ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 16, No. 2 Copyright 1986, Institute for Clinical Science, Inc. Serum Calcitonin in Thyroid Disorders in Pheochromocytoma Kindred* F. J. H ORNICEK, P H.D.,t$ T. S. DANOWSKI, M.D., " S. M. ROBINSON, B.S., K. T. SW EEN EY, G. I. MALININ, P h.d. 11 tsurgical Research Laboratories, Department of Surgery (R-12), University of Miami School of Medicine, Miami, FL 33101 Department of Medicine, Shadyside Hospital, Pittsburgh, PA 15232 University of Pittsburgh School of Medicine, IDepartment of Medicine, Pittsburgh, PA 15121 HDepartment of Physics, Georgetown University, Washington, DC 20057 ABSTRACT Serum calcitonin was determ ined by RIA in 59 healthy subjects (Group 1), 49 romly selected patients with treated or untreated thyroid disorders (Group 2), in 12 kindred of a pheochromocytoma index case (Group 3). A lthough m ost subjects in Group 2 had norm al calcitonin levels, there w ere significant (p < 0.001) differences betw een all three groups. O f the five patients in Group 2 with high serum calcitonin, one had medullary cancer of the thyroid, one had m ultiple endocrine neoplasia, one had acromegaly, two rem ained undiagnosed. Increased serum calcitonin levels w ere also found in seven of 12 normotensive relatives of a patient w ith pheochrom ocytom a. It is therefore concluded that high serum calcitonin levels in patients with thyroid disorders strongly suggest the presence of C-cell neoplasia or medullary cancer of the thyroid. * Aided by grants from the Addison H. Gibson Foundation; Mr. Mrs. John Redfern; Pennsylvania Lion s Sight Conservation Eye Research Foundation, Inc. t Address reprint requests to Dr. Francis J. Hornicek, Surgical Research Laboratories, Departm ent of Surgery (R-12), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101. Introduction E le v a te d h u m an calcito n in (hct) levels in the thyroid in the systemic circulation are frequently associated with medullary thyroid cancer (MTC).7,8,910 12 103 0091-7370/86/0300-0103 $00.90 Institute for Clinical Science, Inc.
104 HORNICEK, DANOWSKI, ROBINSON, SWEENEY, AND MALININ It was also rep o rted that elevated calcitonin levels could be associated with non-thyroid carcinom as.11 Nonetheless, the normal serum calcitonin range is not well defined, owing to w ide calcitonin fluctuations ranging from undetectable levels to 580 pg p er m l.19 For instance, basal serum calcitonin levels m easured by radioim m unoassay (RIA) in a large group of healthy adults w ere less than 250 pg per ml in 25 percent, below 100 pg p er ml in 75 p ercen t of tested subjects.21 The immunological heterogeneity of hct probably accounts for some of th e re p o rte d variances in h ealth y hum ans.34 13'14151617 Therefore, additional data contributing to the definition of normal hct levels are useful. In this communication are reported the results of hct determ inations by RIA in normal hum an sera, in sera of p atien ts w ith thyroid disorders, in relatives of a patient with pheochromocytoma. M aterials Methods Fifty-nine healthy hum an volunteers (Group 1), 49 patients with thyroid disorders (Group 2), 12 healthy relatives of a patient with pheochromocytoma (Group 3) w ere used in this study. Serum calcitonin levels w ere m easured in an endocrine survey. The endocrine indices in th e h ealthy k indred of the pheochrom ocytom a p atien t included: thyroxine, triiodothyronine, oral glucose tolerance test, follicle stim ulating horm one, th y ro id stim u latin g horm one, luteinizing horm one, prolactin, insulin, grow th horm one, gastrin, catecholamines, vanillymelic acid. E ig h t- to ten -p o u n d albino rabbits were purchased.