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The effect of parathormone on the mineral content of various vertebrates Rampone, Alfred J. 1950

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THE EFFECT OF PARATHORMONE ON THE MINERAL CONTENT OF VARIOUS VERTEBRATES by Alfred J . Rampone A Thesis Submitted i n Partial Fulfilment of the Requirements for the Degree of Master of Arts i n the Department of Zoology The University of British Columbia May, 1950 ABSTRACT The effect of parathormone ( L i l l y ) on the total mineral content of bones and muscles (percentage of ash per gram of dry tissue) was used as a measure of hormone activity. Goldfish^ froga, mice, chicks and pigeons were compared,. The effect of the para-thormone varied with the species. Goldfish showed no response even with massive doses at high temperatures. Frogs, mice and chicks showed a rise in percent bone ash. In chicks an i n i t i a l rise was followed by a depression. Mice showed the greatest increase in bone ash. Frogs were less responsive than mice but more res-ponsive than chicks. Pigeons showed a distinct softening of the bones. There was no change in muscle ash in any animals except pigeons, which showed a distinct rise* The parathyroid mechanism, whether it acts directly on bones or kidneys, does not seem to operate in aquatic animals. TABLE OF CONTENTS Page 1. Introduction....... ^  * * 1 2. Materials and Methods 13 a. Care and Treatment of Animals..... IS b. Source of Parathormone 16 c. Ashing Technique 16 3. Results.... 18 a. Goldfish......... 18 hi. Frogs.. 18 o. Mice..;.. 19 a . Chicks.,.......................... 20 e. Pigeons. 4. Discussion.. 23 5• Summary and Conclusions.................... 30 6. Literature Cited........................... 31 7. Appendix i THE EFFECT OF PARATHORMONE ON THE MINERAL CONTENT OF VARIOUS VERTEBRATES Sandstrom in 1880 was the f i r s t to distinguish sharply between the thyroids and the parathyroids, desig-nating the latter "Glanduae Parathyroideas n because of their positioja (Bard,1941) „ He regarded these glands as displaced, undeveloped fragments of thyroid tissue. His work was overlooked and Gley *** 1891 rediscovered them and demonstrated that they were physiologically distinct from, the thyroids (Bard, 1941). Grosohuff and Verdun later demonstrated, that they were also distinct embryologically (Waggener, 1930). The mammalian parathyroid glands are small compact bodies, usually four i n number, and closely apposed to or within the thyroid. I f they are removed the level of serum calcium f a l l s ; this in turn causes neuromuscular hyperexoitability, tremors of skeletal muscle, and panting which produces alkalosis. With calcium deficiency this leads to violent clonic and tonic convulsions In which the animal sooner or later dies from arrest of respiration. These symptoms are beat produced experimentally i n the dog and were at one time frequently seen i n humans following thyroidectomies which included the parathyroid glands. Colllp (1925) vas the f i r s t to obtain an active extract of the parathyroid glands. He dissolved ox glands i n boiling 5$ hydrochloric acid and after cooling removed the congealed fat mechanically. The proteins were then preolpitated out twice and the combined f i l t r a t e s constituted the desired extract. Since dogs react to the extract i n a more predictable manner than most other animals they have been used i n the biological assay. The bioassay involves the conventional graded dose-response method i n which the criterion of response i s an elevation in the blood calcium level. One (Hanson) unit i s one-hundredth the amount re-quired to raise the serum calcium level of normal 20 kg. dogs by 1 mg. per 100 ml. of serum. One ox parathyroid contains approximately ten (Hanson) units;. U n t i l Colllp obtained this extract knowledge of the parathyroid glands did not extend much beyond the faot that these glands were essential to l i f e . Boss and Wood (1942) gave evidence that the active principle of the parathyroid glands i s probably of protein nature or else i s closely associated with a protein carrier. This Is supported by the fact that the hormone Is Inactivated by both acidic and basic hydrolysis and by digestion with proteolytic enzymes. Alteration of the functional group of the protein moleoule, such as esterification of the earboxyl - 3 -group, also results in inactivation. Ultracentrifugation shows the parathyroid hormone to he heterogeneous, consisting of at least two components, one of molecular weight roughly 20,000 and another of 500,000 to 1,000,000. The activity i s believed to be associated with the low molecular weight material. The hormone i s dependent on the presence of free amino groups since the liberation of the phenolyo hydroxyls by alkaline hydrolysis does not restore activity. Kahlau C1940) says that the parathormone obtained by Colllp cannot be completely identical with the substance of the parathyroid glands - either i t possesses only partial function or i t represents a modified hormone. It i s probably identical with the substanee formed by abnormally enlarged parathyroids (tumors, hyperplasia) which leads to generalized osteitis fibrosa. Possibly the parathyroids possess two hormones, one promoting the calcification of osseold tissue and the other maintaining the blood calcium at a constant le v e l or raising i t i n hyperfunction. There are two main theories regarding the actual mechanism of action of parathyroid hormone. One school supports the theory that the hormone acts directly on the bones, stimulating osteoclastic activity and thus bringing about a decalcification of osseold tissue. Because of the reciprocal relationship between calcium and phosphorous the increase i n blood calcium as a result of this decalcification i s thought to bring about a decrease In blood phosphorous by excretion of the latter through the kidneys. The other school states that parathormone acts directly on the kidneys and that a l l other conditions are secondarily