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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  A l f r e d J . Rampone  A Thesis Submitted i n P a r t i a l Fulfilment of the Requirements for the Degree of Master of Arts  i n the Department of Zoology  The U n i v e r s i t y of B r i t i s h Columbia  May, 1950  ABSTRACT  The effect of parathormone ( L i l l y ) on the t o t a l mineral content of bones and muscles (percentage of ash per gram of dry tissue) was used as a measure of hormone a c t i v i t y .  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 r i s e was followed by a  depression. ash.  Mice showed the greatest increase in bone  Frogs were less responsive than mice but more r e s -  ponsive than chicks. of the bones.  Pigeons showed a d i s t i n c t softening  There was no change in muscle ash in any  animals except pigeons, which showed a d i s t i n c t  rise*  The parathyroid mechanism, whether i t acts d i r e c t l y on bones or kidneys, does not seem to operate i n aquatic animals.  TABLE OF CONTENTS  Page 1.  Introduction....... ^ * *  2.  Materials and Methods  3.  1 13  a.  Care and Treatment of Animals.....  IS  b.  Source of Parathormone  16  c.  Ashing Technique  16  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.  L i t e r a t u r e Cited...........................  31  7.  Appendix  i  THE EFFECT OF PARATHORMONE ON THE MINERAL CONTENT OF VARIOUS VERTEBRATES  Sandstrom i n 1880 was the f i r s t to d i s t i n g u i s h sharply between the thyroids and the parathyroids, designating the l a t t e r "Glanduae Parathyroideas because of n  t h e i r positioja (Bard,1941) „  He regarded these glands as  displaced, undeveloped fragments of thyroid t i s s u e . His work was overlooked and Gley *** 1891 rediscovered them and demonstrated that they were p h y s i o l o g i c a l l y d i s t i n c t from, the thyroids (Bard, 1941).  Grosohuff and Verdun l a t e r  demonstrated, that they were also d i s t i n c t embryologically (Waggener, 1930). The mammalian parathyroid glands are small compact bodies, u s u a l l y four i n number, and c l o s e l y apposed to or within the thyroid.  I f they are removed the l e v e l of  serum calcium f a l l s ; t h i s i n turn causes neuromuscular hyperexoitability, tremors of s k e l e t a l muscle, and panting which produces a l k a l o s i s .  With calcium deficiency t h i s  leads to v i o l e n t clonic and tonic convulsions In which the animal sooner or l a t e r dies from arrest of r e s p i r a t i o n . 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. C o l l l p (1925) vas the f i r s t to obtain an active extract of the parathyroid glands.  He dissolved ox glands  i n b o i l i n g 5$ hydrochloric a c i d and a f t e r cooling removed the congealed f a t 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 b i o l o g i c a l assay. the conventional  The bioassay involves  graded dose-response method i n which the  c r i t e r i o n of response i s an elevation i n the blood calcium level.  One  (Hanson) unit i s one-hundredth the amount r e -  quired to r a i s e the serum calcium l e v e l of normal 20 dogs by 1 mg. per 100 ml. of serum.  One ox  contains approximately ten (Hanson) u n i t s ; .  kg.  parathyroid Until Colllp  obtained t h i s 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 p r i n c i p l e of the parathyroid glands i s probably of protein nature or else i s c l o s e l y associated with a protein c a r r i e r . T h i s Is supported by the f a c t that the hormone Is Inactivated by both a c i d i c and basic hydrolysis and by digestion with p r o t e o l y t i c enzymes.  A l t e r a t i o n of the functional group of  the protein moleoule, such as e s t e r i f i c a t i o n of the earboxyl  - 3 group, also r e s u l t s i n i n a c t i v a t i o n . U l t r a c e n t r i f u g a t i o n shows the parathyroid hormone to he heterogeneous, consisting of a t least two components, one of molecular weight roughly 20,000 and another of 500,000 to 1,000,000.  The a c t i v i t y  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 l i b e r a t i o n of the phenolyo hydroxyls by alkaline hydrolysis does not restore a c t i v i t y .  Kahlau  C1940) says that the parathormone obtained by C o l l l p cannot be completely i d e n t i c a l with the substance of the parathyroid glands - either i t possesses only p a r t i a l function or i t represents a modified hormone.  I t i s probably i d e n t i c a l with  the substanee formed by abnormally enlarged parathyroids (tumors, hyperplasia) which leads to generalized o s t e i t i s fibrosa.  P o s s i b l y the parathyroids possess two hormones,  one promoting the c a l c i f i c a t i o n of osseold tissue and the other maintaining the blood calcium at a constant l e v e l or r a i s i n g i t i n hyperfunction. There are two main theories regarding the actual mechanism of a c t i o n of parathyroid hormone.  One school  supports the theory that the hormone acts d i r e c t l y on the bones, stimulating o s t e o c l a s t i c a c t i v i t y and thus bringing about a d e c a l c i f i c a t i o n of osseold t i s s u e .  