hypophysectomized rat. Marenzi & Gerschman [1934] studied six of the University and Royal Infirmary, Glasgow (Received 13 December 1937)

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1 124 J. Physiol. (I938) 92, I i :6I2.I26 THE EFFECT OF HYPOPHYSECTOMY ON THE BLOOD CALCIUM AND PHOSPHORUS OF THE RAT BY A. B. ANDERSON AND E. G. OASTLER From the Biochemical Laboratory, Department of Pathology of the University and Royal Infirmary, Glasgow (Received 13 December 1937) THE presence of a parathyrotropic hormone in the pituitary was suggested by Anselmino et al. [1933, 1934], and by Hertz & Kranes [1934] on histological evidence of parathyroid hypertrophy in rats and rabbits after the injection of anterior pituitary extract. A rise of p.c. in the blood calcium in rats and dogs after the injection of anterior pituitary extract has been reported by Hoffmann & Anselmino [1934], and a rise in both the blood calcium and phosphorus has been reported in dogs by Gerschman & Marenzi [1935]. Shapiro [1934] found an increase of over 40 p.c. in the serum calcium level of cats, 5 hours after the injection of "Antuitrin". Friedgood [1936] and Friedgood & McLean [1937] have reported a rise in the blood calcium in rats and guinea-pigs after several daily injections of anterior pituitary extract. If a rise in the serum calcium is found after injection of anterior pituitary extracts, it might be expected that a fall would result after hypophysectomy. This has been reported in the toad [Charles, 1931; Hogben et al. 1931; Shapiro & Zwarenstein, 1933], but it does not appear to occur in mammals. Collip [1934] reported that the serum calcium of the hypophysectomized rat was normal or slightly increased. Wallace [1935] also found normal values for the blood calcium in the hypophysectomized rat. Marenzi & Gerschman [1934] studied six dogs at times varying from 12 to 297 days after hypophysectomy, and could find no alteration either in the blood calcium or phosphorus.

2 HYPOPHYSECTOMY AND BLOOD CALCIUM 125 Houssay [1936] also found no changes in the plasma calcium of fortyseven hypophysectomized dogs. Kusunoki [1927], on the other hand, described a striking increase in the serum calcium after hypophysectomy in the rabbit, although Cannavo & Beninato [1935] reported that there was no change in the blood calcium of rabbits after irradiation of the pituitary sufficient to produce severe damage in the anterior lobe. In the foregoing experiments, where observations were made on the serum calcium or inorganic phosphorus after hypophysectomy, no details have been given of the dietary intake of calcium and phosphorus. Shelling [1932] showed that the calcium and phosphorus of the diet was a factor determining the level of serum calcium and the onset of tetany in parathyroidectomized animals. It seems necessary, therefore, to control the amount of calcium and phosphorus in the diet of hypophysectomized animals, before it is justifiable to claim that hypophysectomy has no effect on the parathyroids as indicated by the serum calcium and the plasma phosphorus levels. The experiments to be described were undertaken with this consideration in view. METHODS The animals used were exclusively males of the London strain of Wistar rat. At ages of from 8 to 12 weeks they were hypophysectomized under ether anaesthesia, using the technique of Smith [1927] with slight modifications. The testicular atrophy formed a convenient sign in the living animal of the completeness of the removal of the hypophysis, which was also shown by cessation of growth. More than 80 p.c. of the animals survived the operation, a few dying some weeks later in hypoglyceemic coma. Control animals of the same age and, where possible, litter mates, were kept on the same diet and under identical conditions. Blood was drawn by heart puncture under ether anaesthesia, the animal killed, and the testes examined. Marked atrophy of the testes of the hypophysectomized animals was always found. Calcium was determined in the serum by Clark & Collip's [1925] modification of Kramer & Tisdall's method. As the rats yielded only small quantities of blood, it was not possible to estimate both calcium and inorganic phosphate in the blood of a single rat. The plasma inorganic phosphate was therefore estimated in a second series of animals by the method of Briggs [1922]. The blood was drawn as described above, oxalated and centrifuged immediately.

3 126 A. B. ANDERSON AND B. G. OASTLER RESULTS The preliminary experiments were carried out on animals fed on a stock diet containing 30 p.c. of dried milk, with occasional change to bread and fresh milk. The animals were killed at periods varying from 14 to 70 days after operation. The serum calcium of ten hypophysectomized animals varied from 9-3 to 11-8 mg./100 ml. with a mean of 10-6 mg., and that of eight controls varied from 9*6 to 11-3 mg./100 ml. with a mean of 10-3 mg. Therefore hypophysectomy had no effect on the serum calcium of rats fed on the stock diet. Animals were next given a low calcium diet of the same composition as one used by Shelling [1932] and designated I-K in his paper. In his experiments parathyroidectomized rats fed on this diet developed tetany and showed serum calcium levels of mg./100 ml. According to Shelling the diet contained calcium g./100 g., and phosphorus 0*475 g./100 g. Our diet prepared with a purified casein (Glaxo ashless) gave on analysis calcium g./100 g. Rats fed on this diet for a few weeks in our experiments developed a considerable degree of osteoporosis, which was evident at operation when the bone at the base of the skull was found to be very soft and vascular, in contrast to the hard dry bone of the normal rat. This osteoporosis was also shown by skiagrams of the rats taken after death. The results obtained with this diet are given in TABLE I. Rats fed on diet containing calcium g./100 g. Hypophysectomized Controls (A) (B) Difference Days Serum Serum (A) No. after Days calcium No. Days calcium and of rat operation on diet mg./looml. of rat on diet mg./100 ml. (B) 79* * * * *3 69* , * X * X1 75* * * * * X X Mean difference (A) and (B) * Thyroxine 3 mg./kg. injected at 10 and 5 days before blood drawn.

