429 Horm. Metab. Res. 7 (1975) 429-432

© Georg Thieme Verlag Stuttgart

Plasma Immunoreactive Calcitonin Levels in Pregnant Mares and Newborn Foals J.-M. Garei! . W. Martin-Rosset 2 and J.-P. Barlet 2 ! Laboratoire de Physiologie COmparEle. Universite Paris VI. 9. quai St Bernard. Paris Cedex 05. and 2 1.N .R.A . Theix. Beaumont. France

at the end of pregnancy and at the time of parturition in mares and their foals. Material and Methods

Animals: Nine 7-year-old Comtoise mares carrying their 3rd or 4th pregnancy were used. These animals were kept outdoors all the time. During the experimental period they stayed in a paddock. They were fed hay ad libitum and each received In Q-day and 7-day-old foals plasma calcium levels were signi- 3.5 kg of concentrate daily. After foaling, foals stayed with their dams and suckled maternal milk. ficantly higher than in their mothers, but plasma calcitonin levels were not significantly different from those observed Blood sampies were obtained by puncture from a jugular in their dams at the time of parturition. vein. They were collected in heparinized tubes containing a peptidase inhibitor (Iniprol, Laboratoires Choay, Paris) to Key-Words: Calcium - Calcitonin - Horse - Newborn prevent any hormone degradation. After centrifugation plasPregnancy - Parturition ma sampies were kept at -200 C for subsequent analysis.

Introduction Calcitonin has been shown to exert a physiological role against post-prandial hypercalcemia (Gray and Munson 1969, Care, Bates, Swaminathan and Ganguli 1971, Cooper, Schwesinger, Mahgoub, Ontjes, Gray and Munson 1972). It has been suggested that CT may protect the skeleton du ring pregnancy and lactation (Konopka, Klotz and Delorme 1971, Lewis, Raf [erty, Shelley and Robinson 1971). In goats fed high or low calcium diets during pregnancy and lactation, CT protects the skeleton of the female against an excessive demineralization (Barlet and Garel 1975). In sheep, pregnancy is associated with plasma CT levels higher in pregnant animals than in control ewes (Garel, Care and Barlet 1974, Barlet and Garel 1975). In cows afflicted with parturient paresis a simultaneous decrease in plasma calcium and increase in plasma CT is observed at the time of parturition (Bar/et 1969, Black and Capen 1973, Garel and Barlet 1975). On the contrary parturient paresis is very uncommon in ewes (Braithwaite, Glascodc and Riazuddin 1970), and at the time of lambing there is no increase in plasma CT levels and no variation in plasma calcium concentration (Barlet and Garel 1975). Using aporeine radioimmunoassay which has been al ready described in details (Gare/, Care and Barlet 1974, Garel, Savajol and Bar/et 1975) and an antibody which cross-reacts significantly with horse CT, we have measured plasma immunoreactive CT levels Received: 20 Apr. 1975

Accepted: 18 June 1975

Plasma analysis: Plasma calcium and plasma magnesium concentrations were estirnated by atomic absorption spectrophotometry (Perkin-Elmer 303). Plasma inorganic phosphorus levels were estimated colorimetrically (Kalekar 1947).

Plasma CT concentrations were measured by radioimmunoassay (Garei, Care and Barlet 1974, Garei. Savajol and Barlet 1975). Porcine CT antiserum (GP2 s ) used at a final dilution of 1: 3000 was shown to cross-react significantly with horse CT (Fig. 1). Purified horse CT was a gift from Professor G. Milhaud and the biological potency of this preparation estimated by rat bioassay was 50 MRC units/mg

o ,. . ~~.==~p;.:;.'::O~s=:==:~o~'1=:==:===.----::'0'2 mU o 200 400 600 800 1CXXJ 1200 1400 pg _L..'

Fig. 1. Cross-reactivity of antiporcine calcitonin antiserum from guinea-pig no 2 (GP2, fifth bleeding) with purified horse ca1citonin (........, unlabelled porcine calcitonin in pg; 0- - - 0, unlabelled horse calcitonin in MRC mU).

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Summary Plasma calcium and calcitonin levels were measured periodically during the two last months of pregnancy and at the time of parturition in 9 pregnant mares and their foals. In pregnant animals, there was an increase in plasma calcitonin levels in the days before parturition, wh ich was not due to any change in plasma calcium. This result indicates that in the mare, as in the cow, in the days before parturition CT secretion escapes from its control by plasma calcium.