* Synthetic hc T,t 125Ilabeled hum an calcitonin (sp. act 0.3 mci per pg [1.3 X 10~7 Bq per pg]),$ * Hilltop Animal Laboratories, t Ciba-Geigy. Í Immuno Nuclear Corp. kallikrein trypsin inhibitor (Trasylol) w ere obtained. Antibodies to hct w ere supplied. Calcitonin free sera, $ Norit A charcoal,h Dextran 250** were purchased. A n t ib o d y P r o d u c t io n A ntibodies to hc T w ere induced in rabbits using conventional procedures.18- Briefly, rabbits received weekly subcutaneous injections of 0.5 mg synthetic hc T d isso lv ed in 0.001 N HC1 e m u lsified w ith an e q u al v o lu m e of Freunds adjuvant, until sufficiently high titer (1:10000) of antibodies was detected by RIA. R a d io im m u n o a s s a y f o r H u m a n C a l c it o n in in S e r a Radioimmunoassay of hct was p erformed using stard RIA m ethod18 22 as follows: 1 0 0 jjli of stards, ranging from 10 to 1500 pg per ml of synthetic hct, serial dilutions of samples w ere pipetted into ice-cold tubes. Following the 1 0 0 il addition of the antibody, the tubes w ere vortexed then refrigerated for three days. Thereafter, 100 xl of 125I-la b e le d hc T w ere ad d e d to th e tubes. The solutions w ere mixed the tubes refrigerated for an additional three days. The antigen-antibody complex was separated from the labeled antigen-antibody by dextran-charcoal suspension as described elsew here.2'6 Briefly, a five percent w/v N orit A charcoal 0.5 percen t w/v dextran in 0.13 M borate buffer w ere diluted 1:25 with a 0.05 M tris H C l buffer. T he tris-h C l buffer (ph -7.5) contained 10 p e rcen t hum an se ru m from an a th y ro tic o r n o rm al p a tie n t (i.e., one w ith u n d e te c ta b le FBA Pharmaceuticals. *Calbiochem. 11Amend Drug & Chemical Co., Inc. ** Pharmacia Fine Chemicals, Inc.
hct), HC1, tris (hydroxymethyl) aminoethane, 5000 KI units of Trasylol per 10 ml. The diluted dextran-charcoal suspension was stirred for one hour then 1.0 ml of the suspension was p ipetted into each tube. All tubes were then centrifuged at 4 C for at least 30 m inutes at 3000 rp m. T h e ra d io a c tiv ity o f th e supernatants precipitates was m easured in a gam m a scintillation counter for a tim e sufficient to insure a counting accuracy of m ore than 2 percent. The percent of bound calcitonin in each sample was then calculated as follows: % bound calcitonin = From these data, a stard curve was plotted (10 1000 pg per ml) the calcitonin concentrations in the test samples determined. Calcitonin levels ranging from undetectable to 200 pg per ml were considered to fall within the normal range. Statistical Analysis of Data Statistical significance of RIA data was evaluated by conventional m ethods CALCITONIN LEVELS IN HUMAN SERA 105 1000 (Wicoxon s, Kruskal-Wallis, D unn s tests). Non p aram etric m ethods w ere used on account of the markedly skewed data, a high degree of differences in variances (B artlett s test; p < 0.001). O nly th e em p lo y m en t of logarithm ic transformation20 stabilized the variances resulted in an approximately normal distribution of data points (Kolmogorov- Sm irnov goodness of fit te s t ; p < 0.025). Results Serum calcitonin (hct) levels w ere m easured by RIA in healthy male cpm of precipitate X 100% total cpm of precipitate + supernatant female adults, in patients with a variety of thyroid disorders, kindred of a p a tie n t w ith pheochrom ocytom a. As show n in figure 1, th e average hc T levels for th ese groups w ere 40, 116, 108, 248, while the m edian values w ere 12, 15, 48, 255 pg p e r ml, resp ectiv ely. T h e d ifferen ce in hc T lev els b e tw e e n m ales (n = 17) females (n = 42) w ere not statistically 500 E a a. 250 F ig u r e 1. Serum hct levels were m easured in -JOO four groups of subjects. O E ach data p o in t is th e _ mean of three values. The 3 average m edian of g these points is shown by 0) ------... respectively. *Exclusive of propos- 25 itus. 10 MEN WOMEN THYROID PHEOCHROMOCYTOMA CO N TRO LS DISORDERS KINDRED *
