6I2.492:6I2.44. Cushing [1912] noted that in dogs there occurred a transient active

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1 11 6I2.492:6I2.44 STUDIES ON THE PHYSIOLOGY OF THE THYREO- TROPIC HORMONE OF THE ANTERIOR PITUITARY. BY EVELYN M. ANDERSON AND J. B. COLLIP. (Department of Biochemistry, McGill University, Montreal, Canada.) (Received April 4, 1934.) THE close relationship between the anterior pituitary and the thyroid gland has been clearly demonstrated both experimentally and clinically. Cushing [1912] noted that in dogs there occurred a transient active hyperplasia of the thyroid 24 hours after complete hypophysectomy, which was ultimately succeeded by involution of the gland with an excess of colloid and a low epithelium. Allen [1920] reported an atrophy of the thyroid in tadpoles following removal of the anterior pituitary, which could be completely repaired by implanting pituitary from a frog. P. E. Smith [1927] performed a corresponding experiment in the rat with similar results. On the clinical side it is known that in hypopituitary conditions patients tend to have subnormal metabolism. Graubner [1925] found in a review of thirty-four cases of hypophyseal cachexia (Simmonds' disease) reported in the literature up to 1924, that in all cases the thyroid was small and atrophic and sometimes even sclerotic. On the other hand, in acromegaly the metabolism is often high and the thyroid gland is so hyperplastic that it may be operated upon because of symptoms suggesting thyreotoxicosis. Hyperplasia of the thyroid gland has been produced by injections of anterior pituitary extracts in a number of laboratory animals such as the guinea-pig, cat, dog, rabbit, pigeon and duck [L o eb and B assett, 1929; Aron, 1929; Krogh, Lindberg and Okkels, 1932; Janssen and Loeser, ; Grab, 1932a; Schittenhelm and Eisler, 1932; Houssay, Biasotti and Magdalena, 1932; Baumann and Marine, 1932; Riddle and Polhemus 1931; Schockaert, 1932]. Three of these workers, Aron, Loeb and Schockaert, reported failure to produce such a condition in the rat by this means. In our early work, using crude

2 12 E. M. ANDERSON AND J. B. COLLIP. extracts of anterior pituitary combined with killed cultures of Staphylococcus vaccine, we were able to produce a severe hyperplasia of the thyroid with hyperthyroidism in the rat. We have since obtained extracts of the thyreotropic hormone of sufficiently high potency to cause hyperthyroidism in the rat without the simultaneous use of the Staphylococcus vaccine. Signs of hyperthyroidism are associated with the hyperplasia of the thyroid thus produced. There is an increase in the metabolic rate [Siebert and Smith, 1930; Verzar and Wahl, 1931], increased heart rate [Schittenhelm and Eisler, 1932], exophthalmos [Schockaert, 1932; Loeb and Friedmann, 1932], a reduction of the iodine content of the thyroid gland [Loeser, ; Schockaert and Foster, 1932], an increase in the alcohol insoluble iodine of the blood [Clo ss, L o eb and MacKay, 1932; Grab, 1932b; Schittenhelm and Eisler, 1932], a depletion of liver glycogen [Eitel and Loeser, 1932] and an increase in acetone bodies in the blood [Eitel, Lohr and Loeser, 1933]. It has been shown by Verzar and others that none of the signs of hyperthyroidism following the injection of anterior pituitary extracts can be produced in thyroidectomized animals. METHODS. A Benedict multiple chamber respiration apparatus, adapted for rats and young guinea-pigs, has been used for measuring the energy metabolism [Benedict, ]. Table I gives the metabolic rates, expressed in litres of oxygen per square metre body surface per 24 hours, of 601 normal white rats taken under resting conditions. In the first group the animals were fasted 17 hours before measuring the oxygen consumption. The basal metabolic rate was then taken with the animal at a temperature of 280 C., which is the critical temperature for the rat. It did not prove feasible to fast the animals before measuring the oxygen consumption, for in many cases it was necessary to determine the metabolic rate daily. Furthermore, in cases of severe hyperthyroidism the animals could not tolerate fasting for any length of time. In the second group of animals in Table I the oxygen consumption has been measured without submitting the animal to a long period of fasting. Comparing the upper and lower sections of Table I, the difference in 02 consumption between the fasted animals and those not fasted is not striking. There are differences, however, according to sex and season. The males have a slightly higher rate than the females in the same weight group. During the cold months the rates are higher than during the

