6I2.442:6I20*I5* i6D. Montreal, Canada.) greatly increased, although according to Harris and Innes [1931] and

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1 6I2.442:6I20*I5* i6D THE EFFECT OF PARATHYROID HORMONE AND OF IRRADIATED ERGOSTEROL ON CALCIUM AND PHOS- PHOROUS METABOLISM IN THE RAT. BY L. I. PUGSLEY. (From the Department of Biochemistry, McGill University, Montreal, Canada.) I. INTRODUCTION. THE rat is commonly regarded as a species relatively refractory to the parathyroid hormone, although Tweedy and Chandler [1929] were able to show that continued large doses were lethal, and Rose and Stucky [1930] found that the serum calcium could be raised by two or three days' treatment. An increased calcium excretion has been observed by Waltner [1928], and very recently by Biilbring [1931], the calcium being presumably derived from the bones, which according to Waltner [1928] and to Lambie, Kermack and Harvey [1929] soon display osteoporosis. The object of the present research was to reinvestigate the action of parathyroid hormone in the rat, and to compare it with the action of large doses of irradiated ergosterol'. The latter is known to decrease calcium excretion in the feces and increase it in the urine [Brown and Shohl, 1930; Watchorn, 1930] and the interpretation usually offered is that the absorption of calcium from the intestine is greatly increased, although according to Harris and Innes [1931] and others mobilization of calcium from the bones must also take place. T a y l o r, Weld, Branion and Kay [1931] affirm that in the dog the effects of overdosage with irradiated ergosterol are identical with those of parathyroid overdosage, and consider that in large doses vitamin D stimulates secretion from the parathyroid glands. It will be shown, however, that in the rat the metabolic effects of the two substances are entirely dissimilar, and recent studies in this laboratory by Selye [1932] prove that different histological changes are produced in the skeleton by these two agents. The problem first investigated in this research was the effect of parathyroid hormone on the excretion of calcium and phosphorus in the urine and faeces. Greenwald and Gross [1926] found that the administration of parathyroid hormone to dogs led to an increased excretion of 1 The writer is indebted to Eli Lilly and Co. for the parathormone and to Mead, Johnson and Co. for the ergosterol used in these experiments. PH. LXXVI. 21

2 316 L. I. PUGSLEY. calcium in the urine, which was not maintained throughout the period of treatment and declined to subnormal levels in the recovery period; the phosphorus excretion in the urine followed a similar course, while the excretion of these elements in the feeces was little affected. These results have been confirmed by many workers, especially in studies of human subjects, in whom, according to Albright, Bauer, Ropes and Aub [1929] the rise in urinary calcium appears later than, and is probably secondary to, the rise in urinary phosphorus. Other references may be found in a recent review of the literature [Thomson and Collip, 1932]. II. GENERAL METHODS. Albino rats of Wistar stock were used. They were kept in Hopkins' metabolism cages, and the urine and faeces were collected separately. In order to obtain sufficient urine for calcium analyses it was necessary to keep two rats (litter-mates) in a cage, and allow the urine to collect for 48 hours; the results, however, are expressed in mg. per rat per day throughout this paper. The diet used was McCollum's stock diet, except that no powdered bone was included, as it was felt undesirable to run the risk of contamination of the exereta with this ingredient; as compounded, the diet contained p.c. phosphorus and p.c. calcium. Male and female rats were used indifferently, and the animals weighed from 180 to 205 g. at the beginning of the experiments. Analytical methods. The urine was ashed by a modification of the technique described by Ku tz [1931] from this laboratory for dried tissue. It was evaporated to dryness in the 125 c.c. Pyrex Erlenmeyer flasks in which it was collected, and boiled for 3 or 4 hours with 3 to 5 c.c. of concentrated nitric acid (redistilled in glass to get rid of the traces of calcium usually present) until the digestion mixture was a clear straw-yellow; the nitric acid was then allowed to evaporate, and successive small amounts of nitric acid were added and evaporated until a white ash was obtained; the heating was carried out on a sand bath at about 2000 C., and the whole process took about 24 hours. The ash was dissolved in HCl, calcium was determined on an aliquot portion by the method of Clark and Collip [1925], phosphorus on another portion (after heating with ION H2SO4 to convert metaphosphate into orthophosphate, and to drive off excess HCI) by the method of Fiske and Subbarow [1925]. It was shown that known amounts of phosphate added to the urine could be quantitatively recovered by this method. The faeces were collected and dried in a silica dish and ashed in an electric muffle furnace at 5000 C. for 3 hours; the ash was analysed by the method described above.

