Immunoassay for human calcitonin. II. Clinical studies

Immunoassay By

for Human

Calcitonin.

II. Clinical Studies

L. J. DEFTOS,A. E. BURY, J. F. HABENER, F. R. SINGER, AND J. T. POTTS, JR.

A sensitive and specific radioimmunoassay for human calcitooin has ben developed and applied to clinical studies. Under optimal conditions, the assay can be used to detect 50-100 pg of calcitonin per milliliter of human plasma. Patients with medullary thyroid carcinoma had concentrations of plasma calcitonin that could be readily detected by the assay and ranged from 60 to over 100,000 pg per milliliter. In these patients, hypercab cemia induced by calcium infusion resulted in up to a 20-fold increase in calcitonin concentration. Hypocalcemia induced by EDTA infusion suppressed the secretion of calcitonin in each patient

tested. Glucagon infusion had a variable effect on calcitonin secretion. Calcitonin could not be detected with confidence in any basal peripheral sample of plasma taken from uormal adults or hypercalcemic patients. Calcitonin could not be unequivocally detected in the majority of thyroid venous plasma samples taken from patients with hypercalcemia. These results suggest that the concentration of calcitonin in the peripheral blood of adults is much lower than previously reported. Further studies are required to determine if calcitonin circulates in peripheral blood even after hypercalcemic stimulation in normal human subjects.

HE DEVELOPMENT of sensitive and specific radioimmunoassays for calcitonin has provided much information about the secretion of this hormone.1-8 After the initial development of an immunoassay for porcine calcitoninl and its application to physiological studies,2-4 subsequent assays were described for human calcitonin5-8 and for bovine, ovine, and salmon calcitonin.“r1° The animal studies demonstrated that the peptide is continuously secreted at physiologic concentrations of blood calcium, that hormone secretion is under the directly proportional control of blood calcium, and that peripheral degradation of calcitonin occurs rapidly. Glucagon has also been reported to stimulate, either directly or indirectly, the release of calcitonin from the thyroid gland in mammals and a physiologic role has been postulated for gastrointestinal factors in the control of secretion of calcitonin.4,6,8,11,12The development and application of immunoassays for human calcitonin5-lo confirmed the presence of a hypercalcitonin state in man: medullary thyroid carcinoma. However, the importance of this peptide in human calcium and skeletal homeostasis has not been established. It was recently reported that immunoassayable calcitonin

T

From the Department of Medicine, Massachusetts General Hospital, Boston, Mass. Received for publication June 25, 1971. L. J. DEFTOS, M.D.: Assistant Professor of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, Mass.; Research Scholar, American Cancer Society, Massachusetts Division. A. E. BURY, B.S.: Research Assistant, Massachusetts General Hospital, Boston, Mass. J. F. HABENER, M.D.: Fellow in Endocrinology, Massachusetts MassaGeneral Hospital, Boston, Mass. F. R. SINGER, M.D.: Fellow in Endocrinology, chusetts General Hospital, Boston, Mass. J. T. POTTS, JR., M.D.: Associate Professor of Medicine,

Harvard

Medical

METABOLISM, VOL. 20, No.

School, Boston, Mass. 12 (DECEMBER),

1971

1129

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DEFTOS ET AL.

could be detected in the plasma of a group of normal subjects at a concentration under basal conditions of 20-400 pg per milliliter.6 Our report describes the development of a very sensitive and specific immunoassay for human calcitonin and its application to clinical studies. Although readily detectable in the plasma of patients with medullary thyroid carcinoma, the basal concentration of calcitonin in the plasma of most normal adults is less than 100 pg per milliliter. These observations suggest that if calcitonin is secreted under basal conditions in man, either its concentration is lower than previously reported or it circulates in a form undetectable by this immunoassay system. MATERIALS