4 HYPOPHYSECTOMY AND BLOOD CALCIUM 127 Table I, which shows that the serum calcium of the control rats fell to low levels in most cases where the rats had been on the diet for 40 or more days. None of these animals was in actual tetany, but many showed muscular hyperirritability. The serum calcium was significantly higher in the hypophysectomized animals than in the controls. It was considered possible that the hypothyroid state of the rats after removal of the hypophysis would affect the calcium metabolism, and some of the animals-marked with an asterisk in Table I-were given thyroxine (B.D.H.) subcutaneously in doses of 3 mg./kg. at 10 and again at 5 days before blood was drawn. Control rats received similar injections. These doses were based on the work of Gaddum [1930] on the effect of thyroxine on the metabolism of the rat. The injection of thyroxine had no consistent effect on the serum calcium of either the experimental or control rats (Table I). The inorganic phosphate was first determined in the plasma of a few hypophysectomized rats on the stock diet, where it was found to be lower than in control animals. In another series of animals fed on the low calcium diet, the inorganic phosphate of the plasma was estimated at periods varying from 7 to 42 days after hypophysectomy. As will be seen from Table II, the plasma inorganic phosphate of the hypophysectomized animals-with a mean value of 4-64 mg./100 ml.-was markedly lower than that of their controls-mean 7-46 mg./100 ml. TABix II. Rats fed on diet containing calcium g./100 g. Hypophysectomized ControLs A, -A A Plasma Plasma Days inorganic inorganic No. after Days phosphate No. Days phosphate of rat operation on diet mg./100 ml. of rat on diet mg./100 ml * * * *2 Mean *46

5 128 A. B. ANDERSON AND E. G. OASTLER DISCUSSION The histological evidence for the existence of a parathyrotropic hormone is not by any means conclusive. The results recorded of the effect of hypophysectomy on the parathyroid are not in agreement. Atrophy of the parathyroids after hypophysectomy has been reported in rats by Smith [1927], and in dogs by Koster & Geesink [1928], but in neither case have any details been given as to the type or degree of the atrophy. Houssay [1936] has recorded changes after hypophysectomy in the parathyroids of dogs, which may be localized to one gland only, or to part of one gland, the remainder of the gland being normal. His results do not resemble those seen in other endocrine glands after hypophysectomy, where there is a generalized histological change after removal of the tropic influence. Houssay himself states that these changes may be the result of general nutritive alterations or the result of the lack of parathyrotropic hormone. On the other hand Collip [1935] could find no consistent degenerative changes in the parathyroids either of dogs or rats even several months after removal of their pituitaries. Livon & Peyron [1911] and Aschner [1912] also could find no changes in the parathyroids of dogs after hypophysectomy. The other evidence adduced in support of a parathyrotropic factor, namely the changes in the blood calcium after injection of anterior pituitary extracts, is also inconclusive. Only comparatively crude extracts of the anterior pituitary have been used, and no separation of a parathyrotropic from the other factors has been obtained. The rises in the serum calcium after these injections in most cases were small, of the order of p.c., and are not comparable to those found after injection of parathormone. Hogben & Charles [1932] found that a saline extract of the anterior lobes of ox pituitaries produced a marked fall in the serum calcium when injected into normal female rabbits. They also showed that this action was not through the ovaries since it occurred to the same degree after ovariectomy. The results showing the effects of anterior pituitary extracts on the plasma phosphorus are also contradictory. Friedgood & McLean [1937] could find no significant changes in the level of the blood phosphorus in guinea-pigs after daily injection of anterior pituitary extract for 9 days. On the other hand Gerschman & Marenzi [1935] reported a rise in the plasma phosphorus in dogs after inj'ection of anterior pituitary extract.