3 PARATHYROID AND ERGOSTEROL ON METABOLISM. 317 III. EXPERIMENTAL RESULTS. The effect of parathyroid hormone upon the excretion of calcium and phosphorus by adult rats. The first problem studied was the effect of o CON TROL I I. 0" / DAYS Fig. 1. Fig. 1. The effect of administration of 5, 10 and 20 units of parathyroid hormone daily upon the excretion of calcium in the urine of adult rats. 1Thff 9 Thb pffx,.4 -f s -. f A - A*5. i. v&a.-0vlcvv %s %...n.. U Vy AU VIU units of parathyroid hormone daily upon the DAYS excretion of calcium in the faces of adult rats. Fig. 2. varying amounts of parathyroid hormone ("Parathormone" Lilly) on the excretion of calcium and phosphorus. The experiments were divided into three periods (after the animals had become accustomed to the cages and the diet), during which the urine and feces were collected from

4 318 L. I. PUGSLEY. each pair of rats every second day; a preliminary control period of days was followed by a period of days in which the extract was is~~~~~~~~~~~~~~~~/ 20 UNI/ S /6 /0 /2O 6 10 /O UNITS ~ ~ ~ ~ ~~~/ 5UNT a 14~~~~~~~~~~~~~~~~~/ /2 AV/Vf TS 14S /O~~~~~~~~~~~~~~~~~~/ 6 /6 CONT /0 / / /2 /6202o 428 DAYS DAYKS Fig. 3. Fig. 4. Fig. 3. The effect of administration of 5, 10 and 20 units of parathyroid hormone daily upon the excretion of phosphorus in the urine of adult rats. Fig. 4. The effect of administration of 5, 10 and 20 units of parathyroid hormone daily upon the excretion of phosphorus in the foeces of adult rats. injected subcutaneously in daily doses of 5, 10, or 20 units, and a recovery period. The results are shown in Figs. 1, 2, 3, and 4. Each curve gives the excretion per rat per day in mg. and each represents the

5 PARATHYROID AND ERGOSTEROL ON METABOLISM. 319 average excretion of three pairs of rats; three pairs of untreated controls were also followed. The most striking effects are seen in the increase in urinary calcium, which on the largest dose attains some fifty times the control value. It will be observed that the effect varies with the dose, and that it is not maintained throughout the period of treatment (the beginning and end of which are shown in the figures by arrows) but attains a peak on the fourth day and thereafter rapidly declines. In fact the rats appear to become immune rapidly or refractory to the extract, a phenomenon which has also been observed in dogs and in human subjects, and which is the object of further study in the later experiments. The calcium excretion in the faeces is also increased, but to a much less striking extent, in the period of treatment. Since the total "extra calcium" excreted, even on the largest dose, is not over 70 mg., it is not surprising that Day [1930] was unable to obtain significant reductions in the total body calcium of rats receiving much smaller doses of the hormone. The excretion of phosphorus in the urine is somewhat increased during the period of treatment, but the relative change is not nearly as great as that in calcium excretion, nor is there any evidence that it occurs more rapidly; the faecal phosphorus does not appear to be significantly affected. None of these rats showed any ill-effects from the series of injections except a slight loss of weight; the administration of 40 units daily, however, was found to produce cachexia in 3 days and death in 5 or 6; the observation of Tweedy and Chandler [1929], that the congestion and hyperaemia of the stomach characteristic of dogs receiving excessive doses of parathyroid extract does not appear in rats, was confirmed by post-mortem examinations. The effect of parathyroid hormone upon the serum calcium of adult rats. Blood was collected from rats by severing the carotid artery and jugular vein on one side and allowing the animals to bleed to death into a centrifuge tube; in order to obtain sufficient serum for the determination of calcium by the technique of Clark and Collip [1925], it was necessary to pool the blood from two rats. The average serum calcium of 40 pairs of rats on the diet described was found to be 10-3 mg. p.c., with a standard deviation of only 0-22 mg. p.c. Rats receiving 10, 20, or 40 units of parathyroid hormone daily were killed at various intervals and the serum calcium values obtained are plotted in Fig. 5, in which each point represents the average of determinations made on from three to eight pairs of rats. It is clear that marked hypercalcaemia can be produced, that it is to some extent proportionalto the dose injected, andthatit is not maintained throughout treatment but tends to return towards normal levels.