3 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 13 TABLE I. Metabolism of normal white rats. Oxygen 1./sq. m. body surface per 24 hours (number of animals in italics) Body wt... From From From From 30 to 49 g. 50 to 99 g. 100 to 149 g. 150 to 199 g. Male Female Male Female Male Female Male Female Metabolic rate after 17 hours' fasting: Jan (14) (13) (5) Feb (37) (44) (8) (71) (25) March (17) (26) (50) (14) (4) April (5) (38) (9) (13) Sept (30) (35) (4) (25) Metabolic rate without fasting: Jan (2) (5) Feb (12) (24) (26) (11) March Sept (8) Oct (4) (8) Nov (5) (5) (4) The average oxygen consumption for the total number of rats (601) is /sq. m. body surface per 24 hours. warm months. The average value for the oxygen consumption of all the animals in Table 1 (601) is Of 02 per sq. m. body surface per 24 hours. This figure has been taken as the normal metabolism for the rats used in this study, and the metabolic rates of the hypophysectomized and of the goitrous rats have been calculated as a percentage of this value. In the case of normal animals the oxygen consumption was measured in the preexperimental period, and the metabolic rate has been expressed in percentage of that value for the individual animal. Table II gives the oxygen consumption of normal prepubertal guineapigs, taken under resting conditions but without fasting. When the animals were kept in an environment at 310 C., which is the critical temperature for the guinea-pig, for one or two weeks before measuring the oxygen consumption, a greater uniformity of readings was obtained; furthermore, the oxygen consumption was lower.

4 14 E. M. ANDERSON AND J. B. COLLIP. TABLE IL Metabolic rates of normal young guinea-pigs. Oxygen 1./sq. m. body surface per 24 hours (number of animals in italics) Body wt.... From From From From 90 to 150 g. 151 to 200 g. 201 to 250 g. 251 to 300 g. Temp. 0C. Male Female Male Female Male Female Male Female Nov (20) (20) (12) (12) (3) (4) (2) (4) Jan (15) (10) (11) (8) (11) (8) (3) Feb (14) (9) (8) (9) (4) (1) It will be noted that when the animals have been kept at a temperature of 31 C. for 1-2 weeks previously the metabolic rate is lowered. Effect of thyreotropic hormone on the metabolism of the guinea-pig. Since the guinea-pig is exceedingly sensitive to the thyreotropic hormone, it was used in testing for the presence of the hormone in the earlier work of extraction. In a group of thirty-two prepubertal guinea-pigs, shown in Table III, sixteen received extracts containing the thyreotropic TABLE III. Extract of anterior pituitary (bovine and sheep) containing thyreotropic hormone Extracts of anterior pituitary free from thyreotropic hormone Extract containing growth hormone Anterior-pituitary-like principle of pregnancy urine (APL) Controls (not injected) Effect of thyreotropic hormone on the metabolism of the guinea-pig. Metabolic rate p.c. of normal Thyroid, Histology NC. of Daily Body mg./100 g. 6th-8th of animals dose wt. g. body wt. 4th day day thyroid [6 4 c.c Severe hyperplasia 8 4 c.c Normal 2 4 c.c Normal u Normal Normal hormone-which caused an average increase in metabolic rate to 129 p.c. by the 4th day of injection and 134 p.c. on the 6th to 8th day. The thyroid glands in these animals after a week of injections were more than twice the weight of the controls and histologically showed severe hyperplasia such as is seen in the thyroid of Graves' disease. The growth hormone, Q12 [Collip, Selye and Thomson, 1933] and APL, given for

