. H., Howland, B. G., Melvin, K. W., and Hill, C. S., Jr.: N. Engl. J. Med. 283: 890, 1970. Deftos, L. J., Bury, A. E., Habener, J. F., Singer, F. R., and Potter, J. T., Jr. : Metabolism 20: 1129, 197 1. Lequin, R. M., Hackeng, W. H. L., and Schopman, W.: Acta Endocrinol. (Kbh.) 63: 655, 1970. Tsang, R. C., Chen, I. W., Freidman, M. A., et al.: J. Pediatr. 83: 728, 1973.
A.
GARLAND
SAMAAN, D.
MAYBELLE Houston,
M.D.,
ANDERSON, E.
PH.D. M.D.
ADAM-MAYNE,
PH.D.
Texas
Immunoreactive calcitonin (iCT) was measured in umbilical arterial and venous blood and in maternal peripheral blood in 32 normal deliveries. The results were comparedwith values found in nonpregnant adult females. The umbilical arterial blood contained significantly higher concentrations of iCT than umbilical venous blood (p < 0.001). The serum iCT in maternal peripheral blood was significantly higher than in normal nonpregnant subjects ranging
(p < 0.001). Serum iCT was also in age from 1 hour to 60 years. Serum
measured in 342 male and female subjects iCT was found to be high early in life and
to diminish with age. Our data suggest that calcitonin may be of physiologic bone formation during intrauterine life and childhood. High serum iCT responsible for the hypocalcemia seen in the neonatal period.
C ALC ITO N I N is a hormone secreted by the c-cells of the human thyroid.5 Its physiologic function, if any, is unknown, since its normal level in serum has until recently been unmeasurable.12‘14 We have recently reported development of a specific, sensitive radioimmunoassay of iCT in unextracted serum which is capable of measuring normal levels.x With this technique high serum calcitonin has been found in maternal and mixed arterial and venous cord blood at the time of
Supported by United States Public Health National Institutes of Health Grant No. CA-05831-13, and American Cancer Society No. CI-78B. Presented at the Twenty-first the Society for Gynecologic Angeles, California, March Receilved
for publication
Accepted
September
Service, Grant
Annual Meeting of Investigation, Los 27-29, 1974. May
12, 1974.
5, 1974.
Reprint requests: Naguib A. Samaan, M.D., Ph.D., Chief, Section of Endocrinology, M. D. Anderson Hospital, 6723 Bertner Ave., Houston, Texas
77025.
in
delivery. 6rr We now wish to report studies on serum calcium and calcitonin concentrations in umbilical arterial and venous blood collected at 32 normal deliveries. This work was undertaken to determine the site of origin of the high iCT level found in mixed cord blood. ICT was also measured in 342 subjects varying from 1 hour to 60 years of age to determine the normal levels of serum calcitonin at various ages. Material
From the Section of Endocrinology, Departments of Gynecology and Pathology, The Univesrity of Texas System Cancer Center M. D. Anderson Hospital and Tumor Institute, Hermann Hospital, and the Texas Children Hospital.
significance
may also be
and
methcrds
Blood was collected at time of delivery selectively from the umbilical arteries and vein in each of 32 normal term babies. Serum iCT was measured in 342 male and female subjects (208 male and 134 female) ranging in age from 1 hour to 60 years: Blood was obtained by heel prick from babies and children at the same time that they were having blood collected for the study of metabolic or respiratory disturbances and by venous puncture from healthy adult volunteers. Serum was separated and stored at -20° C. until time of the assay. Serum was available for measurement of total serum calcium in someof thesesamples. The serum iCT concentrations were measured by a specific sensitive radioimmunoassay, which can
Volume Number
121 5
lmmunoreactive
Cord
Blood
Percent of subjects with Colcilonin
f32 Pis) Vem
calcitonin
623
level above normal range
Artery
T
1 Fig. 1. Serum iCT levels (mean + SD.) in nonpregnant subjects (32) compared to those found in the peripheral maternal serum and the umbilical venous and arterial serum at time of delivery in 32 patients studied.