6 320 L. I. PUGSLEY. Ij ~I Fig. 5. DA YS The effect of administration of 10, 20 and 40 units of parathyroid hormone daily upon the serum calcium of adult rats. 8 6 ai 4 ~io I. oii '2 2 0 Fig / O A Y' The effect of administration of 2000 to 4000, 20,000 and 50,000 units of vitamin D daily upon the excretion of calcium in the urine of adult rats.

7 PARATHYROID AND ERGOSTEROL ON METABOLISM. 321 The effect of irradiated ergosterol upon the excretion of calcium and phosphorus by adult rats. This experiment was carried out in the same manner as the first experiment. The irradiated ergosterol used was a sample prepared by Mead, Johnson and Co. for research purposes and was stated to have the potency 10,000 X ± 10 p.c., assayed by the Fig /I /6 '4 /2 1. /8 ; /6 a- '4 t /2 z /6 /6 14 /2 20 '8 /6 /4 /2 0 4 a /2 / DAYS The effect of administration of 2000 to 4000, 20,000 and 50,000 units of vitamin I daily upon the excretion of phosphorus in the urine of adult rats. method of Bills, Honeywell, Wirick and Nussmeier [1931J; according to the figures given by these workers, it therefore contained one million international rat units per c.c. Various dilutions in olive oil were prepared and were administered orally by means of a pipette in doses of 2000, 4000, 20,000 and 50,000 units daily, in each case in a volume of 0*1 c.c. per dose. The results of the experiments are shown in Figs. 6, 7, 8 and 9; as they are on the whole in harmony with those of other workers,

8 322 L. I. PUGSLEY. they do not require extended discussion. With the larger doses, there is a great increase in the excretion of calcium in the urine with no regular change in urinary phosphorus, and, in contrast to the experiments with J 6 4 Fig a DA YS The effect of administration of 2000 to 4000, 20,000 and 50,000 units of vitamin D daily upon the excretion of calcium in the feces of adult rats. parathyroid extract, a very distinct decrease in the foecal excretion of both elements. Except on the highest doses, the rats did not appear to suffer any ill effects from the treatment. These results are not readily brought into line with the theory that the chief effect of large doses of irradiated ergosterol is to stimulate the parathyroid glands.

9 W / W A T~~~~~~~O 4IN 7h T PARATHYROID AND ERGOSTEROL ON METABOLISM. 323 The administration of irradiated ergosterol to rats refractory to parathyroid hormone. In this experiment a study was made of the calcium and phosphorus excretion of a group of rats which received first 10 units of parathyroid hormone daily for 12 days on two separate occasions, then dt, /2 6 4? /4 X OT '4- /0 -X I X -TO- 400 XIN J2 16 Z Fig. 9. The effect of administration of 2000 to 4000, 20,000 and 50,000 units of vitamin D daily upon the excretion of phosphorus in the faeces of adult rats. received 50,000 units of irradiated ergosterol daily for 30 days, and lastly received 10 units of parathyroid hormone daily for 14 days. The results are shown in Figs. 10 and 11, in which the parathyroid periods are marked by a shaded block and the vitamin period by a black block. The urinary calcium increased in the usual way at the beginning of the first parathyroid period, but showed almost no response in the second and third parathyroid periods, though it increased as usual, with the usual