6 HYPOPHYSECTOMY AND BLOOD CALCIUM 129 There is the possibility that the alterations recorded in the serum calcium may have been due primarily to a tropic factor other than a possible parathyrotropic hormone, since the anterior pituitary extracts used had not been freed from all the other factors. Such a factor may have acted through its respective endocrine gland directly on the serum calcium or indirectly through the action of its own endocrine gland on the parathyroids. Our results show that hypophysectomy has no effect on the serum calcium of rats on a normal diet. This finding agrees with other results reported in the literature regarding the effect of hypophysectomy in mammals. In our experiments with a low calcium diet, it was found that after some weeks most of the control rats had a serum calcium of the order of 5-6 mg./100 ml. Hypophysectomy did not produce a lower serum calcium; in fact the calcium was higher in the serum of the hypophysectomized animals. Many of these rats had been fed on the low calcium diet for 2-3 weeks before operation, and therefore should have been particularly sensitive to any parathyroid deficiency. Shelling [1932] has shown the importance of this factor, because fatal tetany ensued within hours after he had removed the parathyroids of animals which had been fed on this diet for a few days before operation. The plasma inorganic phosphate of the rats fed on this diet was markedly lower after hypophysectomy. Whatever the explanation may be for our low figures for plasma phosphorus after hypophysectomy, they are in striking contrast to the raised plasma phosphorus found after parathyroidectomy. The diminished intake of food by the hypophysectomized animal may account in part for the fall in phosphate. There is the further complicating factor of cessation of growth in these animals. Perla & Sandberg [1936] have shown that, after hypophysectomy in the rat, not only was there a very much increased excretion of calcium but also of phosphorus. Pugsley & Anderson [1934] have also found a negative calcium balance in the rat after hypophysectomy, and noted that this was changed to a positive balance after the administration of growth hormone. The lack of effect of hypophysectomy on the parathyroid function as shown in our experiments is in marked contrast to the effect seen on the other endocrine glands when their respective tropic factors are removed. It is difficult to accept the existence of a parathyrotropic hormone, the removal of which appears to have no effect on the functional activity of the parathyroids.

7 130 A. B. ANDERSON AND E. G. OASTLER SUMMARY 1. Hypophysectomy had no effect on the serum calcium of rats fed on a normal diet. 2. After some weeks on a low calcium diet, control rats showed a fall in serum calcium to 5-6 mg./100 ml. Hypophysectomy did not produce a lower serum calcium, but the plasma inorganic phosphate of the hypophysectomized rats-with a mean value of 4-6 mg./100 ml.-was markedly lower than that of the controls-mean 7-4 mg. 3. In view of our results, and of the conflicting accounts in the literature, it is concluded that the existence of a parathyrotropic hormone is not proved. We are indebted to the Medical Research Council for a grant for expenses to E. G. O., and also to the Rankin Fund of the University of Glasgow for a similar grant to A. B. A. REFERENCES Anselmino, K. J., Hoffmann, F. & Herold, L. (1933). Klin. W8chr. 12, Anselmino, K. J., Hoffmann, F. & Herold, L. (1934). Ibid. 13, 45. Aschner, B. (1912). Pftugere Arch. 144, 1. Briggs, A. P. (1922). J. biol. Chem. 53,13. Cannavo, L. & Beninato, R. (1935). Endokrinologie, 15, 389. Charles, E. (1931). Proc. Roy. Soc. B. 107, 504. Clark, E. P. & Collip, J. B. (1925). J. biol. Chem. 63, 461. Collip, J. B. (1934). J. Mt Sinai Hosp. 1, 28. Collip, J. B. (1935). J. Amer. med. A , 916. Friedgood, H. B. (1936). Endocrinology, 20, 159. Friedgood, H. B. & McLean, R. (1937). Amer. J. Phy8iol. 118, 588. Gaddum, J. H. (1930). J. Phy8iol. 68, 383. Gerschman, R. & Marenzi, A. D. (1935). C.R. Soc. Biol., Pari8, 120, 817. Hertz, S. & Kranes, F. (1934). Endocrinology, 18, 350. Hoffmann, F. & Anselmino, K. J. (1934). Klin. Wechr. 13, 44. Hogben, L. & Charles, E. (1932). J. exp. Biol. 9, 139. Hogben, L., Charles, E. & Slome, D. (1931). Ibid. 8, 345. Houssay, B. A. (1936). New England J. Med. 214, Koster, S. & Geesink, A. (1928). Arch. nderland. Phy8iol. 13, 601. Kusunoki, G. (1927). Folia endocrin. japon. 3, 34. Livon, C. & Peyron (1911). C.R. Soc. Biol., Pari, 71, 49. Marenzi, A. D. & Gerschman, R. (1934). Ibid. 117, 56. Perla, D. & Sandberg, M. (1936). Endocrinology, 20, 481. Pugsley, L. I. & Anderson, E. M. (1934). Amer. J. Physiol. 109, 85. Shapiro, H. A. (1934). Quart. J. Pharm. Pharmacol. 7, 223. Shapiro, H. A. & Zwarenstein, H. (1933). J. exp. Biol. 10, 186. Shelling, D. H. (1932). J. biol. Chem. 96, 195. Smith, P. E. (1927). J. Amer. med. A88. 88, 158. Wallace, E. W. (1935). J. Pharmacol., Baltimore, 54, 161.