5 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 15 only a short period, had no effect on the metabolic rate or on the histological picture of the thyroid. With prolonged injections of APL it has been shown by Collip, Selye, Thomson and Williamson [1933] that an enlargement of the thyroid occurs in females, together with enlargement of the ovary and pituitary gland. A study was made of the changes in metabolism followed from day to day in guinea-pigs injected with the thyreotropic hormone (Table IV). TABLE IV. Daily changes in the metabolism of the guinea-pig during injections of thyreotropic hormone. Metabolic rate (p.c. of normal) Daily Body Days Thyroid dose Wt.,_ A It mg./100 g. Animal C.C. g body wt. Experimental: Thyreotropic hormone, Extract H * Av Controls (not injected) Av In a group of eight guinea-pigs receiving the extract, five showed an increase in metabolic rate by the second day. All the guinea-pigs showed a preliminary rise sometime within 4 days, followed by a secondary rise, reaching a peak in about a week, and followed by a decline in metabolism. This type of curve for the metabolic rate is seen also in the normal and hypophysectomized rats. It has been shown by a number of workers [Loeser, ; Grab, 1932b; Schockaert and Foster, 1932; Closs, Loeb and MacKay, 1932] that following the injection of the thyreotropic hormone there occurs an outpouring of thyroid secretion as indicated by a reduction of the iodine content of the thyroid gland and an increase in the alcohol insoluble iodine of the blood. Grab [1932b] showed that the increase in blood iodine appeared immediately following the injection of thyreotropic hormone in the dog. We might postulate the first rise in metabolism to be due to the sudden discharge of existing stores of thyroid hormone into the blood stream, while the

6 16 E. M. ANDERSON AND J. B. COLLIP. second rise follows after hyperplasia of the thyroid has advanced sufficiently to give an increased production of its hormone. Effect of thyreotropic hormone upon the normnal rat. With the use of a highly purified extract of the thyreotropic hormone an increase in metabolic rate can be obtained in the rat [Anderson and Collip, 1933]. Table V represents a group of thirty-five animals which had received the TABLE V. Daily dose Extract c.c. TG TGB Controls (not injected) Effect of thyreotropic hormone on the metabolism of the adult rat. No. of animals Age days Metabolic rate (p.c. of normal) Days f1l thyreotropic hormone in varying doses. It will be seen that amounts of extract as low as 0-16 c.c. a day have produced a significant rise in the metabolic rate of the rat. We have repeated our earlier work in which we obtained hyperthyroidism in the rat by means of Staphylococcus vaccine in conjunction with crude extracts of anterior pituitary [Anderson, 1933], using the purified extracts of the hormone (Table VI). There occurred an increase in the metabolic rate in both groups of rats receiving the hormone, but TABLE VI. The effect of thyreotropic hormone in conjunction with intraperitoneal injections of Stqphyloooccu8 aureu8 vaccine. Extract of thyreotropic hormone, 2 c.c. daily. StaphylocoCCU8 aureus vaccine, 20 million intraperitoneauly daily Extract of thyreotropic hormone, 2 c.c. daily Staphylococcus aureus vaccine, 20 million intraperitoneally daily Metabolic rate (p.c. of normal) Days No. of A._ animals

7 THYREJOTROPIC HORMONE OF ANTERIOR PITUITARY. 17 these effects were more pronounced in the group receiving Staphylococcus vaccine in addition to the hormone. There was no metabolic response to the Staphylococcus vaccine alone. It would seem that the Staphylococcus vaccine in some way increases the sensitivity of the animal to the thyreotropic hormone. The effect of hypophysectomy upon metabolism ofthe rat and replacement therapy with the thyreotropic hormone. In a series of 118 rats studied after hypophysectomy, it was found that the metabolic rate falls to an average of 74 p.c. of the normal (Table VII). The thyroid gland decreases in TABLE VII. The metabolism of untreated hypophysectomized rats. No. of days after hypophysectomy P.c. of normal metabolism ' weight after hypophysectomy and the histological examination shows marked involution of the cellular elements. The hypophysectomized animal is very sensitive to minute doses of the thyreotropic hormone. In Table VIII it is seen that the metabolic rate of the hypophysectomized animal can be brought back to normal with amounts of the purified extracts of the hormone as low as 0 01 c.c. a day. We have chosen as an arbitrary unit of the hormone the least TABLE VIII. Effect of thyreotropic hormone on the metabolism of the hypophysectomized rat. Days Metabolic rate during the course of treatment (p.c. of normal) after Daily Days opera- dose,ak Animal tion Extraot C.C BB 14 TG YH 14 TG YB 14 TG YB 37 TGS YT 37 TGS 0* PH 37 TGS 0* PB 14 TG YT 22 TGS 0* B PH 20 TGB B PT 20 TGB C PH-YB 20 TGB OPT-YB 20 TGB PT 20 TGS YT 11 TGS BB 28 TGPB BT 28 TGPB PH-T 28 TGPB 0* VH 28 TGA YT 28 TGA PH. LXXX2II. 2