10 324 L. I. PUGSLEY. concomitant decrease in feecal calcium, in the intervening vitamin period. This experiment shows firstly, that rats which have once become immune or refractory to parathyroid hormone do not readily regain sensitivity, and secondly, that such rats respond as usual to the treatment with irradiated ergosterol. O A? /8 JO J 364X ? 75 "s x S NO =ilifillifl D fawrs Fig. 10. Comparison of the effect of successive administration of parathyroid hormone with that of irradiated ergosterol upon the excretion of calcium of adult rats: 10 units of parathyroid hormone daily. ~MM 50,000 units of vitamin D daily. The effect of urine of immune rats receiving parathyroid hormone on the calcium excretion of responsive rats. Since it was felt that the animals which had developed "immunity" to parathyroid hormone might be excreting the injected hormone in the urine, an investigation of the point was undertaken. Five pairs of rats which had previously received 10 units of parathyroid hormone daily in three periods, and which no longer

11 PARATHYROID AND ERGOSTEROL ON METABOLISM. 325 showed any response to the injections, were treated with 20 units of parathyroid hormone daily. One pair ("A") was kept as a control, the urine and feces being analysed for calcium, which did not increase during the treatment. The urine of the other four pairs was injected into to - *i-- Fig / 44S Xz8X OIIJIflhI DAYS Comparison of the successive administration of parathyroid hormone with that of irradiated ergosterol upon the excretion of phosphorus of adult rats. three pairs of normal rats (X, Y and Z) whose calcium excretion was followed. The results are summarized in Table I. It is seen that though the amount of urine injected into each of the normal (and presumably responsive) rats daily might have contained 26-6 units of parathyroid hormone, if quantitative excretion occurred, and though the urine was collected in the presence of acetic acid and chloroform to prevent destruction of any hormone present, yet none of the rats receiving injections

12 326 L. I. PUGSLEY. TABLE I. Effect of administration of urine of rats immune to the action of parathyroid hormone, upon the excretion of calcium of normal adult rats expressed in mg. per day. Urine Fseces Date A A 1932 Control X Y Z Control X Y Z Feb * Control Experimental 24 0* * period * O08 0* of this urine showed any significant increase in the excretion of calcium in the urine or faeces. It may be concluded that certainly not more than one-fifth of the parathyroid hormone injected into the immune rats appeared in the urine. The effect of the injection of the serum of immune rats upon the response of normal rats to parathyroid hormone. It was also thought possible that in "immune" animals some substance antagonistic to the parathyroid extract might be present in the blood serum. Normal responsive rats were therefore treated with 20 units daily of parathyroid hormone and simultaneously with 2 c.c. of serum from either normal, responsive rats or previously treated " immune " rats, and the excretion of calcium in the urine of the injected animals was studied. The results are shown in Table II. Both groups of injected rats showed the typical great increase TABLE II. Effect of administration of 20 units of parathyroid hormone and serum of rats immune to the action of parathyroid hormone, upon the excretion of calcium in the urine of normal adult rats. Injected with Injected with normal serum immune serum Date + parathyroid + parathyroid 1932 hormone hormone Jan Control period,, ,, 7 1V Experimental period,, I i in urinary calcium during the first part of the period of parathyroid treatment, and neither normal serum nor "immune" serum inhibited this effect. IV. DIsCUSSION. Although the rat is relatively refractory to parathyroid hormone, when compared with the dog or the man, nevertheless it is possible to produce an increased serum calcium and an increased excretion of