Because of the  r e c i p r o c a l r e l a t i o n s h i p between calcium and phosphorous the increase i n blood calcium as a r e s u l t o f t h i s d e c a l c i f i c a t i o n i s thought to bring about a decrease In blood phosphorous by excretion o f the l a t t e r through the kidneys.  The other  school states that parathormone acts d i r e c t l y 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 r e c i p r o c a l r e l a t i o n s h i p between calcium and phosphorous the lowered serum phosphate l e v e l r e s u l t s i n 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  d a i l y to r a t s and found that numerous osteoclasts formed i n the bone two days a f t e r the beginning of treatment. treatment continued osteoblasts began to form and i n number along with osteoclasts. fourth day of treatment.  As  increased  This was noted on the  At t h i s time also the  excretion l e v e l was noticeably elevated.  calcium  On the ninth  day  the osteoclasts began to disappear and i t was further noted that t h i s period coincided exactly with the return of c a l cium excretion and serum calcium to normal l e v e l s .  On  the  fourteenth day no osteoclasts were present at a l l but the number of osteoblasts had increased greatly. was  continued  was produced.  I f treatment  f u r t h e r the condition known as "marble bone" This experiment c e r t a i n l y gives support to  the idea that parathormone acts d i r e c t l y on the bones, but i t does not i n v a l i d a t e the theory that the hormone might act by d i r e c t l y a f f e c t i n g the kidney.  Is osteoclast form-  a t i o n stimulated by parathormone i t s e l f or i s i t the r e s u l t  of the lowering of the phosphate l e v e l of the blood ? C o l l l p In an a r t i c l e written i n conjunction with Kewfeld (1942) favours the hypothesis that parathormone aots by a f f e c t i n g the excretion of phosphate by the kidney. These investigators established that the hormone has l i t t l e or no e f f e c t on the serum calcium i n nephrectomlzed animals. After l i g a t l n g the renal vessels o r ureters the serum c a l cium remained unchanged under the influence o f parathormone administration.  Re-establishment of urine flow of the  animals i n j e c t e d with parathormone resulted i n a subsequent r i s e i n serum calcium.  These observations associate the  hvperealeaemia of parathyroid extract with the excretory function of the kidney.  They are i n almost d i r e c t contra-  d i c t i o n to the e a r l i e r investigations of Pugsley, C o l l i p 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* e i t h e r com-  plete nephrectomy d i f f e r s i n some way from merely tying o f f 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  C o l l l p i n the same a r t i c l e gave further evidence to show that parathormone has a d i r e c t e f f e c t upon the excretion of phosphates, followed successively by hypophosphataemia, hypercalcaemia and hypercalcurea. Chanutin and Ludewig (1940) investigated the r e l a t i o n -  ship between r e n a l disease and the parathyroid glands.  It  was evident that parathyroid hyperplasia I s proportional to the degree of kidney damage i n patients; that r e n a l damage i s frequently associated with hyperparathyroidism  i n patients  that the parathyroids are enlarged i n cases of renal r i c k e t s ; that p a r t i a l nephrectomy i n r a t s leads to an increase i n the s i z e of the parathyroid glands; that complete nephrectomy protects the dog from the e f f e c t s of large doses of parathyroid hormone; and that p a r t i a l nephrectomy i n r a t s decreases, the e f f e c t of parathormone i n r a i s i n g the serum calcium. Tweedy and Campbell (1944) noted an Immediate increase i n the urinary excretion of l a b e l l e d phosphorous on administ e r i n g parathyroid extract. afreets i n young dogs.  Logan (1939) observed the same  He also noted that the increase of  phosphate excretion i n urine occurs i n advance of the blood calcium increase.  maximum  He interpreted t h i s to mean that the  s o l u t i o n of bone i s caused by a depletion of blood and tissue inorganic phosphate r e s u l t i n g from Increased phosphate excretion by the kidney. The hormone has been administered dogs by Tweedy et a l ( 1 9 3 a ) .  to nephrectomlzed  They found that large doses  produce no greater Increase of blood calcium than that observed as a r e s u l t of nephrectomy alone.  The phosphate  retention resulting, from nephrectomy increased the inorganic phosphate 80$ i n two to 3 i x hours.  blood  This increase  was greater than that observed i n the terminal stages  r e s u l t i n g from the administration of f a t a l doses of parathyroid hormone.  As a r e s u l t of the r a p i d 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  r e s u l t s are therefore not conclusive evidence i n support of the t h e s i s that the a c t i o n of parathormone i s e x c l u s i v e l y on the kidney. Logan (1939) made determinations of urinary phosphate, blood calcium, phosphorous and magnesium at one hour i n t e r v a l s f o r four hours a f t e r the administration of a single large dose of parathormone In an e f f o r t to decide whether the changes are due to increased phosphate excretion or to bone resorption.  