I.M. Garei, W. Martin-Rossert and 1.-P. Bartlet

(Raulais, Merle and Milhaud 1974). Porcine CT (Lot K600072 F-2; 116 MRC units/mg; Armour Pharmaceutical Co; Kankakee, Illinois, USA) 13'I-labelled was supplied by the Centre National de Transfusion Sanguine (Paris, France). Porcine CT (Lot K600-072 F-2; 116 MRC units/mg) was used as a standard. Bound and free CT were separated after 6 days of incubation in an equilibrium system by talcum powder as aiready described (Garei, Savajol and Barlet 1975). The sensitivity of the technique was 100 pg porcine CT/ml of plasma and allowed CT to be measured in 50 [J.l duplicate sampies. The results were expressed in ng/ ml equivalents of porcine CT and given as means ± S.E.M. Statistical analysis was done by impaired Student's t-test.

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In pregnant mares, the mean of plasma CI levels increased significantly between -50 and -25 days before parturition (P < 0.05) ~nd then decreased between -25 and -15 days (P < 0.05). A sharp increase in plasma CI levels was observed between -15 and -5 days before parturition (P < 0.001). At the time of parturition plasma CI levels became more unstable (Fig. 2).

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Days be fore or af/er parfurifion Fig. 3. Developmental changes in plasma concentrations of calcitonin (...........). calcium (e - - - - e), inorganic phosphorus (0 ........ 0) and magnesium (O--C) in two pregnant mares showing an obvious increase in plasma calcitonin level at the time of parturition.

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In some pregnant mares there was an obvious increase in plasma CI levels in the five days before and at the time of parturition (Fig. 3). Ihis elevation was associated with a simultaneous decrease of plasma calcium and plasma inorganic phosphorus levels (Fig. 3). In one of these animals plasma magnesium concentration was also decreased at the time of parturition.

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At each stage studied the mean of plasma calcium concentration was not changed (Fig. 2). Means of plasma inorganic phosphorus and magnesium concentrations showed no significant variation during the course of the experiment.

Ihe mean of plasma CI levels was significantly higher in newborn foals than in the mothers during the first Doys before or after parfurition stage studied (-50 days). However, there was no significant difference in plasma CI levels of both mothers Fig. 2. Changes in plasma concentrations of calcit on in (CT) and newborns at the time of parturition (Fig. 2). and calcium at the end of pregnancy and during parturition ()'day-old newborns were found to have a plasma calin mares and their foals. Each point indicates a mean value, the vertical bars ± SEM, and the integer the number of anicium concentration higher than in their mothers mals. (P < 0.05). Iwo-day-old newborns exhibited a small ~ pregnant mares; 0 - - - 0 newborn foals. For statistical but significant decrease (0.84 mg/100 ml; P < 0.01) analysis see the text. -50

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430

Calcitonin in Pregnant Mares

431

robin, Jöchle and Steingruber 1973), in normal delivery PGE 2 may be stimulus increasing plasma CT levels. In newborn foals plasma CT levels are higher than those found in mothers -50 days before parturition Discussion but not significantly different from those of partuIn cows and in goats parturition is associated with dent mares. At the time of birth in man the serum hypocalcemia and hypophosphatemia, a deep hypoCT level is higher in the umbilical cord blood (5.39 calcemia and hypophosphatemia inducing parturient ng/ml) than in the mother (1. 79 ngjml) (Samaan, paresis (Linzel/ 1965, Barlet 1968). It has been deWigoda and Castillo 1974). In lambs before weaning monstrated that thyroidectomized goats supplemented plasma CT level remains high (Garei, Care and Barlet with thyroxine and fed a low calcium diet do not de- 1974). Hesch, Woodhead, Huelner and Wolf (19'/3) velop hypocalcemia and hypophosphatemia during have also observed higher CT levels in 5-day-old baparturition (Barlet 1974). In co ws CT secretion apbies than in adults. In newborn calves plasma CT pears to account for hypocalcemia observed at the levels increase 48 h after birth with the simultanetime of parturition: a syndrome clinically and bioous development of hypercalcemia (Gare I and Bar/et, chemically similar to parturient paresis can be inunpublished results). Like in newborn calves or lambs duced by CT infusion in nonnal lactating cows (Bar- (Barlet et al. 1971) plasma calcium levels are higher let 1968). Moreover plasma CT levels measured by in newborn foals than in their mothers. In foals a bioassay in rats (Barlet 1969, Black and Capen 1973) slight neonatal hypocalcemia was observed 48 h afor immunoassay (Garel and Barlet 1975) du ring par- ter birth (Fig. 2). Neither lambs nor calves developed turition were higher than in control animals or besuch neonatal hypocalcemia. fore calving. On the contrary parturient paresis is Hesch et al. (1973) have suggested that CT may be very uncommon in ewes (Braithwaite, Glascock and important in early human life protecting against nuRiazuddin 1970, Bar/et, Michel, Larvor et Theriez tritive hypercalcemia at this period. We have demon1971). In these animals nonnal plasma calcium and strated in newborn lambs that calcium given oraJly CT levels were simultaneously observed at the time (10 mgjkg b.w.) - i.e. calcium contained in 15 ml of lambing (Barlet and Garel 1975). In the 9 pregof colostrum, absorbed during 2 or 3 consecutive nant mares studied there was an increase in plasma meals by a I-day-old lamb - induced a significant CT level during the last two months of gestation; rise in plasma CT level, without changing plasma calmoreover a peak in plasma CT concentration was observed five days be fore parturition. These changes cium concentration (Barlet, Michel, Theriez, Savajol and Garel 1974). The metabolic effects of CT in newin plasma CT concentration were independent of plasma calcium variations since means of plasma cal- born rats (Garei, Kervran, Ricquier, Rieutort and Girard 1973) and in newborn lambs (Barlet et al. cium levels were not changed du ring the same time. 1974) suggest that the high levels of endogenous CT In some pregnant mares (animals 6624 and 6809) observed in newboms are able to protect the animal plasma CT is obviously increased du ring parturition against any important fluctuation in plasma calcium despite the simultaneous hypocalcemia and hypoand nu trient levels foJlowing the numerous colostrum phosphatemia (Fig. 3). Thus in parturient mares, as in goats and cows, CT secretion escapes from its con- meals ingested in the postnatal period. This protection involves the regulation of gastric emptying and trol by blood calcium. It has been recently dem onintestinal absorption, possibly mediated via the gastrostrated that prostagiandin PGE 2 is the most potent secretagogue in monolayer C-cell-cultures from trout intestinal honnones. ultimobranchial glands (Roos, Bundy, Bailey and Acknowledgement Deltos 1974). Since prostaglandins - especially This work was supported by A.T.P. A 655 1310 from the PGE 2 - are involved in parturition in the cow (ZeCentre National de Ia Recherche Scientifiq ue.