8 18 E. M. ANDERSON AND J. B. COLLIP. amount of extract per day which will cause a rise of 20 p.c. in the metabolism of the hypophysectomized rat by the 4th day. We have found this method of assaying it more satisfactory than the method of Junkmann and Schoeller [1932]. In Table IX a number of extracts have been tested for potency in this way. TABLE IX. Effect of small doses of thyreotropic hormone on the metabolism of the hypophysectomized rat. Metabolic rate (p.c. of normal) Days t after Daily Before Animal operation Extract dose c.c. injection 4th day Difference 1158 VB 14 TGPB3 0' YH-T 19 TO7 0' YH 19 TG '5 +27' PH 19 TG7 0' PH 1102 PT TG9 TG '01 65' ' ' VT 19 TGP2 0' '5 + 3' VH-T 19 TGP2 0' ' YH 19 TGP2 0'01 75' YH 10 TGP3 0'04 73'5 89' BT 24 PH TGROB TGROB 0'04 0' ' ' YT 12 PH TGB TGB 0'04 0' YB-T 19 TGB 0' '5 +11'5 16 VH 14 TGB1 0'04 70' VB 14 TGB1 0' '5 + 4X5 16 VT 14 TGB1 0' Prolonged treatment with thyreotropic hormone. It was seen in Tables IV and V that the elevated metabolic rate produced by the hormone is not maintained in spite of continued injections, that the peak is usually reached in a week or 10 days and is followed by a decline. The results of treatment of a large group of normal rats over a long period of time are shown in Table X. In 3 or 4 weeks the metabolic rate has returned to the TABLE X. Prolonged treatment of normal rats with large doses of thyreotropic hormone. No. In- Metabolic rate (p.c. of normal) of itial Daily Days ani- age dose,_ i- _ Sex mals Days Extract c.c M TG, M TGB M TGPS F. 4 5ot {TTGA 0'25} F TGPB F TGA F TGA

9 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 19 normal level, and if the injections are continued the rate may fall to the level of the hypophysectomized animal. In Table X three rats, which received large doses of the extract TGPS, showed an average metabolic rate of 61 p.c. of the normal on the 37th day of injection. This is the level of metabolism which one finds in the hypophysectomized rat (Table VII). Histologically the thyroid at this stage resembles the thyroid of the hypophysectomized rat. Increasing the dose of hormone to two or three times the original dose does not cause any rise over this low level, nor does transplantation of thyroid tissue from normal rats into the injected animals have any effect upon the metabolic rate. The serum from these resistant animals has been shown to contain a substance which has an inhibitory effect upon the action of the thyreotropic hormone. The results of these findings have been reported previously [Collip and Anderson 1934; Anderson and Collip, 1934], so that only a brief summary will be given here. The antithyreotropic substance has been extracted from the serum of a horse which had been injected with thyreotropic hormone for a period of two months. This extract is capable not only of inhibiting the action of a dose of thyreotropic hormone in the normal rat which is five times the minimum effective dose, but at the same time of inhibiting the thyreotropic hormone of the animal's own pituitary by lowering the metabolic rate to the hypophysectomized level. Hypophysectomized rats which have been injected with the antithyreotropic substance together with the thyreotropic hormone show hyperplastic changes in the thyroid gland without any rise in the metabolic rate. We have seen in several instances hyperplasia still present in the thyroid of rats on the 14th day of injection of thyreotropic hormone when the metabolic rate had returned to normal. Friedgood [1934] has reported similar observations in the guinea-pig. Animals which have received the thyreotropic hormone for one or two months until the metabolic rate is below normal are as sensitive to thyroxine as the normal rat. The pituitary glands of the resistant rats are lacking in thyreotropic hormone, although the growth hormone is still present. A group of thirty hypophysectomized rats have been injected with thyreotropic hormone over a long period of time (Table XI). The hypophysectomized animal seems to develop a resistance to the thyreotropic hormone like the normal rat. After a month of injections the metabolic rate returns to the level of the untreated hypophysectomized rat. This is evidence that the antithyreotropic substance is not formed in the 2-2