brought about by a lowering of the kidney threshold for phosphate; Because of the reciprocal relationship between calcium and phosphor-ous the lowered serum phosphate level results in an increased serum oaloium l e v e l , the excess calcium coming from the bones i n the form of calcium phosphate. Some of the evidence In support of each theory w i l l now be summarized: Pugsley and Selye (1933) administered parathormone daily to rats and found that numerous osteoclasts formed in the bone two days after the beginning of treatment. As treatment continued osteoblasts began to form and increased i n number along with osteoclasts. This was noted on the fourth day of treatment. At this time also the calcium excretion level was noticeably elevated. On the ninth day the osteoclasts began to disappear and i t was further noted that this period coincided exactly with the return of cal-cium excretion and serum calcium to normal levels. On the fourteenth day no osteoclasts were present at a l l but the number of osteoblasts had increased greatly. If treatment was continued further the condition known as "marble bone" was produced. This experiment certainly gives support to the idea that parathormone acts directly on the bones, but i t does not invalidate the theory that the hormone might act by directly affecting the kidney. Is osteoclast form-ation stimulated by parathormone i t s e l f or i s i t the result of the lowering of the phosphate level of the blood ? Colllp In an ar t i c l e written i n conjunction with Kewfeld (1942) favours the hypothesis that parathormone aots by affecting the excretion of phosphate by the kidney. These investigators established that the hormone has l i t t l e or no effect on the serum calcium i n nephrectomlzed animals. After ligatlng the renal vessels or ureters the serum cal-cium remained unchanged under the influence of parathormone administration. Re-establishment of urine flow of the animals injected with parathormone resulted i n a subsequent ris e i n serum calcium. These observations associate the hvperealeaemia of parathyroid extract with the excretory function of the kidney. They are in almost direct contra-diction to the earlier investigations of Pugsley, Collip et a l C1934) which showed that bone resorption took place under the influence of parathormone i n spite of nephrectomy. What, then, happened to the oalcium which was removed from the bones i n these experiments ? The serum calcium was found to remain unchanged. Two p o s s i b i l i t i e s arise* either com-plete nephrectomy differs i n some way from merely tying off the renal vessels, or else osteoclastic a c t i v i t y does not involve the removal of calcium from the bones. Newfeld and Colllp i n the same ar t i c l e gave further evidence to show that parathormone has a direct effect upon the excretion of phosphates, followed successively by hypophosphataemia, hypercalcaemia and hypercalcurea. Chanutin and Ludewig (1940) investigated the relation-ship between renal disease and the parathyroid glands. It was evident that parathyroid hyperplasia Is proportional to the degree of kidney damage i n patients; that renal damage i s frequently associated with hyperparathyroidism i n patients that the parathyroids are enlarged i n cases of renal rickets; that partial nephrectomy i n rats leads to an increase in the size of the parathyroid glands; that complete nephrectomy protects the dog from the effects of large doses of para-thyroid hormone; and that p a r t i a l nephrectomy i n rats de-creases, the effect of parathormone in raising the serum calcium. Tweedy and Campbell (1944) noted an Immediate increase i n the urinary excretion of labelled phosphorous on adminis-tering parathyroid extract. Logan (1939) observed the same afreets i n young dogs. He also noted that the increase of phosphate excretion i n urine occurs i n advance of the m a x i m u m blood calcium increase. He interpreted this to mean that the solution of bone i s caused by a depletion of blood and tissue inorganic phosphate resulting from Increased phosphate ex-cretion by the kidney. The hormone has been administered to nephrectomlzed dogs by Tweedy et a l (193a). They found that large doses produce no greater Increase of blood calcium than that observed as a result of nephrectomy alone. The phosphate retention resulting, from nephrectomy increased the blood inorganic phosphate 80$ i n two to 3 i x hours. This increase was greater than that observed i n the terminal stages resulting from the administration of f a t a l doses of para-thyroid hormone. As a result of the rapid accumulation of phosphate i n the blood and tisane following nephrectomy, the solution of bone would be d i f f i c u l t to demonstrate. The results are therefore not conclusive evidence i n support of the thesis that the action of parathormone i s exclusively on the kidney. Logan (1939) made determinations of urinary phosphate, blood calcium, phosphorous and magnesium at one hour intervals for four hours after the administration of a single large dose of parathormone In an effort to decide whether the changes are due to increased phosphate excretion or to bone resorption. He found that the solution of bone occurred immediately since the blood calcium rose In the f i r s t hour; but he found also ah Increase i n phosphate ex-cretion and a decrease i n blood phosphate i n the f i r s t hour; Observations at twelve to twenty-four hours showed that the inorganic phosphate increased but the blood calcium remained at the same high l e v e l . Logan interprets this as being the result of phosphate retention by the kidney, but i t would seem more lo g i c a l to say that i t might be the result of the mobilization of calcium phosphate (Ca 3 (PO^g from the bones to the blood. This would increase both the calcium and the phosphate content of the blood. The results obtained here must be interpreted to indicate that parathormone acts by increasing the urinary excretion of phosphorous as well as by causing a