References chevre gestante ou aUaitante. Ann.Biol.anim.Bioch.BioBar/et, J.P.: Induction experimentale d'un syndrome analogue phys. 14: 447-457 (1974) a Ja fi~vre vitulaire par administration de thyrocalcitonine ades vaches en cours de lactation. C.r.hebd. S~anc.Acad. Barlet, J.P., J.M. Garei: Physiological role of calcitonin in ScL Paris serie D 267: 2010-2013 (1968) pregnant goats and ewes. In: R.V. Talmage, D.H. Copp Barlet, J.P.: Mise en evidence d'un facteur hypocalcemiant (Eds.): Calcium regulating hormones. Proceedings of the et hypophosphatemiant dans le plasma des vaches 5th Parathyroid Conference, Excerpta Medica, Amsterlaitieres; variations du taux de ce facteur au moment du dam (in press) (1975) velage et dans les cas de syndrome vitulaire. C.r.hebd. Barlet, J.P., M.C. Michel, P. Larvor, M. Theriiz: Calcemie, Seanc.Acad.Sci. Paris serie D 268: 1864-1867 (1969) phosphatemie, magnesemie et glycemie comparees de Ia Barlet, J.P.: Role physiologique de la calcitonine chez Ia mere et du nouveau-ne chez les ruminants domestiques (vache, chevre, brebis). Ann.Biol.anim.Bioch.Biophys. 11: 415-426 (1971)

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in their plasma calcium level. In 7-day-old newborns plasma calcium level was also higher than in the mothers (Fig. 2).

Helena P. Selawry, K.L. Becker, L.E. Bivins, R.H. Snider and O.L. Silva

8arlet, J.P., M.e. Michel, M. Theriez, H. Savajol, J.M. Garel: Ca1citoninemie et effets metaboliques de la calcitonine chez l'agneau nouveau-ne. J .Physiol. Paris 68: 519-529 (1974) Black, H.E., e.e. Capen: Plasma calcitonin-like activity and urinary cyclic adenosine monophosphate during pregnancy, parturition and lactat ion in cows with parturient hypocalcemia. Horm.Metab.Res. 5: 297-502 (1973) Braithwaite, G.D., R.F. Glascock, S. Rillzuddin: Calcium metabolism in pregnant ewes. Br.J.Nutr. 24: 661-670 (1970) Care, A.D., D.F.L. Bates, R. Swaminathan, P.e. Ganguli: The role of gastrin as a calcitonin secretagogue. J.Endocr. 51: '/35-744 (1971) Cooper, e. w., W.H. Schwesinger, A.M. Mahgoub, D.A. Ontjes, T.K. Gray, P.L. Munson: Regulation of secretion of thyrocalcitonin: In: R. V. Talmage, P.L. Munson (Eds.): Calcium, parathyroid hormone and the calcitonins. Proceedings of the 4th Parathyroid Conference, Excerpta Medica, Amsterdam 128-139 (1972) Garei, J.M., J.P. Barlet: Plasma immunoreactive calcitonin and parathyroid hormone levels in parturient cows. J.Endocr. (in press) (1975) Garei, J.M., A.D. Care, J.P. Barlet: A radioimmunoassay for ovine calcitonin: an evaluation of calcitonin secretion during gestation, lactation and foetal life. J.Endocr. 62: 497-509 (1974) Garei, J.M., A. Kervran, D. Ricquier, M. Rieutort, J.R. Girard: Effets metaboliques de la calcitonine de saurnon chez le rat apres la naissance. C.R. hebd.Seanc.Acad. Sci. Paris Serie D 277: 2757-2760 (1973) Garel, J.M., H. Savajol, J.P. Barlet: Plasma immunoreactive calcitonin levels in pregnant ewes and their lambs. Biol. Neonate (in press) (1975)