AND METHODS

The details of the immunoassay for human calcitonin used in these studies has been described separately.13 A synthetic preparation of human calcitonin was used for radioiodination and as a reference standard. Also used for reference was a preparation of human calcitonin supplied by the Medical Research Council (MRC human calcitonin Standard A). The antibody to human calcitonin was made against synthetic human calcitonin. Calcitonin was labeled with 1251 by the method of Hunter and Greenwood and purified on silica.13 Prior to use, further purification of labeled calcitonin was accomplished by gel filtration on Bio-gel P-10.13 Phase separation was accomplished by dioxane precipitation.15 Both the supernate and the precipitate of each sample were counted in a gamma counter for a time sufficient to provide a counting accuracy of 1.5%. Peripheral blood samples were collected from normal subjects; from patients with hypercalcemia, including hyperparathyroidism; and from patients with medullary thyroid carcinoma. The samples were collected under basal (fasting) conditions and/or during calcium, ethylenediaminetetraacetic acid (EDTA), and/or glucagon infusions.*JaJs Thyroid vein samples were collected at surgery or by selective venous catheterization from patients with hyperparathyroidism.16 Thyroid vein (and thymic vein) samples were also collected from one patient with idiopathic hypercalcuria. The blood samples were heparinized and kept on ice until the plasma could be separated from the cells (usually less than 30 min). An aliquot was taken for calcium determination17 and each sample was then subdivided into multiple aliquots and kept frozen until calcitonin assay. Replicate determinations of multiple dilutions of each sample were assayed for calcitonin. A mean calcitonin concentration with standard error could be therefore calculated for each sample. Inter- and intraassay variation were 20% and lo%, respectively. Four normal hemithyroid glands, removed at surgery during laryngectomy, were extracted and assayed for calcitonin content.4 Since plasma samples can produce nonspecific displacement of tracer from antibody in immunoassay systems, certain precautions were taken to evaluate this possibility. Some plasma samples were mixed with charcoal (Norit A) at a concentration of 1 g of charcoal per 20 ml of plasma for 3 hr at 4OC. This procedure is known to adsorb small peptides from plasma.ls Charcoal was removed by centrifugation and calcitonin concentration was determined for these samples before and after charcoal adsorption. RESULTS Figure 1 summarizes the peripheral calcitonin concentrations in 33 samples from 18 patients with medullary thyroid carcinoma, in 36 samples from 25 normal subjects, in 20 samples from 15 patients with hypercalcemia (five with hyperparathyroidism) , and the calcitonin concentration in 21 plasma samples taken during selective venous catheterization or at surgery from the thyroid veins (and one thymic vein) of nine patients with hyperparathyroidism and one patient with idiopathic hypercalcuria. The concentration of calcitonin in most of the patients with medullary thyroid carcinoma could be readily detected at plasma dilutions usually exceeding 1: 10,

IMMUNOASSAY

FOR HUMAN

MEfU$U$;Y CARCINOMA

CALCITONIN.

HYPEA-

NORMAL

CALCEYIA

T%‘D

-;

. . t ,,i

1131

II

Fig. l.-Calcitonin concentration in basal (fasting) plasma samples. Open symbols represent samples that appear to displace labeled calcitonin spuriously from specific antibody. All thyroid venous samples, except those from patient with idiopathic hypercalcuria (open squares), were collected from patients with hyperparathyroidism. Open triangle: thymic vein sample from same patient. Broken line: assay detection limits.

’

L.-

Lower dilutions of sample were necessary for other subjects. When a 1:2.5 dilution of plasma was used (200 ~1 of sample in a final volume of 500 ,pl), calcitonin was apparently detectable in most normal subjects (Fig. 2). However, to preclude any nonspecific displacement of tracer from antibody (which can be introduced into immunoassays by high concentrations of plasma proteinlg), the maximum concentration of plasma sample in the assay incubations was kept at 20% (100 ~1 of sample in 500 ~1 final volume). Since scatter occurs at the low-concentration end of immunoassay curves,zo,B1 only those samples whose mean concentrations of calcitonin was significantly (p < 0.05) different from the mean (lo-15 determinations) of the zero point of the standard curve by Student’s t test were considered as detectable (Fig. 2).21 These criteria meant that under optimal conditions, 100 pg of calcitonin per milliliter of plasma could be measured in the assay. Figures 1 and 2 illustrate that, under these conditions, the majority of normal subjects had undetectable concentrations of calcitonin (less than 100 pg per milliliter) but that some normal subjects did have apparently detectable concentrations of the hormone, However, the apparent concentration of calcitonin was so low in the plasma of most normal subjects

with detectable

levels that the progressive

Fig. Z.-Displacement of labeled human calcitonin from specific antibody by loo-p1 and 200-~1 aliquots of plasma from normal adults in basal (fasting) state. Apparent displacement of tracer by 200-~1 samples is nonspecific and probably due to high concentration of plasma protein (40%) in incubation mixture of these samples. Only three subjects have detectable concentrations (significantly different from zero by Student’s t test) when loo-/11 aliquots of plasma are used, but this displacement of tracer also appears to be artefactual.