106 HORNICEK, DANOWSKI, ROBINSON, SWEENEY, AND MALININ significant, although significant differences b e tw e e n th e young m ales fem ales th e old m en w om en have been reported.1 Therefore, calcitonin values from the normal subjects of both sexes w ere pooled to comprise the c o n tro l g ro u p. C om p ariso n of th e se three groups of subjects indicated significant differences betw een hct levels of all groups. (Kruskal-Wallis one-way classification; p < 0.001). The respective m ean ranks of th e pheochrom ocytom a kindred, thyroid control groups, w ere 90.6, 66.2, 49.7, respectively. Using these values all three groups were found to differ from each other at the p < 0.05 level of significance (Dunn s m ultiple comparison.) The diagnoses of patients with thyroid disorders are sum m arized in table I. The endocrine indices in the kindred of the p h eo ch ro m o cy to m a p a tie n t w ere all w ithin norm al range, except for elevated serum calcitonin in seven out of 12 subjects. In the group of 49 patients with thyroid disorders, serum calcitonin levels w ere within normal range in 44 patients. In the rem aining five, one was found to have m edullary cancer of the thyroid, one had m ultiple endocrine neoplasia, one had acrom egaly, two rem ain undiagnosed. Exclusive of the index case, calcitonin levels w ere distinctly increased in seven out of 12 kindred members of a patient T A B L E I Status of Patients with Thyroid Disorders* (Group 2) Multiple endocrine neoplasia 2 Acromegaly 1 Hyperthyroidism treated or untreated 5 Hypothyroidism or myxedema treated or untreated 13 Goiter - euthyroid 7 Non-toxic adenoma 7 Thyroiditis treated or untreated 3 Cancer of thyroid 4 Medullary cancer of thyroid 2 Hypercalcemia 3 Renal calculi 3 Miscellaneous 7 Includes multiple disorders in some patients with pheochromocytoma (figure 2). The endocrine indices w ere otherw ise normal in all m em bers of this group. Discussion O ur data indicate that m ost patients u nder treatm ent for thyroid disorders had norm al serum calcitonin levels as was the case with the majority of control subjects (figure 1). However, in a group of 49 such patients, five subjects w ere found to have above normal serum calcitonin concentrations. These data seem to suggest that thyroid abnorm alities are not associated per se with the changes in se ru m c a lc ito n in le v e ls. H o w ev er, patients with thyroid abnormalities an elevated serum calcitonin probably are in a high risk group with respect to F ig u r e 2. Pedigree o f t h e p a t i e n t w i t h p h e o c h r o m o c y to m a! Shaded areas re p re sen t e l e v a t e d s e r u m h C T betw een 200 726 pg per ml. *Index Case. ( ) Age. (9) (6 ) (2) (11) (10) (6) 355 386 315
m edullary carcinom a of th e th y ro id, m ultiple endocrine neoplasia, or other disorders attributable to calcitonin-producing C-cells. In general, patients with MTC had hc T levels twice above the norm al level.5 O ur findings pertaining to the relatives of th e pheochrom ocytoma index subject seem to indicate that serum calcitonin may be increased in a majority of the kindred. References 1. B o d y, J. J. H e a t h III, H. : Estimates of circulating monom eric calcitonin: physiological studies in normal thyroidectomized man. J. Clin. Endocrinol. Metab. 57:897 903, 1983. 