He found that the s o l u t i o n 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 excretion 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 i n t e r p r e t s t h i s as being the  r e s u l t o f phosphate retention by the kidney, but i t would seem more l o g i c a l to say that i t might be the r e s u l t of the mobilization o f calcium phosphate ( C a t o the blood.  3  (PO^g from the bones  This would increase both the calcium and the  phosphate content of the blood.  The r e s u l t s obtained here  must be interpreted to indicate that parathormone acts by increasing the urinary excretion of phosphorous as well as by causing a s o l u t i o n of bone. The work just summarized i s devoted e n t i r e l y to  8 mammals.  Lower vertebrates have been, r a r e l y studied.  It  would be i n t e r e s t i n g to investigate t h i s problem i n 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 r e g u l a t i o n . Parathyroid glands are not generally recognized In f i s h although Sqhereschewsky (1940.) described what she thought was parathyroid tissue i n the Lebistes larvae (Family Gyprlnodontldae).  She  described  In some d e t a i l glandular c e l l s i n the pharyngeal g i l l region of these f i s h which resembled the c e l l s 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 t h e i r appearance i n amphibians.  Even In  these animals the Indications are that they serve merely as accessory organs f o r calcium regulation.  Zhenevskaya (1948)  c a r r i e d out some rather b r i l l i a n t experiments using Rana temporarla  (the grass frog) and Rana ridibunda (the lake f r o g ) .  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 h i s t o r y of vertebrates, has not yet become an organ e s s e n t i a l to l i f e .  He attempted  to determine evolutionary changes i n the mechanism f o r c a l cium regulation and to show that the r o l e of the parathyroids In the regulation of calcium In amphibia was merely subordinate 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  a f t e r 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 r e s t o r a t i o n of the calcium, l e v e l by some other mechanism.  In Rana catesbiana, on the other hand,  death i n v a r i a b l l y followed parathyroidectomy  (Waggener,193Q).  In order to determine the i n t e n s i t y of t h i s other calcium regulating agent Zhenevskaya subjected  parathyroidectomized  and normal frogs to "washing out" experiments i n v o l v i n g the replacement of the e n t i r e blood volume with calcium-free Ringer's solution and noting the time required for the calcium l e v e l i n the vascular f l u i d to restore i t s e l f to the normal l e v e l .  Both parathyroidectomized and normal frogs  restored t h e i r calcium l e v e l s at the same time and same r a t e .  at the  This i s rather conclusive evidence that amphibia'  a c t u a l l y possess, besides parathyroid glands, some other powerful factor f o r calcium r e g u l a t i o n . . Amphibians are descendants of t r a n s i t i o n a l forms which bridged the gap between the water and the land vertebrates. I t would be i n t e r e s t i n g to f i n d that, from the time they put i n t h e i r appearance, the parathyroids have been i n d i s pensable i n the evolution of the organism.  I t 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 o f making the ascent to land.  In the amphibians the parathyroids are absent i n the  e a r l y aquatic l i f e of the i n d i v i d u a l .  In the Anura the  parathyroids have been reported by Mauer to develops^ during the l a r v a l 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  o r i g i n a l l y concerned  with r e s p i r a t i o n . I f the above observations are correct, the place of o r i g i n , the time, and the circumstances of t h e i r appearance may  eventually be found to f i t into the  ultimate explanation of the physiological function and b i o l o g i c a l s i g n i f i c a n c e of the parathyroids. ceivable that they may  owe  I t i s con-  t h e i r functional behavior to  their place of o r i g i n and that they may  have some pertinent  connection with the mode of r e s p i r a t i o n found i n the land vertebrates.  I f t h i s were true the e f f e c t s of parathyroid-  ectomy on amphibians would be problematical by v i r t u e of the dual mode of r e s p i r a t i o n found i n these forms. Waggoner (1930) further suggests that the parathyroids may  function to render toxic substances harmless  since such substances have been shown to accumulate i n the •  . . . . .  , •  .  .  ^  .  blood and urine of animals which have been parathyroidectomized.  These t o x i c agents probably a r i s e from the  metabolic substances of the animal and the need f o r t h e i r removal may have a r i s e n i n land animals which of necessity have a l e s s permeable surface. There are a number of d i f f e r e n t p o s s i b i l i t i e s of making a study of the e f f e c t of parathyroid hormone; one  may  study the e f f e c t of the hormone on calcium and phosphorous excretion; or the l e v e l of calcium and phosphorous i n the bones; or i n the blood serum, or other t i s s u e s of the body. I t has been well established that parathormone i s a regulator  of mineral metabolism i n mammals.  