displacement

of tracer

from

1132

DEFTOS ET AL.

antibody by these samples, indistinguishable from the displacement produced by authentic human calcitonin, did not exceed 20%, a minimum value for significant tracer displacement suggested by Berson and Yalow.20 Therefore, the plasma samples from normal subjects that were available in sufficient quantity and that had apparently significant concentrations of calcitonin by statistical criteria (Student’s t test) but did not displace more than 20% of tracer from antibody were assayed before and after charcoal adsorption. With this treatment, up to 50,000 pg of calcitonin per milliliter of plasma could be removed from plasma. Suitable samples from patients with medullary thyroid carcinoma and plasma samples enriched with synthetic human calcitonin were also treated similarly with charcoal. Table 1 and Fig. 3 illustrate results of such charcoal treatment. When the samples from patients with medullary thyroid carcinoma and the calcitonin-enriched samples were assayed before and after charcoal treatment, there was a striking decrease in the calcitonin concentration measured in the immunoassay. However, when the plasma samples from normal subjects with “detectable calcitonin were assayed before and after charcoal adsorption, there was no significant decrease in apparent calcitonin concentration, despite the fact that there was a large excess of charcoal sufficient to adsorb all apparent calcitonin. We concluded the apparent displacement of tracer produced by these samples (open circles in the normal category in Fig. 1) was nonspecific and did not represent calcitonin. Figure 1 also shows that most of the patients with hypercalcemia also had undetectable plasma calcitonin concentrations (less than 100 pg per milliliter) not differing from the normal subjects. Even when thyroid venous samples were used, a large percentage of patients with hypercalcemia had less than 100 pg Table l.-Calcitonin Concentration (pglml) in Human Plasma Samples Before and After Charcoal Adsorption to Remove CaIcitonin* Control

After Charcoal Adsorption

Decrease in Calcitonin

Normal Human Plasma 1

4 5 6 Calcitonin-rich 1 2 3 4 5 6 7

256 110

120 126 278 235 266 390

2 3

245 322 162 540

0 0 0 0

100 0

Plasma

56,500 > 6,400 3,200 800 200 100 50

< < < < <

425 724 50 50 50 50 50

56,000 6,200 3,200 800 200 100

50

* Calcitonin-rich plasma samples (samples from medullary thyroid carcinoma patients and samples to which synthetic cakitonin was added) showed decreases in calcitonin concentration. Normal human plasma samples with apparently detectable concentrations of calcitonin did not show any decrease in calcitonin following charcoal adsorption, suggesting that displacement of labeled cakitonin from antibody by these samples was nonspecific and did not represent authentic human calcitonin.

IMMUNOASSAY

,u/

80

60

FOR HUMAN

CALCITONIN.

1133

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/u/

PLASMA

PLASMA

THYROID CARCINOMA CONTROL POST CHARCOAL ADSORPTION

0

POST CHARCOAL ADSORPTION

CAL C/ TO/V/N, pg / m I Fig. 3.-Effect of charcoal absorption on immunoassayable calcitonin concentration in plasma sample from patient with medullary thyroid carcinoma and on apparent immunoassayable calcitonin concentration in plasma sample from normal adult. Curved line: standard curve of tracer displacement produced by human calcitonin assay standard. Charcoal absorption removes all calcitonin activity from medullary thyroid carcinoma plasma sample and eradicates progressive displacement of tracer from antibody produced by increasing aliquots of this plasma sample before charcoal absorption. Charcoal absorption has no significant effect on displacement of tracer produced by aliquots of plasma from normal adult. Because of sensitivity of assay, this latter sample can be estimated to have calcitonin concentration of 200 pg per milliliter despite its only slight displacement of tracer. Since this displacement of tracer cannot be eradicated by charcoal absorption as in medullary thyroid carcinoma plasma sample, it is probably artefactual and does not represent authentic human calcitonin. per milliliter of calcitonin. Sufficient sample from thyroid veins was not available for charcoal adsorption. When the apparent calcitonin concentration in