Fig. 2. Serum iCT levels (mean k S.D.) in peripheral serum at different age groups. There was progressive fall of serum iCT level with age.
quantify as little as 0.032 ng. per milliliter of unextracted serum or 0.0032 ng. per incubating tube in terms of synthetic human calcitonin M MRC standard. The assay procedure, sensitivity, and reproducibility have been described in detail previously.” We also measured the effect of dilution on endogenous calcitonin in three umbilical cord serum samples from three patients. The serum samples were diluted with pooled serum from athyreotic patients in such a way as to have equal concentration of serum proteins in each diluted sample. Degree of recovery was measured by adding various concentrations of synthetic human calcitonin standard to four serum samples from the umbilical cord. Total serum calcium was measured by atomic absorption spectrophotometry.
Table I. The effect of dilution of serum from umbilical cord. The measured iCT multiplied the dilution factor remains constant Calcitonin
Umbilical cord serum
In
Dilutions
assay tube
undiluted serum 6.4 6.2 6.8 5.6
1:l 1:4 1:a
3.2 1.8 0.7 0.5
6.25 3.2 3.6 2.8 4.0
1:l 1:4 i:a
4.5 2.1 1.0 0.6
3.4 4.5 4.2 4.0 4.8
2
Mean
In
6.4 3.1 1.7 0.7
Mean
Mean 3
(ng./ml.)
1:l 1:4 1:a
1
Results In each of the 32 newborn infants, the serum calcitonin level in the umbilical artery was significantly higher than that in the vein (with the use of the pair test, p < 0.001). The mean level of serum iCT in the umbilical artery was 7.27 + S.D. 2.18 . .. ng. per mllhhter, whereas the umbilical vein concentration was 4.11 + SD. 1.19 ng. per milliliter (p < 0.001). The serum iCT concentrations both in the umbilical artery and vein were also significantly higher than that in the maternal peripheral blood, 1.39 k S.D. 0.47 ng. per milliliter (p < 0.001). This latter value, however, was in turn significantly higher (p < 0.001) than that found in normal
1 1
the by
4.4
nonpregnant subjects, 0.49 + S.D. 0.25 ng. per milliliter (Fig. 1 j . The serum samples from the umbilical cord which were diluted showed that the concentration of iCT in the serum obtained by reference to the standard curve fell linearly with the dilution and that the measured iCT multiplied by the dilution factor remained constant (Table I). This indicates that there is no immunologic difference between the endogenous hormone in serum found in the newborn infant and the synthetic calcitonin stan-
624
Samaan,
Anderson,
and
March 1, 1975 Am. J. Obstet. Gynecol.
Adam-Mayne
dard. There was 90 to 105 per cent recovery of synthetic calcitonin standard when it was added to serum from the cord. This is further evidence that there is no significant interference of plasma proteins in our calcitonin immunoassay system. In the group of patients studied at time of delivery the maternal serum calcium was 8.6 + SD. 0.65 mg. per 100 ml., the umbilical arterial serum calcium 9.4 k S.D. 0.42 mg. per 100 ml., and the umbilical venous serum calcium 10.6 + SD. 0.77 mg. per 100 ml. These differences are significant. For maternal serum calcium/umbilical vein serum calcium p < 0.001; and for umbilical arterial serum calcium/umbilical venous serum calcium p < 0.01. Among the 342 normal subjects studied serum iCT was greater than 1 ng. per milliliter (the upper limit for normal adults) in 96 per cent of 47 newborn infants 1 to 24 hours of age, 68 per cent of infants of 1 day to 1 month, 42 per cent of 77 infants of 1 month to 5 years, 24 per cent of 65 children 5 to 10 years of age, and 12 per cent of 70 subjects between 10 and 20 years of age (Fig.
2). In 20 newborn infants 1 to 24 hours of age the total serum calcium level was 6.6 + S.D. 1.3 mg. per 100 ml. In 20 neonates of 1 day to 1 month the serum calcium level was 7.2 f S.D. 1.3 mg. per 100 ml. In 18 children of 1 month to 5 years, the serum calcium was 10.1 + S.D. 1.3 mg. per 100 ml., in 23 children 6 to 10 years of age the serum calcium was 9.4 ? S.D. 1.4 mg. per 100 ml., and in 52 subjects 10 to 20 years of age the serum calcium was 9.5 t S.D. 0.86 mg. per 100 ml. In the 32 normal adult subjects the serum calcium was 9.7 + S.D. 0.24 mg. per 100 ml.