solution of bone. The work just summarized i s devoted entirely to 8 mammals. Lower vertebrates have been, rarely studied. It would be interesting to investigate this problem in the lower forms to determine the p o s s i b i l i t y of evolutionary trends i n the methods of calcium regulation. Parathyroid glands are not generally recognized In f i s h although Sqhereschewsky (1940.) described what she thought was parathyroid tissue in the Lebistes larvae (Family Gyprlnodontldae). She described In some detail glandular c e l l s i n the pharyngeal g i l l region of these f i s h which resembled the cells of the parathyroid glands of higher animals. I t i s generally held, i n spite of a few claims that f i s h possess parathyroids, that these glands f i r s t make their appearance i n amphibians. Even In these animals the Indications are that they serve merely as accessory organs for calcium regulation. Zhenevskaya (1948) carried out some rather b r i l l i a n t experiments using Rana temporarla (the grass frog) and Rana ridibunda (the lake frog). His work aimed to prove the statement of Studitsky (Zhenevs-? kaya, 1941) that the parathyroid apparatus, which appears i n amphibia for the f i r s t time In the history of vertebrates, has not yet become an organ essential to l i f e . He attempted to determine evolutionary changes i n the mechanism for cal-cium regulation and to show that the role of the parathyroids In the regulation of calcium In amphibia was merely subordin-ate to the vitamin D mechanism of calcium metabolism found In f i s h . Zhenevskaya found l i t t l e change In blood calcium after parathyroidectomy In almost a l l cases. Tetany was observed; i n one or two instances but death did not ensue possibly due to the restoration of the calcium, le v e l by some other mechanism. In Rana catesbiana, on the other hand, death invariablly followed parathyroidectomy (Waggener,193Q). In order to determine the intensity of this other calcium regulating agent Zhenevskaya subjected parathyroidectomized and normal frogs to "washing out" experiments involving the replacement of the entire blood volume with calcium-free Ringer's solution and noting the time required for the calcium level i n the vascular f l u i d to restore i t s e l f to the normal le v e l . Both parathyroidectomized and normal frogs restored their calcium levels at the same time and at the same rate. This i s rather conclusive evidence that amphibia' actually possess, besides parathyroid glands, some other powerful factor for calcium regulation.. Amphibians are descendants of transitional forms which bridged the gap between the water and the land vertebrates. It would be interesting to find that, from the time they put i n their appearance, the parathyroids have been indis-pensable i n the evolution of the organism. It i s conceivable that the parathyroid glands may have developed as a means of overcoming; one of the d i f f i c u l t i e s of making the ascent to land. In the amphibians the parathyroids are absent in the early aquatic l i f e of the individual. In the Anura the parathyroids have been reported by Mauer to develops^ during the larval period (Waggener, 1930), while i n the Urodeles they appear at the time of transformation. These glands are not encountered In amphibians which do not transform, such ^ - 10 -the Axojot0l Nejjturus. Also, these glands are derived from the branchial epithelium which was originally concerned with respiration. I f the above observations are correct, the place of origin, the time, and the circumstances of their appearance may eventually be found to f i t into the ultimate explanation of the physiological function and biological significance of the parathyroids. It i s con-ceivable that they may owe their functional behavior to their place of origin and that they may have some pertinent connection with the mode of respiration found i n the land vertebrates. I f this were true the effects of parathyroid-ectomy on amphibians would be problematical by virtue of the dual mode of respiration found i n these forms. Waggoner (1930) further suggests that the para-thyroids may function to render toxic substances harmless since such substances have been shown to accumulate in the • , • . . . . . . . . ^ blood and urine of animals which have been parathyroidec-tomized. These toxic agents probably arise from the metabolic substances of the animal and the need for their removal may have arisen i n land animals which of necessity have a less permeable surface. There are a number of different p o s s i b i l i t i e s of making a study of the effect of parathyroid hormone; one may study the effect of the hormone on calcium and phosphorous excretion; or the level of calcium and phosphorous i n the bones; or i n the blood serum, or other tissues of the body. It has been well established that parathormone i s a regulator of mineral metabolism i n mammals. Its general action seems to maintain the serum calcium at a constant level either by mobilizing calcium from the bones or by lowering the renal threshold for phosphorous. How i s mineral metabolism regulated in\ forms which have no parathyroid glands ? What i s the effect of parathormone on mineral metabolism i n general ? The problem of this thesis i s to test the effect of parathormone on the total mineral content of lower vertebrates rather than to study the more specific effects on calcium and: phosphorous regulation. The study includes forms in which parathyroids are not recognized, transitional forms which appear to have a dual mode of mineral metabolism and those forms i n which the parathyroids are essential for the proper regulation of minerals. To make the problem more specific the work has been confined to determining the effect of parathormone on the total mineral content of bones and muscle. - 18 ACKHOWLEDQMENTS The writer extends his thanks to Dr. ¥. A. Clemens for making this work possible; and to Dr.. I.i MoT; Cowan and Mr. Charles Guiget for the generous loan of the beetle