10 20 E. M. ANDERSON AND J. B. COLLIP. Days after Daily oper- dose Animal ation Extract c.c. iio 1130 PH PH 1 PH_YB t2 PT-YB 380 PH YB PT VH BT VB VTr YB PB YT I14 H H H H I II I I II TGB TGB TGB TGB TG TG TG TG TGS TG TG TGS TG 2-*0 2* *0 2*0 2* * ] * * *05 0*02 0* Experimental _ Controls TABLE XI. Prolonged treatment of hypophy- Metabolic rate * Injections of thyreotropic hormone pituitary gland. If the injections of the hormone are stopped after prolonged treatment when the metabolic rate is very low, a temporary elevation of the rate occurs which may even rise to the normal level. Animals resistant to thyreotropic hormone may still respond to growth hormone. Seven hypophysectomized rats were given the thyreotropic hormone for 65 days. During this time they had lost considerable weight. Small doses of growth hormone were then given; all the animals showed an increase in body weight in 4 days, the average increase being 10 g. Thyreotropic hormone and spontaneous goitre. A study has been made of the effect of thyreotropic hormone upon the metabolism of a group of rats with spontaneous goitre which were furnished us by Dr C. N. H. Long of the George S. Cox Medical Research Institute, Philadelphia.

11 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 21 sectomized rats with thyreotropic hormone. during the course of treatment (p.c. of normal) Days Experimenta * * * * * * * - Controls * 76* 69* 84* omitted from the 46th to the 61st day. The thyroid gland from one of the rats of this group which was sacrificed at the beginning of the experiment weighed 142 mg. The thyroid of a normal Wistar rat of the same body weight (180 g.) which was chosen for comparison, weighed 20 mg. Histologically the goitrous thyroid showed severe hyperplasia. The average metabolic rate of the six goitrous rats was 91 p.c. of the normal before the injections were given. Immediately following the thyreotropic hormone injections the metabolic rate rose very steeply. One animal showed a 95 p.c. increase in 24 hours. During the 9 days of injections the metabolism rose to extraordinarily high levels, the highest rate going to 262 p.c. of the normal. The animals showed definite signs of hyperthyroidism, e.g., irritability, weakness, exophthalmos and excessive sweating. An average loss of 20 p.c. in body

12 22 B. M. ANDERSON AND J. B. COLLIP. TABLE XII. The influence of thyreotropic hormone upon rats with spontaneous goitre. Autopsy Metabolic rate (p.c. of normal) Wt. of Wt. of Body Daily Days thy- adrewt. Ex- dose, roid nals Animal g. tract c.c mg. mg. Experimental 1143 YH 318 TGO (Dead on 10th day) YB 298 TG, (Dead on 10th day) YT 300 TG * VB 274 TGg (Dead on 9th, ~~~~~~~~~~day) VT 287 AIA f 6J TGi AIA cx- Control VH * Animals VH and YT were given potassium iodide, mg. daily, starting on the 29th day of the experiment. The metabolic rate rose from an average of 87 to 100 p.c. in 10 days. The addition of adrenotropic hormone (AIA4) did not seem to influence the effect of the thyreotropic hormone. weight occurred during these 9 days. Within 5 days after stopping the injections of the one experimental rat which survived, the metabolism was practically normal. Iodine seemed to have only a slight effect on the metabolic rate of these goitrous rats. Two of the animals received mg. potassium iodide daily for 10 days. During this time the rates rose from an average of 87 to 100 p.c. Preparation of thyreotropic hormone extracts. The presence of the thyreotropic principle can be detected in almost any simple extract of anterior pituitary glands if the very sensitive test object, the untreated hypophysectomized rat, is used. Loeser [1932] has described a method of preparing an active extract in the form of a dry powder. His process consists in extracting with aqueous ammonia glands dehydrated by acetone, and then removing proteins with trichloroacetic acid. The active principle is removed from the trichloroacetic acid filtrate by acetone. Alcohol and acetone are subsequently used as precipitating reagents. Junkmann and Schoeller [1932] have reported on the chemical properties of their thyreotropic extracts. We have used the following method in preparing highly active and concentrated extracts of the thyreotropic hormone. The combined filtrates obtained following the removal of the growth principle by treatment of the concentrate of the original extracts with Ca3 (PO4)2 [Collip, Selye and Thomson, 1933] are concentrated at low temperature and