2. C l a r k, M. B., B y f i e l d, P. G. H., B o y d, G. W., F o s t e r, G. V.: A radioim m unoassay for human calcitonin M. Lancet 2:74-77, 1969. 3. C o o m b e s, R. C., H i l l y a r d, C., G r e e n b u r c, P. B., M a c I n t y r e, I. : Plasma-immunoreactive-calcitonin in patients with non-thyroid tumours. Lancet 1:1080-1083, 1974. 4. D e f t o s, L. J.: Immunoassay for hum an calcitonin. I. M ethod. Metabolism 20:1122-1128, 1971. 5. D e f t o s, L. J., Roos, B. A., B r o n z e r t, D., P a r t h e m o r e, J. G.: Immunological heterogeneity of calcitonin in plasma. J. Clin. Endocrinol. Metab. 40:409-412, 1975. 6. D i e t r i c h, F. M. R i t t e l, W.: Antigenic site(s) of synthetic human calcitonin M. Nature 225:75-76, 1970. 7. E k e l a n d, A., G a u t v ik, K. M., U n d e r d a l, T.: Calcitonin producing tumour. Effects on fractu re rep air norm al bone in rats. Acta Orthop. Sc. 54:760-767, 1983. 8. E m m e r t s e n, K., N i e l s e n, H. E., M o s e k i l d e, L., H a n s e n, H. H. : N o effect o f Cimetidine on calcitonin secretion from medullary thyroid carcinoma. Acta M ed. Sc. 207:367-369, 1980. 9. G a c e l, R. F., Z e y t i n o g l u, F. N., V o e l k e l, E. F., T a s h j i a n, A. H.: Establishment of a calcitonin-producing rat medullary thyroid carcinoma cell line. II. Secretory studies of the tu m o r cells in cu ltu re. E ndocrinology 107:516, 1980. CALCITONIN LEVELS IN HUMAN SERA 107 10. H i r s c h, P. F. M u n s o n, P. L.: Thyrocalcitonin. Physiol. Rev. 49:548-622, 1969. 11. H o m, D. W a x m a n, K.: Calcinonin as a tumor marker for nonthyroid neoplasia. J. Surg. Oncol. 29:59-60, 1985. 12. N o r t o n, J. A., D o p p m a n, J. L., B r e n n a n, M. F.: Localization resection of clinically inapparent medullary carcinoma of the thyroid. Surgery 87:616 622, 1980. 13. S a m a a n, N. A., H i l l, C. S., B e c i e r o, J. R., S c h u l t z, P. N.: Im m unoreactive calcitonin in medullary carcinoma of the thyroid in maternal cord serum. J. Lab. Clin. Med. 81:671-681, 1973. 14. S il v a, O. L., S n i d e r, R. H., B e c k e r, K. L.: R adioim m unoassay of calcitonin in hum an plasma. Clin. Chem. 20:337 339, 1974. 15. S i n g e r, F. R. H a b e n e r, J. F.: M ultiple im munoreactive forms of calcitonin in hum an plasm a. Biochem. Biophys. Res. C om m un. 61:710, 1974. 16. S i z e m o r e, G. W., V a y, L. W. G., K a p l a n, E. L., S a n z e n b a c h e r, L. J., H o l t e r m u l l e r, K. H., A r n a u d, C. D. : Relations of calcitonin gastrin in the Zollinger-Ellison syndrom e medullary carcinoma of the thyroid. New Engl. J. Med. 288:641-648, 1973. 17. T a s h j i a n, A. H. T., H o w l a n d, B. G., M e l v i n, K. E. W. N., H i l l, C. S.: Immunoassay of human calcitonin: clinical measurement, relation to serum calcitonin studies in patients with m edullary carcinom a. New Engl. J. M ed. 283:890-895, 1970. 18. T a s h j i a n, A. H. V o e l k e l, E. F.: Human calcitonin: Application of affinity chromatography. Methods of Hormone Radioimmunoassay, New York, Academic Press, 1974, pp. 119 214. 19. T a s h j i a n, A. H., W o l f e, H. J., V o e l k e l, E. F.: Hum an calcitonin. Immunologic assay, cytologic localization studies on medullary thyroid carcinoma. Am. J. Med. 56:840 849, 1974. 20. W in e r, B.: Statistical Principles in Experimental Design. New York, M cgraw-hill Book Co., 1971. 21. W r ig h t, D. R., V o e l k e l, E. F., T a s h jia n, A. H.: M easurem ent of hum an calcitonin by affinity chromatography radioimmunoassay. Hbook of RIA, 1977, p. 391. 22. Ya l o w, R. S. B e r s o n, S. A.: Radioimmunoassays. Statistics in Endocrinology. M c A r t h u r, J. W., C o l t o n, T., eds. Cam bridge, MA, The MIT Press, 1970, pp. 327-344.