colony. A special note of thanks goes to Dr. W. S. Hoar, under whose able supervision t h i s work was carried out, for his invaluable aid and his constant reassurance through-out the year. The author i s indebted to fellow students: Mr. G. D. Potter for his kindly help and criticisms throughout the yearj Miss Aline Redlioh for help in translating a German art i c l e and to both Miss Redlioh and Mr• M. Jampolsky for help i n caring for the experimental animals The Slavonics department under Dr. G. St. Clair-Sobell deserves a special note of appreciation for trans-lating Zhenevskaya*s very pertinent Russian a r t i c l e . - 13 MATERIALS AND METHODS In this series of experiments the effects of para-thormone on the total mineral content of bones and muscles was used as a measure of hormone act i v i t y . In a l l 225 goldfish (Carasslus auratus), 12; frogs (Rana pl.pl.ens), 30 mice (Mus domestlous). 12 chicks (Gallus domesticus). white leghorn cockerels and 2 pigeons (Columba ectopistes migra- torius) were studied. The most complete experiments were carried out on goldfish since the effect of parathyroid ex-tract on forms having no parathyroid glands was to be clearly established. The numbers of animals used appear in the various tables of results. Goldfish were acclimitized to definite temperatures and maintained in thermostatically controlled aerated aquaria. They were fed on brine shrimp eggs and injected Intraperlt-oneally with the hormone. Details of temperatures and dosages appear i n the tables of results. Large numbers of f i s h were required to obtain suf f i c -ient bone and muscle tissue to carry out the ashing proced-ures. In order to standardize the ashing methods and deter-mine individual variations preliminary experiments involving approximately twenty/ goldfish were carried out. A l l f i s h were k i l l e d by tapping on the head and the bones and muscles separated as follows: the skin was removed by outting behind the g i l l s and pulling away by means of a pair of fine forceps. - 14 -The muscles along the back were taken: by undercutting with one point of a pair of fine forceps and drawing the forceps along between the muscle and the vertebral column. The skeleton with i t s adhering tissue was placed i n warm (not boiling) water for 80 minutes to half an hour. The bones were thea picked clean with a pair of fine forceps. Only the cranium, the vertebral column and the fi n s were l e f t behind. The bones from each group of f i s h were mixed to-gether and re-divided into as many portions as seemed per-missahle. Muscle was treated in the same manner. Determin-ations were usually done i n t r i p l i c a t e . Frogs were maintained unfed In a tank throughout the experimental period. Injections were Intraperitoneal. Details of dosages, number of frogs used and variations due to sex appear in the tables of results. One group was studied i n February and another i n Apr i l of the same year. The f i r s t group received no parathormone and were not seg-^ regated as to sex. A l l frogs were k i l l e d by pithing and bleeding from the heart. A l l bones except the cranium, vertebral column and phalanges were taken and were prepared as described above. Muscles were taken from the limbs only. Mice were maintained on a standard diet of fox chow and water. Ten were kept In one cage to be used as controls and twenty In a second cage to be used as experimental animals. Injections were intraperitoneal, the dosages and the number of animals used appearing i n the tables of results. The animals were k i l l e d by hitting sharply on the head with a blunt instrument and cutting the throats to Insure maximum bleeding. Muscles were taken from the legs and along the back. The entire skeleton was used but the process of cleaning was different from that i n goldfish and frogs. Beetles (Bermestes maoulatus Degeer 1774 (B. vulpinus Fab. 1781) } which clean bones completely i n two or three days, were used because the other procedure proved too tedious. These beetles were maintained In a constant temperature incubator. 'Oncleaned bones had to be dried i n a i r before placing them i n the beetle colony. Chicks were maintained i n a large incubator and fed the usual diet of chick starter and water. Seven chicks served as controls and five received subcutaneous injections of parathormone as indicated i n the tables of results. Chicks were k i l l e d by gas. The muscles taken were those of the limbs and those above and around the pectoral and pelvic girdles. The bones used were those of the pectoral and pelvic girdles and the limbs except those above the humeri of the forelimbs and below the tibiae and fibulae of the hind limbs. Individual variations i n ash content, the age of the chicks and the dosages of parathormone used are indicated i n the tables of results. A l l injections were subcutaneous. Only two pigeons were available. One served as a control and the other received intraperitoneal injections of parathormone as indicated In the tables of results. The birds were the same age. Both birds were k i l l e d by gas. 16 -The muscles taken were those of the breast only. The same bones were taken as for the chick but with the breast bone included. The cleaning method was as for the chicks and mice. Parathormone ( L i l l y ) was used as a source of para-thyroid hormone. This i s an active extract of beef para-thyroids and contains twenty units per cc. One unit of this extract obtained by Gollips method i s defined as one-hundredth the amount required to raise the serum calcium of normal 10, - 12 kg. dogs by 5 mg. per 100 cc. of serum i n 16 - 18 hours. Because of the relative intolerance of other animals to the hormone, average, dosages given were approximately ten times that required i n dogs when calculated on a units per gm. basis. Goldfish weighed approximately 10 gms. each, frogs 25 gms., mice 20 gms., chicks 100 gms. and pigeons about 150 gms. Total mineral content was determined by ashing as follows: the fresh tissue was placed