four thyroid vein samples from two patients with hyperparathyroidism

(open

circles in the thyroid vein category of Fig. 1) and in three thyroid vein samples from a patient with idiopathic hypercalcemia (open squares Fig. 1) was compared to the apparent calcitonin concentration in a matched peripheral sample, it was found that there was no marked difference in calcitonin concentration between the thyroid venous and the peripheral sample. This is in contrast to the 20- to 30-fold gradient in calcitonin between porcine peripheral and thyroid venous blood4 and suggests that the apparent concentration of calcitonin in thyroid venous samples from humans was also artifactual. A thymic vein sample from the patient with hypercalcuria (open triangle Fig. 1) similarly had an apparent concentration of calcitonin not markedly different from a matched peripheral sample. Figure 4 summarizes the changes in plasma calcitonin concentrations found in 13 patients with medullary thyroid carcinoma before and after calcium, gluCalcium infusion led to an increase in the pecagon, and EDTA infusions. ripheral concentration of calcitonin in every patient. EDTA-induced hypo-

DEFTOSET AL. EDTA

CALCIUM

Fig. 4.-Changes in calcitonin concentration in patients with medullary thyroid carcinoma after calcium, glucagon, and EDTA infusion. Maximum change that occurred after infusion was started is represented. Each change illustrated was statistically significant by Student’s t test (P < 0.05).

calcemia suppressed the secretion of calcitonin in the four patients tested. Glucagon, however, had a variable effect on calcitonin concentration. In each patient the induced concentration of calcitonin was significantly different (p < 0.05) from the basal concentration by Student’s t test. Table 2 compares the concentration of calcitonin in four normal human hemithyroid glands to the calcitonin concentration of four porcine thyroid glands.4 This concentration of calcitonin in the human glands is several orders of magnitude less than that detected in the thyroid gland of the porcine species by immunoassay. DISCUSSION Elevated concentrations of calcitonin have been demonstrated by both bioassay and immunoassay in the thyroid gland and in the peripheral blood of patients with medullary carcinoma of the thyroid, a tumor often associated with multiple endocrine neoplasias.j-1°J2 The current studies confirm these observations (Fig. 1). Calcium uniformly stimulated the release of calcitonin in every patient with medullary thyroid carcinoma in this study, and EDTA-induced Table

Z.-Calcitonin Thyroid

Concentration (rig/g Tissue f SE) of Human Glands as Measured by Specific Immunoassays*

and Porcine

Human thyroid glands 1 2 3 4 5 Porcine 1 2 3

58.96 2.35 20.12 59.18 1.88 thyroid

(L (c (c (k (k

9.3) 0.6) 2.7) 2.8) 0.2)

glands 35,500 (5 3800) 49,600 ( rt 4600) 21,200 (? 1900)

* Calcitonin was extracted from thyroid glands with dilute hydrochloric represents mean of at least five determinations.

acid.4 Each value

IMMUNOASSAY

FOR HUMAN

CALCITONIN.