Gray, T.K., P.L. Munson: Thyrocalcitonin: evidence for a physiological function. Science 166: 512-513 (1969) Hesch, R.D., S. Woodhead, M. Huefner, H. Wolf' Gastrointestinal stimulation of calcitonin in adults and newborns. Horm.Metab.Res. 5: 235 (1973) Kalckar, H.M.: The enzymatic synthesis of purine ribosides. J.bioI.Chem. 167: 477-486 (1974) Konopka, P., H.P. Klotz, M.L. Delorme: L'litat calcitoninique au cours de la gravidite. In: Les hormones et le calcium. Problemes actuels d'endocrinologie et de nutrition, Serie 15, ExpansionScientifique, Paris, 253-269 (1971) Lewis, P.. B. Rafferty, M. Shel/ey, e.J. Robinson: A suggested physiological role of calcitonin: the protection of the skeleton during pregnancy and lactation. J.Endocr. 49: lX-X (1971) Linzel/, J.L.: Milk-fever in goats. Vet.Record 77: 767-768 (1965) Raulais, D., M. Merle, G. Milhaud: Horse calcitonin: isolation and biological properties. In: Endocrinology 1973. Proceedings of the 4th International Symposium, W. Heinemann, London, 177-183 (1974) Roos, B.A., L.L. Bundy, R. Bailey, L.J. Deftos: Calcitonin secretion in vitro. I. Preparation of monolayer C-ceU cultures. Endocrinology 95: 1142-1149 (1974) Samaan, N.A., e. Wigoda, S.G. Castillo: Human serum calcitonin and parathyroid hormone levels in the maternal, umbilical cord blood and postpartum. In: Endocrinology 1973 - Proceedings of the 4th International Symposium, W. Heinemann, London, 364-372 (1974) Zerobin, K., W. Jöchle, Ch. Steingruber: Termination of pregnancy with prostaglandins E 2 (PGE 2 ) and F 2 Q (PGFuv in cattle. Prostaglandins 4: 891-902 (1973)

Requests for reprints should be addressed to: J.P. Barlet, I.N.R.A. Theix, F-63110 Beaumont (France)

Horm. Metab. Res. 7 (1975) 432-437

© Georg Thieme Verlag Stuttgart

In Vitro Studies 01 Calcitonin Release in Man Helena P. Selawry*, K.L. Becker, L.E. Bivins, R.H. Snider and O.L. Silva Section of Metabolism, Veterans Administration Hospital, Washington, O.C., and Oepartment of Medicine, George Washington and Georgetown University, Washington, O.C., U.S.A.

Summary gon were potent stimulators of calcitonin release. TheophylThe influence of various agents on calcitonin release from line and dibutyryl cyc\ic AMP were also potent stimuli. human thyroid was studied in vitro. Under the conditions A highly purified preparation of pancreatic glucagon was of this investigation, calcium, magn~sium and phosphate did not stimulate calcitonin release from short-term incubated without an effect. Those agents which stimulated calcitonin slices of human thyroid. However, pentagastrin and USP gluca- release were associated with augmented cyclic AMP accumulation. Although maximal discharge of calcitonin required the ·Present address: Division of Endocrinology and Metabolism, presence of calcium, our in vitro experiments raise the quesDepartment of Medicine, University of Miami School of Med- tion as to whether a gastrointestinal hormone, rather than calcium, might not be the principal agent affecting calcitonin icine, P.O. Box 520875, Biscayne Annex, Miami, Florida release. 33152, USA. Key-Words: Calcitonin - Human Thyroid - Calcium - GastroReceived: 17 Dec. 1974 Accepted: 21 Apr. 1975 intestinal Hormone

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432