i n an oven at 95 - 100eC for not less than 48 hours to extract a l l moisture. The tissues were then placed in s i l i c a crucibles previously weighed to 4 plaees on the chalnomatic balance. The cruc-ibles containing the tissue were cooled for exactly one-half hour i n a vacuum dessicator before being re-weighed. They were weighed i n the same sequence i n which they were removed from the oven. Preliminary /carbonizing was done oyer a small bunsen flame u n t i l a l l smoke was expelled. The crucibles were then placed In a small muffle furnace at - 17 -525 - 550° C and l e f t overnight. Constant ash weight oould usually be obtained in less than 8 hours. The cruc-ibles were removed from the muffle furnace and when s u f f i c -i e n t ly cool a few drops of water were added to each. They were then gently heated over a hot plate u n t i l dry and replaced in the muffle furnace for one or two more hours. Cooling and weighing of ash were exactly as for dry tissue. A l l crucibles were re-weighed before each set of determin-ations. A l l animals were k i l l e d and determinations carried out twenty-four hours after the injections designated in the tables. With the exception of goldfish skeleta, a l l bones were crushed i n a mortar before drying. 18 -RESULTS GOLDFISH Effect of Temperature The effect of moderate variations in temperature are shown i n Table I. There i s no significant change i n ash content of either bones or muscles. -Effect of Parathormone Injections Tables H and H I shows that the parathyroid hormone has no effect on the total mineral content of bones or musoles. Massive doses given at high temperatures (table HI) produce no particular change i n mineral content. The hormone was found to be absolutely non-toxic since a l l f i s h survived overdoses for a period of four days• FROGS Table IV compares the normal ash content of frogs k i l l e d in,February with frogs taken from the same tank in A p r i l . There is an increase i n bone ash with no change In muscle ash. The difference in total mineral content of bones from males and females (Table IV) i s also quite marked, being higher i n females. There i s no significant difference i n muscles. Parathormone injections as indicated bring about an increase i n bone ash with no change in muscles. MICE Table Y shows that parathormone increases the ash content of bones while i t seems to have no effect on muscle. The slight increase i n muscle ash shown in Table IV i s probably insignificant. A l l mice were males. Fig. 1 shows these changes better. ' I I Control Y/A ^ Units Parathormone Fig. 1 The effect of parathormone on the total mineral content of bones and muscles of micei CHICKS - 20 -The variations in bone and muscle ash content from one individual to another are slight as shown in Table VI • The i n i t i a l effect of parathormone i s to increase the ash content of bones by about 2$. With prolonged administration the effeot i s diminished u n t i l on the fourth day after increasing daily dosages the bone ash i s decreased about 2$ below normal. Muscle i s unaffected. There i s no difference i n ash content between 12 day old chicks and 16 day old chicks. Fig. 2 shows the slight up and down effect pro-duced in bones by parathormone injections. Muscles show no significant change from the normal as shown in Fig. 2. J Control Fig. Z The effect of parathormone on the total mineral content of bones and muscles of growing chicks. - 2 1 -PIGEONS Parathormone evidently softens the bones of pigeons although the survey was not extensive enough to permit con-clusions to be drawn. Only two pigeons were available at the time. These were from the same hatching. A noticeable increase was noted i n the muscle as well as in the bone ash. The beetles, which were used to clean the bones, removed both the adhering soft tissue and a l l the organic material which remains where calcium s a l t s are removed by the para-thormone. The photograph (Fig. 3) ill u s t r a t e s this. The bones from the injected pigeon are perforated and jagged throughout. Fig. 3 The effect of parathormone on the bones of pigeons. Bones on l e f t are from injected bird. Bones on right are from control bird of same age. - 22 Prior to killing the birds i t was noted that the one which had received the injections was not able to support i t s own weight - further evidence that the bones had become softened. Table vTI shows only slight changes in the ash content but, in this case, this did not mean that the bones did not become soft and fibrous. DISCUSSION Several authors have reported species differences among mammals In the aotion of parathormone. Stewart and Percival (192?) found that parathormone raises the serum calcium more i n cats than In rabbits but less than In dogs. Tholldte (1928) found the same species difference regarding the action of parathormone. He found that repeated doses did not show a summation of effect in cats and rabbits as has been reported to occur i n dogs. Jaffe et a l (1932) gave further evidence that rabbits are relatively tolerant to parathormone. He found the same with guinea pigs but the resistance of these animals i s not so strong as In rabbits. Burrows (1938) found that parathyroid extract retards growth i n young rats, the relative retardation being propor-tional to the dally dosages. Over three units per day re-sulted i n a rapid loss i n weight and death In a few days. Par t i a l l y parathyroidectomized rats tolerated the dosages better; Microscopically Burrows found that early injections brought about a resorption of bone, a decrease i n osteoblasts-and their _change to fibrous tissue - and an increase i n osteo-clasts leading to osteitis fibrosa. Continued injections, however, lead to a reversal of these processes, producing hypercalcification of bone leading ultimately to "marble bone" disease. S t i l l further injections cause a return to normal, m the present series of experiments only an Increase - 34 -in the total mineral content was detected except In the