13 PARATHYROID AND ERGOSTEROL ON METABOLISM. 327 calcium in the urine and fseces by the injection of moderate doses. Of these effects, the greatest relatively is that upon the urine calcium in the first few days of treatment. Since this effect is obtainable with doses as small as 5 units daily per rat, and is roughly proportional to the dose, it suggests that a valuable method of biological assay of parathyroid extracts might be founded on this basis, the only objection being the tedious nature of the analysis required'. The apparent immunity to parathyroid extracts which the rats rapidly develop is of great interest. Attempts to demonstrate the presence of an immune body in the serum, or an increased permeability of the kidney to the injected hormone, completely failed. Since these experiments were carried out, however, an entirely new light on the nature of this immunity has appeared in the work of Selye [1932], in this laboratory. He has shown by histological study of the bones of rats receiving parathyroid extract, that while the first response is a formation of numerous osteoclasts with rarefaction of bone (doubtless leading to hypercalcaemia and increased calcium excretion), this is succeeded by the disappearance of the osteoclasts, the formation of numerous osteoblasts, with bone apposition proceeding to such an extent that the picture of "marble-bone disease" can be produced experimentally. In this second phase of the reaction one would expect to find a decrease in calcium excretion and possibly subnormal serum calcium. The metabolic effects of large doses of vitamin D (irradiated ergosterol) are quite unlike those of parathyroid hormone. There is once more an increased excretion of calcium in the urine, but it is accompanied, as others have found, by a decreased excretion in the feces; moreover, animals may be responsive to irradiated ergosterol when they are almost completely "immune" (as far as calcium excretion is concerned) to parathyroid hormone. It may be concluded that if large doses of irradiated ergosterol do stimulate parathyroid secretion, this is but a small part of their pharmacological action. V. SUMMARY. 1. General and chemical methods for following the excretion of calcium and phosphorus in the urine and feeces of rats are described. 2. The injection of parathyroid hormone, in doses of 5 to 20 units daily, leads to an increase in serum calcium, an increase in faecal calcium 1 After this article had gone to press F. J. Dyer reported before the Physiological Society, London, J. Phy8iol. 75, 13 P, 1932, a method for the estimation of parathyroid hormone based on the rise in urinary calcium of male rats.

14 328 L. I. PUGSLEY. and urine phosphorus, and a relatively more striking increase in urine calcium, which however is not long maintained. Experiments on the nature of the apparent immunity to the hormone developed under treatment are described and discussed. 3. Oral administration of irradiated ergosterol, in doses of 20,000 to 50,000 units daily, leads to an increase in urine calcium but a decrease in fecal calcium and phosphorus. This effect is also obtainable in rats which have become "immune" to parathyroid hormone. I wish to express my thanks to Prof. J. B. Collip and to Dr D. L. Thomson for help and advice during the course of this work. REFERENCES. Albright, F., Bauer, W., Ropes, M. and Aub, J. C. (1929). J. Clin. Inve8t. 7, 139. Bills, C. E., Honeywell, E. M., Wirick, A. M. and Nussmeier, M. (1931). J. Biol. Chem. 40, 619. Brown, H. B. and Shohl, A. T. (1930). Ibid. 86, 245. Biulbring, E. (1931). Arch. exp. Path. Pharmak. 162, 209. Clark, E. P. and Collip, J. B. (1925). J. Biol. Chem. 83, 461. Day, P. L. (1930). J. Nutrition, 3, 157. Fiske, C. H. and Subbarow, Y. (1925). J. Biol. Chem. 66, 375. Greenwald, I. and Gross, J. (1926). Ibid. 68, 325. Harris, L. J. and Innes, J. R. M. (1931). Biochem. J. 25, 367. Kutz, R. L. (1931). J. Biol. Chem. 92, lxxii. Lambie, C. G., Kermack, W. 0. and Harvey, W. F. (1929). Nature, 123, 348. Rose, W. B. and Stucky, C. J. (1930). Amer. J. Phy8iol. 91, 513. Selye, H. (1932). Endocrinology, in press. Taylor, N. B., Weld, C. B., Branion, H. D. and Kay, H. D. (1931). Can. Med. A88. J. 24, 763, and 25, 20. Thomson, D. L. and Collip, J. B. (1932). Phy8iol. Rev. 12, 309. Tweedy, W. R. and Chandler, S. B. (1929). Amer. J. Phy8iol. 88, 754. Waltner, K. (1928). M8chr. Kinderhlk. 40, 317. Watchorn, E. (1930). Biochem. J. 24, 631.