13 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 23 pressure to a volume such as is equal to one-half the volume of the original glands used. The solution is saturated with ammonium sulphate and the resultant precipitate is removed by filtration. The moist precipitate is extracted on the paper with distilled water. The insoluble residue is discarded. This solution is saturated with ammonium sulphate and the precipitate is filtered off as before. It is extracted with distilled water and to the solution is added 2-5 volumes of absolute alcohol. The precipitate is removed by filtration and taken up in distilled water. Precipitation with 2-5 volumes of absolute alcohol is again carried out. A distilled water extract of this precipitate is very satisfactory for physiological work. Further purification can be carried out by repeated precipitation from 60 p.c. alcohol or 50 p.c. acetone. If this is done a great amount of the active principle passes into the filtrates along with the adrenotropic hormone. These filtrates are combined and from them very potent extracts containing both the thyreotropic and adrenotropic principles can be made [Collip, Anderson and Thomson, 1933]. Such extracts, if sufficiently concentrated, can be separated by isoelectric precipitation into an almost pure adrenotropic fraction represented by the precipitate and a mixture still rich in both of these principles represented by the filtrate. Of the desiccated thyreotropic extract 1 mg. has represented 200 units of the active principle. The yield in terms of units from the fresh glands is between 100 and 200 g. We have found the glands of cattle, pig and sheep to be excellent sources of potent extracts. We prefer, however, to use the anterior lobes of ox pituitary glands because of the low content of maturity principles in these glands. TABLE XIII. The stability of thyreotropic hormone (tested upon hypophysectomized rats). Thyreotropic Metabolic rate (p.c. of normal) hormone Days daily A dose Maximum c.c change Extract TGS Extract TGS after 1 year (Kept in sterile ampoules in the refrigerator) Extract TGS boiled 2 hours Extract TGP boiled i hour The thyreotropic hormone is apparently stable if kept under sterile conditions in the cold. It does not withstand boiling.

14 24 E. M. ANDERSON AND J. B. COLLIP. The thyreotropic hormone seems to be completely stable if kept under sterile conditions at a low temperature. A sample of the hormone has been tested on the hypophysectomized rat after being kept for one year in sterile ampoules in the refrigerator. Its ability to raise the metabolic rate of the hypophysectomized rat had not changed (Table XIII). The hormone is not, however, heat stable. We have found that if it is heated on a boiling water bath for 3 min. a part of the activity is lost; if it is heated for 2 hours all is lost. This is shown in Table XIII. We have found the thyreotropic hormone to be inactive when given by mouth. This was tested on young guinea-pigs which were fed amounts of the hormone 50 times the minimum effective dose by injection. There was no increase in metabolic rate after 11 days. SUMMARY. Some of the physiological properties of the thyreotropic hormone have been reported. Hyperthyroidism, as indicated by an elevated metabolic rate, has been produced in the guinea-pig and the rat by means of purified extracts of the hormone. The sensitivity of the rat to the hormone may be increased by the injection of Staphylococcus aureus vaccine in conjunction with it. Following hypophysectomy atrophy of the thyroid gland occurs which is associated with a decrease in metabolism. The average metabolic rate of 118 hypophysectomized rats was 74 p.c. of the normal. This may be raised temporarily to the normal level with amounts of the purified thyreotropic extract as low as 0-02 c.c. a day. The effect of prolonged treatment of the normal rat with large doses of thyreotropic hormone is a final lowering of the metabolic rate to the level of the hypophysectomized rat due to the production of an antithyreotropic substance. There is evidence that this substance is not formed by the pituitary gland, since the hypophysectomized rat develops a similar resistance to the hormone. A group of rats with goitre of unknown etiology, in which the metabolic rate was slightly below normal, was given thyreotropic hormone. Very severe hyperthyroidism was produced, associated with an increase in the metabolic rate, going as high as 262 p.c. of the normal in one instance. The method of preparation of a highly purified extract of the thyreotropic hormone has been described. This preparation has been found to be stable if kept in the refrigerator in sterile ampoules. It is not active when administered by mouth. It is destroyed by prolonged boiling.