case of the chicks which showed an Increase and a subsequent f a l l i n bone ash. The logical explanation for the increase In each case i s the time factor. Each series of determinations shown i n the tables represents the ash content twenty-four hours after the animal had received the designated injection, m a l l probability the period when the ash content was low -had been bipassed since parathormone i s known to act quite rapidly. The various degrees of susceptibility to the hormone can probably be attributed to class differences since the dosages were approximately similar with regard to body weight i n each case. Since species differences in mammals have been found by a number of investigators we would expect an even greater divergence among the various classes of animals* In spite of the administration of parathormone there i s always the possibility that the ash content w i l l remain unchanged even though other changes are taking place. One chemical element might take the place of another element removed by the action of the hormone. Cox and Imboden (1936) produced soft, thick, knobbed bones, easily l i a b l e to fracture by injecting parathormone into rats and keeping the animals on a high phosphate diet. But i n spite of this the bones had a normal ash content. Goldfish did not respond to the hormone even in ex-tremely large doses. The mortality rate was not abnormal - 35 -Thus mammalian, parathyroid extract Is completely non toxic in these animals. We might expect such negative results In f i s h In the light of the fact that parathyroid glands are not generally recognized in any class of animals below amphibia. Evidently parathormone does not have any pharma-cological nor specific action on bone or kidney. It Is interesting to find a definite sex difference in frogs with regard to bone mineral content. This d i f f e r -ence was recorded in the spring, at the height of the mating season. It would be interesting to investigate sex difference at other times of the year. Rana plpiens was surprisingly responsive to parathormone. Species differences are quite marked among frogs as reported by earlier investigators (Waggener, 1930.) and Zhenevskaya, (1948). In order of responsiveness frogs rank second to mice in these Investi-gations and the mode of response i s the same i n both. Mice received equivalent doses of parathormone to those given frogs. Their susceptibility i s s l i g h t l y greater using the Increase In mineral content as the criterion. This might be expected since the parathyroids have been definitely established as essential to l i f e in mammals. In amphibians the functional significance of these organs i s problematical. On the other hand the difference in response might be a temperature effect. The mortality rate as a result of parathormone injections i s rather high In mice, seven of the experimental animals dying two to three hours after receiving the injections. Autopsy revealed lesions - 26 -i n the intestine which were not the result of injury from injection. It has been pointed out that f i s h can withstand much larger doses with no apparent 111 effects. There were no deaths among the amphibians during the experimental period. The results obtained with mice are in f u l l agreement with the investigations carried out by Silberberg and Silber-berg (1941). These investigators found that parathormone caused an increased calcification, degeneration and o s s i f i -cation of the epiphyseal cartilage i n 5 weeks old mice. But i n these same mice there was no stimulation of the pro l i f e r -ation of the epiphyseal cartilage at a time when under normal conditions the proliferation of this region i s s t i l l quite marked. There was an increase i n the number of osteoblasts i n the subeplphyseal layer, leading to a temporary increase i n the number and thickness of the trabeculae. No changes reminiscent of ostei t i s fibrosa could be produced even after prolonged administration of the hormone, although this condition was produced i n rats and guinea pigs subsequent to administering equivalent doses. The results obtained in chicks are interesting since other Investigators have failed to obtain any positive re-sponse i n fowls. In our experiments chicks are definitely more tolerant to the action of parathormone than the other higher vertebrates studied but the results Indicate that they are not entirely immune. Spisni (1946) observed that parathormone had a parti a l inhibitory effect on the growth of young rabbits but found i t to have no effect on the growth 2? -of young chicks. Rabbits, as has been pointed out, are among the most resistant animals themselves. Avery et a l (1940) found no significant increase i n the blood calculm of fowl of a l l types even with large doses of parathyroid extract. The same authors (1940) obtained negative re-sults In testing the effeet of the hormone on calcium retention and excretion. No one has studied the ash content of parathyroid Injected fowls. Gbok and Robertson (1940) state that the normal ash content of the bones of young; chicks Is about 36,1$. The author finds i t to be more of the order of 51*3$. The discrepancy l i e s in the method of cleaning, at least in part. The method employed here insured that the bones were stripped completely of a l l adhering tissue as well as their contained marrow. We would expect a higher ash content under these conditions. . Off a l l the animals studied pigeons were probably the most responsive to the parathormone. The bone ash content does not show this but f i g * 3 definitely Indicates a soften-ing of osseold tissue* The increase In ash content of the bones of the experimental animal was due to the fact that the beetles used In cleaning them were able to enter the marrow cavity through the epiphysis which had been eroded. The bones of the control animal, because of their relative hardness prevented the beetles from entering the marrow cavity. This condition combined with the fact that the fibrous material had been eaten