II

1135

hypocalcemia suppressed the release of calcitonin from these tumors. Glucagon has been reported to have a more variable effect on calcitonin secretion in medullary thyroid carcinoma. 6 The present results indicate that glucagon can even suppress the release of calcitonin from these tumors and should therefore not be considered a reliable provocative test of calcitonin secretion from this tumor.8 These observations demonstrate that control of calcitonin secretion in medullary thyroid carcinomas is not autonomous but responds to alteration in serum calcium. The tumors not only release hormone with calcium challenge but can be suppressed by hypocalcemia (Fig. 4). Although calcitonin can be readily detected in the plasma of patients with medullary thyroid carcinoma, secretion of calcitonin in the basal state by normal humans has not been definitely established. Estimates by bioassay have ranged from 200 to 1700 pg per milliliteP”-sz3 but artifacts have been demonstrated in these methods.“” Tashjian et al. reported basal concentrations in normal subjects of 20-400 pg per milliliter of plasma using an immunoassay of sensitivity comparable to the one described in this report.” These workers do not discuss the statistical criteria they used to determine if a sample has detectable calcitonin (significantly differs from zero). A concentration of plasma as high as 40% 200-~1 sample in 500 ~1 final volume) was used in their assay. This concentration of plasma sample, and even less, is known to introduce artifacts into immunoassay systems (Figs. 1 and 2), and no attempts to control for such artifacts (Table 1) are indicated .10-21Assay of thyroid effluent blood for normal thyroid extract is not reported. The results of the present study suggest that in most normal adults and in adults with chronic hypercalcemia, calcitonin does not circulate at peripheral concentrations greater than 100 pg per milliliter of plasma in the basal (fasting) state (Figs. 1 and 2). The circulating peripheral level of hormone is probably even much less, since no hormone was detected even in thyroid effluent plasma of most of the patients tested, despite the presence of hypercalcemia (Fig. 1) . These observations are consistent with the low concentration of calcitonin found by both bioassay and immunoassay in human thyroid glands (Table 2) when compared to other mammalian thyroids and by the difficulty with which C cells are identified in normal human thyroids.““*?” Tashjian et al6 stated the calcium infusions resulted in a two- to threefold increase in calcitonin in 60% of normal subjects studied. In preliminary studies with this immunoassay, calcitonin appears to become detectable in the peripheral plasma of some subjects other than those with medullary thyroid carcinoma during provocative tests, lo but because of the methodologic considerations discussed above, further careful and rigorously controlled studies are necessary to’ confirm these observations. The reasons for the discrepancy between the current results in normal subjects and those of Tashjian et al. are not absolutely clear, although the differences could largely be explained by the methodologic considerations discussed above. A differing specificity of antibodies cannot be ruled out.27 It may be that the antibody used in this assay system reacts with a labile region of the calcitonin molecule, but the stability of immunoassayable calcitonin in the present assay system makes this unlikely.13

1136

DEFTOSETAL.

The significance of calcitonin in adult humans in calcium and skeletal homeostasis remains to be established. At present, the assay does seem useful in diagnosis and evaluation of success of therapy in patients with medullary carcinoma of the thyroid.5-8 For support of the thesis that calcitonin plays a hormonal role in man, it is essential to demonstrate secretion of the hormone during basal conditions or in response to physiologic stimuli. With the present highly sensitive assay for human calcitonin, no hormone could be unequivocally detected in peripheral or thyroid effluent blood of adults even with the stimulus of hypercalcemia. The hormone may be secreted at concentrations or in a form not detectable by the present assay. If the former is true then at least it must be observed that calcitonin concentrations are much lower than those found by immunoassays of comparabIe sensitivity in the porcine, bovine, rabbit, and salmon species.1*‘~4~g*10 Increased secretion of calcitonin in man may occur in response to factors other than hypercalcemia such as gastrointestinal hormones,4qgJo or calcitonin may be a developmental hormone in man, participating in calcium and skeletal metabolism only in infants and children. The development of even more sensitive immunoassays for human calcitonin will be necessary to study calcitonin secretion further and thereby assist in defining the significance of calcitonin in human physiology. ACKNOWLEDGMENT The following physicians kindly supplied some of the plasma samples used in these studies: A. D. Goodman (Albany, N.Y.), B. D. Weintraub (Bethesda, Md.), K. Engelman (Philadelphia, Pa.), A. L. Steiner, (Albany, N.Y.), R. G. Wieland (Cleveland), F. M. Chu (Hong Kong), J. L. Doppman (La Jolla, Calif.), T. M. Deftos (Athens), and H. B. Wilson (London). REFERENCES 1. Deftos, L. J., Lee, M. R., and Potts, J. T., Jr.: A radioimmunoassay for thyrocalcitonin. Proc. Nat. Acad. Sci. U. S. A. 60:293, 1968. 2. Lee, M. R., Deftos, L. J., and Potts, J. T., Jr.: Conrol of secretion of thyrocalcitonin in the rabbit as evaluated by radioimmunoassay. Endocrinology 84:36, 1969. 3. Deftos, L. J., and Potts, J. T., Jr.: Radioimmunoassay for parathyroid hormone and calcitonin. Brit. J. Hosp. Med. Nov. 1969, p. 1813. 4. Cooper, C. W., Deftos, L. J., and Potts, J. T., Jr.: Direct measurement of in vivo secretion of pig throcalcitonin by radioimmunoassay. Endocrinology 88:747, 1971. 5. Clark, M. B., Byfield, P. G. H., Boyd, G. W., and Foster, G. V.: A radioimmunoassay for human calcitonin. Lancet 2:74, 1969. 6. Tashjian, A. H., Jr., Howland, B. G., Melvin, K. E. W., and Hill, C. S., Jr.:

Immunoassay of human calcitonin. New Eng. J. Med. 283:890, 1970. 7. Deftos, L. J., and Potts, J. T., Jr.: Plasma calcitonin measurements in medullary thyroid carcinoma and disorders of calcium metabolism. Clin. Res. 18: 673, 1970. 8. -, Goodman, A. D., Engelman, K., Bury, A. E., and Potts, J. T., Jr.: Suppression and stimulation of calcitonian secretion in medullary thyroid carcinoma. Metabolism 20:428, 1971. 9. Potts, J. T., Jr., Keutmann, H. T., Deftos, L. J., and Niall, H. D.: The chemistry and immunochemistry of calcitonins. In Lande, S. and Weinstein, B. (Eds.): Peptides: Chemistry and Biochemistry. New York, Gordon and Breach. In press. 10. Deftos, L. J., et al.: Radioimmunoassays for parathyroid hormones in calcitonins. In Proceedings of Fourth International Parathyroid Conference. Amsterdam, Excerpta Medica, 1971. In press. 11. Cooper, C. W., and Deftos, L. J.:

IMMUNOASSAY

FOR HUMAN

CALCITONIN.

Evaluation of thyrocalcitonin in pig thyroid vein blood following oral administration of calcium. Fed. Proc., 29:253, 1970. 12. Potts, J. T., Jr., and Deftos, L. J.: Parathyroid hormone, thyrocalcitonin, vitamin D, bone and bone mineral metabolism. In Bondy, P. K. (Ed.): Duncan’s Diseases of Metabolism. Philadelphia, W. B. Saunders, 1969, p. 904. 13. Deftos, L. J.: An immunoassay for human calcitonin: I. The method. Metabolism 20:1122, 1971. 14. Hunter, W. M., and Greenwood, F. C.: Preparation of iodine-131 labeled human growth hormone of high specific activity. Nature (London) 194:495, 1962. 15. Thomas, K., and Ferin, J.: A new rapid immunoassay for HCG in plasma using dioxan. J. Clin. Endocr. 28:1667, 1968. 16. Reitz. R. E., et al.: Localization of parathyroid adenomas by selective venous catheterization and radioimmunoassay. New Eng. J. Med. 281:348, 1969. 17. Hill, J. B.: Automated fluorometric method for determination of serum calcium. Clin. Chem. 11: 122, 1965. 18. Zabusky, R., and Herbert, V.: Isotope-dilution methods using coated charcoal. In Hayes, R. L., Goswitz, F. A., and Murphy, B. E. P. (Eds.): Radioisotopes in Medicine: In vitro studies. U.S. Atomic Energy Commission, 1968, p. 395. 19. Berson, S. A., Yalow, R. S., Glick,

II

1137 S. M., and Roth, J.: Immunoassay of protein and peptide hormones. Metabolism 13:1135, 1964. 20. -, and -: Radioimmunoassay of ACTH in plasma. J. Clin. Invest. 47:2725, 1968. 21. Rodbard, D., Rayford, P. L., Cooper, J. A., and Ross, G. T.: Statistical quality control of radioimmunoassay. J. Clin. Endocr. 28:1412, 1968. 22. Sturtridge, W. C., and Kumar, M. A.: Assay of calcitonin in human plasma. Lancet, 1: 725-726, 1968. 23. Gudmundsson, T. V., Woodhouse, N. J. Y., and Osafo, T. D.: Plasma calcitonin in man, Lancet 1:443, 1969. 24. Bell, P. H., Dziokowski, C., and Barge, W. F., Jr.: Plasma calcitonin in man. Lancet 2:104, 1970. 25. Aliapoulios, M. A., and Rose, E. H.: Specific localization of calcitonin activity in human thyroid glands. In Taylor, S. and Foster, G. (Eds.): Calcitonin. New York, Springer-Verlag, 1970, p. 295. 26. Kalina, M., Foster, G. V., Clark, M. B., and Pearse, A. G. E.: C cells in man. Zn Taylor, S. and Foster, G. (Eds.): Calcitonin. New York, Springer-Verlag, 1970, p. 268. 27. Cargille, C. M., Rodbard, D., and Ross, G. T.: Radioimmunoassay of human follicle stimulating hormone: Bias due to antisera. J. Clin. Endocr. 28:1276, 1968.