15 THYREOTROPIC HORMONE OF ANTERIOR PITUITARY. 25 REFERENCES. Allen, B. M. (1920). Science, 52, 274. Anderson, E. M. (1933). J. Canad. med. A88. 28, 23. Anderson, E. M. and Collip, J. B. (1933). Proc. Soc. exp. Biol., N.Y. 30, 680. Anderson, E. M. and Collip, J. B. (1934). Lancet, p Aron, M. (1929). C. R. Soc. Biol., Paris, 102, 682. Baumann, E. J. and Marine, D. (1932). Proc. Soc. exp. Biol., N.Y. 29, Benedict, F. (1930-1). J. Nutrition, 3, 161. Closs, K., Loeb, L. and MacKay, E. (1932). J. biol. Chem. 96, 585. Collip, J. B. and Anderson, E. M. (1934). Lancet, p. 76. Collip, J. B., Anderson, E. M. and Thomson, D. L. (1933). Ibid. p Collip, J. B., Selye, H. and Thomson, D. L. (1933). Proc. Soc. exp. Biol., N.Y. 30, 544. Collip, J. B., Selye, H., Thomson, D. L. and Williamson, J. E. (1933). Ibid. 30, 590. Cushing, Harvey (1912). The Pituitary Body and it8 Di8order8. Philadelphia, Pa.: J. B. Lippincott Company. Eitel, H. and Loeser, A. (1932). Arch. exp. Path. Pharmak. 167, 381. Eitel, H., Lohr, G. and Loeser, A. (1933). Ibid. 173, 205. Friedgood, H. B. (1934). Johns Hopk. Ho8p. Bull. 56, 48. Grab, W. (1932a). Arch. exp. Path. Pharmak. 167, 313. Grab, W. (1932b). Ibid. 167, 413. Graubner, W. (1925). Z. klin. Med. 101, 249. Houssay, B. A., Biasotti, A. and Magdalena, A. (1932). Rev. Soc. Argent. Biol. 8, 130. Janssen, S. and Loeser, A. (1931-2). Arch. exp. Path. Pharmak. 163, 517. Junkmann, K. and Schoeller, W. (1932). Klin. W8chr. 11, Krogh, M., Lindberg, A. L. and Okkels, A. (1932). Acta Path. Microbiol. Scand. 9, 37. Loeb, L. and Bassett, R. B. (1929). Proc. Soc. exp. Biol., N.Y., 26, 860. Loeb, L. and Friedmann, H. (1932). Ibid. 29, 648. Loeser, A. (1931-2). Arch. exp. Path. Pharmak. 163, 530. Loeser, A. (1932). Ibid. 166, 693. Riddle, 0. and Polhemus, I. (1931). Amer. J. Phy8iol. 98, 121. Schittenhelm, A. and Eisler, B. (1932). Klin. W8chr. 11, Schockaert, J. A. (1932). Amer. J. Anat. 49, 379. Schockaert, J. A. and Foster, G. L. (1932). J. biol. Chem. 95, 89. Siebert, W. J. and Smith, R. S. (1930). Amer. J. Physiol. 95, 369. Smith, P. E. (1927). J. Amer. Med. As8. 88, 158. Verzar, F. and Wahl, V. (1931). Biochem. Z. 240, 37.