would tent to raise the ash content per gram of dry tissue. - 28 -The effect of parathormone on muscle tissue has been l i t t l e studied. Eong (1930) reported a drop In muscle calcium of dogs after thyroparathyroldectomy. However,this drop was less abrupt and less extensive than the f a l l of serum calcium. This suggested that the diminution in the muscle was the result of the drop in the blood. The same author tried the effect of parathormone on muscle calcium and found the results variable, but in no case did a de-pression of muscle calcium occur. This is in agreement with the results of the present investigation. In no case was there a significant change in muscle ash except in the pigeon which showed a visible increase. Since the survey was not very extensive the difference could be attributed to individual variations but this is not likely In view of the definite effect the hormone had on the bones. The negative results obtained with the other animals in this investigation can be explained In the light of previous work. Under (1935) found that the muscle calcium i s quite variable from one species to the next and even in the same species there is a much greater individual varia-tion than In the serum calcium. These are well known facts for In any series of determinations by one worker using one kind of animal under the same conditions, the highest figure is seldom less than three times the lowest. This makes i t difficult to demonstrate the small changes brought about by such an agent as parathormone. Burns (1933) found the same variations. It is not likely that such a procedure as ashing would detect small changes resulting from parathormone administration unless done In rather large quantities. Dennis and Gorley (1932) attribute these wide variations to the contamination of the tissue with blood and lymph. Few investigators have studied the effect of para-thormone on the ash content of tissues. Pisa (1934) report-ed no change i n the total ash content of rats under para-thormone administration. Frugoni (1934) reported an increased percentage of body ash In thyroid or parathy-roide eternized rats. The present experiments have been the f i r s t to test the effect of parathormone on amphibia and f i s h . Amphibians respond but f i s h are completely immune. The theory of Waggener (1930) that the parathyroids developed with the land habitat should be more f u l l y investigated. It presents an Interesting problem as to how mineral metabolism Is regulated i n aquatic forms. There Is the po s s i b i l i t y that vitamin D i s the sole factor . On the other hand the vitamin D mechanism may be aided by the presence of special mineral secreting and absorbing c e l l s . - 30 -SUMEMRY AND CONCLUSIONS With the exception of goldfish a l l animals showed an effeot of parathormone but the effect varied with the species. The pigeons probably are most reactive but the use of only two birds does not permit definite conclusions to be drawn. Pigeons were the only ones to show a pronounced effect on muscle tissue with a rise i n total mineral content. A l l animals with the exception of chicks and goldfish show a r i s e In percentage bone ash. Goldfish did not re-spond and chicks showed an I n i t i a l r i s e followed by a de-pression. Mice showed the greatest increase In bone ash. Frogs were less responsive than mice but more responsive than chicks. There i s a difference In bone ash content according to sex i n frogs. There Is a difference i n bone ash between winter frogs and spring frogs of the same species. Chicks react to the hormone only slig h t l y . Individual variations i n ash content are only slight. A new method i s described for cleaning bones of adhering tissue for this type of work. Variation i n temperature does not affect the ash content of goldfish. Parathyroid mechanism, whether i t acts directly on bones or kidneys, does not seem to operate i n aquatic animals. The mechanism of mineral balance remains a problem. - 31 -\ LITERATURE CITED Avery, T. B., Scott, HOI., and R. M. Conrad, Effect of parathyroid preparations on the blood calcium of the fowl. Poultry Sci., 19: 321 - 325, 1940. Bard, Philip, Macleod ts physiology In Modern Medicine. 9th ed. C.V. Mosby Co., 1941. Burns, G. M. The calcium'content of muscle. Biochem. J., 27: 22 - 32, 1933. Burrows, R.B. variations produced in bones of growing rats by parathyroid extracts. Am. J . Anat., 62: 23? - 290, 1938. Chanutin, A., and S. Ludewig, The effect of renal Insuffic-iency on the response of serum calcuim after administration of parathyroid hormone in the rat. Am. J> Physiol., 129: 242 - 243;, 1940. Collip, J . B.,The extraction of a parathyroid hormone which w i l l prevent or control parathyroid tetany and which regulates the level of blood cal-oium. J . b l o l . Chem., 65: 395 - 438, 1925. Cook, J.W., and E.I.Robertson, The effect of minerals and vitamin D on the percentage bone-ash of young chicks. Poultry Sci., 19: 385 - 388, 1940. Cox, W; M., and M. Imboden The role of calcium and phosphorous in determining reproductive success. J . N u t r i t i o n , 11: 147 - 175, 1936. Denis, ¥. and R.C.Corley A study of the effect of excessive calcium ingestion on the ealeuim content of tissues with and without the application- of ultra-violet l i g h t . J". b l o l . chem., 66: 609 - 617, 1925. Fong - Yen Hsu and Chtao Isal The calcium content of the skeletal muscles"after thyrdparathyroid-ectomy and parathormone injections. Chinese J. Physiol., 4_: 423; - 29, 1930 (abstract only). Frugonl, Piero The total content of calcium and phosphorous of rats after thyroparathyroidectomy and the effect of an excess of parathormone. Arch. Sol.Med., 58: 237 - 252, 1934 (abstract only). - 32 -Jaffa, H, L., Bodansky, A., and J.E.Blair The effects of parathormone and ammonuim chloride on the hones of rabbits, J . exp. Med. 55:695 -701,1932. Kahlau, G. The parathyroid glands and calcuim metabolism. Frankfurt Z. Path., 54: 474, 1940 (abstract only). Linder, G.C., Effect of parathyroid hormone and of tuber-closis on the serum and tissue calcuim of guinea pigs* Bioehem. J., 29: 8095 - 2100, 1935. Logan, M.A. The early effects of parathyroid hormone on the blood and urine. J*. b i o l . 