That serum calcium in the umbilical vein is higher than that in the maternal blood indicates that there is active transport of calcium through the placenta to the fetus.“, 9 That serum calcium in the umbilical artery was lower than in the umbilical vein shows that calcium is metabolized by the fetus. It has been previously shown by Pearse”! 5 that the human fetal thyroid gland has a greater number of calcitonin-producing cells than does the adult gland. The levels of circulating calcitonin at various ages have not been reported previously. Our data show that serum calcitonin is elevated not only during neonatal life but also during childhood. There is no significant sex difference. Low serum calcium has been reported to occur in term infants during the first few days in life.‘” Premature, low-birth-weight infants and infants with respiratory difficulties have also been found to have lower serum calciums than term neonates.l”t I1 The low serum calcium found in the newborn infants in the present series may be attributed to the fact that most of them were premature with low birth weights or had respiratory difficulties. A sluggish response of the parathyroid glands to hypocalcemia has been proposed as the mechanism of hypocalcemia commonly seen in the first few days of life. I6 The possible significance of calcitonin has not been studied previously. The results of this study suggest that the high level of serum calcitonin in the newborn infant may be important in producing the hypocalcemia commonly seen in the first days of life. Furthermore, the high level of serum calcitonin in the newborn period and early childhood may play an important role in bone formation and calcium metabolism during the growth years.
Comment Serum iCT concentrations in the maternal and cord serum have previously been shown to be significantly higher than those found in normal adult nonpregnant human subjects.G-8 The high calcitonin levels found in the arterial cord blood as compared with the umbilical venous blood indicate that calcitonin is produced by the fetus and not the placenta. Milhaud and associates3 have shown that calcitonin does not cross the placental barrier in rats.
REFERENCES
1.
Fairney, A., Jackson, D., Dis. Child. 48: 419, 1973.
The authors wish to express their sincere gratitude to Dr. Robert Neher, Ciba Limited, Basle, Switzerland, for the synthetic human calcitonin M and the British Medical Research Council for the supply of calcitonin standard. We also wish to thank Drs. W. A. McRoberts, ,Jr., B. Held, and H. S. Gallager for their most helpful suggestions. We are also indebted to Mrs. Socorro Castillo and Mrs. Pamela Schultz for their excellent technical assistance.
2. and
Clayton,
B. E.: Arch. 3.
Hallman, 1953. Milhaud,
N.,
and
Salmi,
G., Maukhtar,
I.: M.
Acta
Pediatr.
S., Perault-Staub,
42:
126,
A. M.,
Volume Number
121 5
and Coutris, G.: In Taylor, S., and Foster, G. V., editors: Calcitonin, Proceedings Second International Symposium, London, 1969, William Heinemann Med. Books, p. 182. 4. Pearse, A. G. E.: In Taylor, S., and Foster, G. V., editors: Calcitonin, Proceedings Second International Symposium, London, 1969, William Heinemann Med. Books, p. 125. 5. Pearse, A. G. E.: Proc. R. Sot. Biol. 164: 478, 1966. 6. Samaan, N. A., Hill, C. S., Jr., Beciero, J. R., and Schultz, P. N.: .J. Lab. Clin. Med. 81: 671, 1973. 7. Samaan, N. A., Wigoda, C., and Castillo, S.: In Tavlor. S.. and Foster. G. V.. editor: Calcitonin, Proceebings Fourth International Symposium, Len: don, 1974, William Heinemann Med. Books. 8. Samaan, N. A., Castillo, S., and Wigoda, C.: Abst. No. 256, Endocrine Society, Chicago, Ill., June 20-22, 1973.
lmmunoreactive
9. 10. 11. 12. 13. 14. 15. 16.
calcitonin
625
Widdowson, E. M., and McCance, R. A.: Pediatr. Clin. North Am. 12: 595, 1965. Acharya, P. T., and Payne, W. W.: Arch. Dis. Child. 40: 430, 1965. Rosh, A., and Fanconi, A.: Helv. Pediatr. Acta 28: 443, 1973. Clark, M. B., Boyd, G. W., Byfield, P. G. H., and Foster, G. V.: Lancet 2: 74, 1969. Tashjian, A. H., Howland, B. G., Melvin, K. W., and Hill, C. S., Jr.: N. Engl. J. Med. 283: 890, 1970. Deftos, L. J., Bury, A. E., Habener, J. F., Singer, F. R., and Potter, J. T., Jr. : Metabolism 20: 1129, 197 1. Lequin, R. M., Hackeng, W. H. L., and Schopman, W.: Acta Endocrinol. (Kbh.) 63: 655, 1970. Tsang, R. C., Chen, I. W., Freidman, M. A., et al.: J. Pediatr. 83: 728, 1973.