8hem., 127: 711-719, 1939i Newfeld, A.H., and J.B.Collip The primary action of parathyroid hormone. Endocrinology, 30: 135 - 141, 1942. "~~ Pisa, Manlio The "total calcium and phosphorous in rats treated with small doses of parathormone. Arch. S c i . Med., 58: 629-632, 1934 (abstract only) • Pugsley, X. I., Colllp, J.B.,, Selye, H;, and D. L. Thomson. . Observations, concerning, the mechanism, of para-thyroid hormone action. B r i t . J.Exp. Path., 15: 335 -336,1934. Pugsley, L. I., and Hans Selye The histological changes i n the bone responsible for the action of parathyroid hormone on the calcium metabolism of the rat. J . Physiol.,,79: 113 - 117,1933. * Ross, W.F., and T.R.Wood The partial purification and some observations oh the nature of the para-thyroid hormone. J. b i o l . Chem., 146: 49-58, 1942. ^ Schereschewsky, Helene Uber den bau des epithelgewebes i n der pharyngeal klemenregion der knochen-fische-. C.R. (Doklady) Acad. Sol. URSS, N.S., 29: 165 - 168,.1940. : V Silberberg, M., and R. SUberberg Effects of hormones on the skeleton of mice, guinea pigs, arid rats. Endocrinology, 89: 4175 - 482, 1941. - 33 -1/ Spisni, Dino Experimental studies on hyperparathyroidism provoked in chickens. Boll.Soo. I t a l . Biol., 21: 71 -75, 1946 (abstract only) Stewart, C. P., and G. H. Percival The action of the parathyroid hormone on the calcium content of the serum and on the absorption and exoretion of calcium. Biochem. J., 21: 301 - 313, 1927. Tholldte, M, Calcium metabolism and parathyroid hormone in different kinds of animals. Krankheitsforseh, 6: 397 - 492, 1928. (abstract only). Tweedy, W.R., and W.W. Campbell The effect of parathy-roid extract upon the distribution, retention and exoretion of labelled phosphorous. J . b l o l . Chem., 154: 339 - 347, 1944. Tweedy, W.R., Templeton, R. D,,, and E.A.MeJunkln, The effeot of complete renal insufficiency on the action of parathyroid hormone in the dog. Amer. J . Physiol., 115: 514 - 519, 1936. Waggoner, R.A.,An experimental study of the parathyroids i n the anura. J . exp. Zool.. 57: 13-55,1930. i / Zhenevskaya, R.P., Restoration of cal culm content in parathyroidectomized frogs. C.R. (Doklady) Acad. S c i . U R S S., 60 (3): 533 -535,1948. - i -TABLE I The e f f e c t of moderate va r i a t i o n s i n temperature on the percent ash composition of bones and muscles of g o l d f i s h . Temp.°C. No,Fish No. Deternu Muscle #Ash Range Bone Np % Ash Bet jrm. ' range" 13 16 go 20 20 20 8 4 Ave-rage 5.72 5.78 5.62 5.30-5.99 5.69-5.92 5.41-5.72 Ave-rage 61.65 SO .30-53.0 62 .53 60.88 51.75-52.97 59.51-63.73 TABLE II The effect of average doses of parathormone on the ash content of bones and muscles of goldfish. Dosages increased 1 unit per day. Fish at 20°C. I o. Fish Muscle I1 # Ash I O . eterm. lone # Ash No. Determ. Ave-rage Range Ave-rage Range 1 Unit Inj. 10 5.56 5;47 -5; 68 3 62.18 61.21 -62.99 3 Control 10 5.67 5.67-5.68 2 61.41 60.86-61.96 2 2 Units In;). a 5.52 5.34-5.71 3 61.62 61.44 -61.93 3 Control 9 5.87 5.62-6.13 3 62.40 61.96 -62.90 3 4 Units InJ. 9 5.65 5.55-5.74 3 61*09 60.62-61.66 3 Control 10 5.57 5.51-5.60 60.86 59;43 -62.43 3 5 Units In j . 10 5.65: 5.59-5.68 3 61.64 61.33-62.11/ 3 Control 9 5.58 5.46-5 .66 3 61.29 60.35-62 .£6 3 10 Units Inj. 5 5.59 1 58.75 1 i i l TABLE III Effect of large doses of parathormone and extreme temperatures on bone and muscle ash content of goldfish, Dosages 10 units per day for 3 days. No.Fish 15° C Control InJ. 10 Unit* 30 Unit s 10 30°C Control Inj. • 10 Unitb 30 Unit 3 10 10 10 10 10 Muscle % Ash Ave-rage 5.59 6.47 5.56 5.46 5.65 5.37 So. Determ. Bange 5.51-5.67 6.18-6.81 5.40-5.67 5.23-5.73 5.55-5.73 5.52-5.84 ;2one fo Ash No. Determ. Ave-rage 60.61 61.49 62.20 61.57 61.97 61.46 Range 60.12-61.29 60.97-62.32 61.59-62.48 61.12-62.11 61.45-62.78 61.20-61.77 TABLE J\T Effect of parathormone on the hone and muscle ash content of frogs. Dosages Increased 5 units per day. Seasonal and sex differences in ash content. No* Frogs Muscle fo Ash ro. -)eterm. Bone $ Ash No; Determ. Average Range > Average Range Group I (February) Control 6 4.44 4.37-4.59 60.93 5 7.29-52.64 ' 4 Males Fem-ales Scales Fem-ales Males. Fern- I ales ales Fem-ales. Group II (April). Gontrol 2 r 4.12 3.77- 4 56.90 : 56.35->7.63 4 InJ. . 6 Units 2 4.35 4.32 4.12-4.58 4.16-4.51 4 64.83 58.37 64.43 65.14 - 67.61 69.14 4 12 Units 2 4.10 3.36-4.51 4 68.62, 67; 69 70.05 * 4 - V -TABLE V Eff e c t of parathormone on the bone and muscle ash content of mice. Dosages increased 2 u n i t s per day f o r 3 days. No.Mice Muscle % > Ash , ro. ieterm. Bone fo Ash Mo. )eterm; Average Range Average Range Control 8 4.46 4.36 -4.53 5 53.74 - 52.7Q-54.50 4 Intf. . 2 Units 4.58 4.52-4.61 3 59.25 57.88-6i.45< 3 6 Units 6 4.79 4;67-4.98 3 59.77 . 59*56 59;88 3 TABLE VI Effect of parathormone on the bone and muscle ash content of chicks. Dosages increased 5 units per day. To.Chieks IS days old •Control 1 *Contro; L 1 13 days old Control. Inj. (20 Units) a 15 days old Control 8 Inj. (.30 uni-ds) a 16 days bid. Control m j , (35 Units)! Muscle % Ash Average Range 4.46 4.72 4.76 4.65 4.80 4.57 4.57 £.75 4.38 4.78 4.53-5 .04 4.'57-4.98 4.45-4.82 4.55-4.93 4.31-4.89 t.53-1.6G U53-5.05 No; Determ. Bone % Ash NO. : Jeter ml. 3 Average Range 51.10 51.54 51.30 53.47 51.28 52.71 51.38 49.38 51.02-51.18 49.92-53.26 51.11-51.67 52.71-54.32 51.13-51.42 51.29-53.72 50.37-52.22 18.76-i9.70 2 2 Kept separate to determine individual variations. - v i i -TABLE VII Effect of parathormone on the bone and muscle ash content of pigeons. Dosages increased 10 units per day. No.Birds Contrc1 (180 q m t for S s days) 1 Muscle % Ash Average 4.60 5.68 -Range 4.46-4.72 5.47-5.91 WTi Determ. I Bone % Ash TJbT Deterktt Average 49.31 51.89 Range 48.49 49*88 3 51.18-52.78 

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