I t s general a c t i o n seems  to maintain the serum calcium a t a constant l e v e l either by mobilizing calcium from the bones or by lowering the renal threshold f o r phosphorous.  How i s mineral metabolism  regulated in\ forms which have no parathyroid glands ?  What  i s the e f f e c t of parathormone on mineral metabolism i n general ? The problem o f t h i s t h e s i s i s to test the e f f e c t of parathormone on the t o t a l mineral content o f lower vertebrates rather than to study the more s p e c i f i c e f f e c t s on calcium and: phosphorous regulation. The study includes forms i n which parathyroids are not recognized, t r a n s i t i o n a l forms which appear to have a dual mode of mineral metabolism and those forms i n which the parathyroids are e s s e n t i a l f o r the proper r e g u l a t i o n of minerals.  To make the problem more  s p e c i f i c the work has been confined to determining the e f f e c t of parathormone on the t o t a l mineral content of bones and muscle.  - 18  ACKHOWLEDQMENTS  The writer extends h i s thanks to Dr. ¥. A. Clemens f o r making t h i s work possible; and to Dr.. I.i MoT; Cowan and Mr. Charles Guiget f o r the generous loan of the beetle colony. A s p e c i a l note of thanks goes t o Dr. W. S. Hoar, under whose able supervision t h i s work was c a r r i e d out, for h i s invaluable a i d and h i s constant reassurance  through-  out the year. The author i s indebted to fellow students: Mr. G. D. Potter f o r h i s k i n d l y help and c r i t i c i s m s throughout the yearj Miss Aline Redlioh for help i n t r a n s l a t i n g a German a r t i c l e and to both Miss Redlioh and Mr• M. Jampolsky f o r help i n caring f o r the experimental  animals  The Slavonics department under Dr. G. S t . C l a i r Sobell deserves a s p e c i a l note of appreciation f o r transl a t i n g Zhenevskaya*s very pertinent Russian a r t i c l e .  - 13  MATERIALS AND METHODS  In t h i s s e r i e s of experiments the e f f e c t s of parathormone on the t o t a l mineral content of bones and muscles was used as a measure of hormone a c t i v i t y .  In a l l 225  g o l d f i s h (Carasslus auratus), 12; frogs (Rana pl.pl.ens), 30 mice (Mus domestlous). 12 chicks (Gallus domesticus). white leghorn cockerels and 2 pigeons (Columba ectopistes migratorius) were studied.  The most complete experiments were  carried out on g o l d f i s h since the e f f e c t of parathyroid ext r a c t on forms having no parathyroid glands was to be c l e a r l y established.  The numbers of animals used appear i n the  various tables of r e s u l t s . Goldfish were a c c l i m i t i z e d to d e f i n i t e temperatures and maintained i n thermostatically controlled aerated  aquaria.  They were f e d on brine shrimp eggs and i n j e c t e d I n t r a p e r l t oneally with the hormone.  D e t a i l s of temperatures and  dosages appear i n the tables of r e s u l t s . Large numbers of f i s h were required to obtain s u f f i c ient bone and muscle tissue to carry out the ashing procedures.  In order to standardize the ashing methods and deter-  mine i n d i v i d u a l v a r i a t i o n s preliminary experiments involving approximately twenty/ g o l d f i s h were c a r r i e d 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 p u l l i n g away by means of a p a i r of f i n e forceps.  - 14 The muscles along the back were taken: by undercutting with one point of a p a i r 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 f o r 80 minutes to h a l f an hour.  The bones  were thea picked clean with a p a i r of f i n e forceps.  Only  the cranium, the v e r t e b r a l column and the f i n s were l e f t behind.  The bones from each group of f i s h were mixed t o -  gether and re-divided i n t o as many portions as seemed permissahle.  Muscle was treated i n the same manner.  Determin-  ations were u s u a l l y 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.  D e t a i l s of dosages, number of frogs used and v a r i a t i o n s due to sex appear i n the tables of r e s u l t s .  One group was  studied i n February and another i n A p r 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 p i t h i n g 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 d i e t 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 i n t r a p e r i t o n e a l , 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 h i t t i n g sharply on the  head with a blunt instrument Insure maximum bleeding. and along the back.  and cutting the throats to  Muscles were taken from the legs  The e n t i r e skeleton was used but the  process of cleaning was d i f f e r e n t from that i n g o l d f i s h and frogs.  Beetles (Bermestes maoulatus Degeer 1774  Fab. 1781)  (B. vulpinus  } which clean bones completely i n two or three  days, were used because the other procedure proved too tedious. These beetles were maintained incubator.  In a constant temperature  'Oncleaned bones had to be d r i e d 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 d i e t of chick s t a r t e r and water.  Seven chicks  served as controls and f i v e received subcutaneous i n j e c t i o n s of parathormone as indicated i n the tables of r e s u l t s . 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 p e l v i c girdles.  The bones used were those of the pectoral and  p e l v i c g i r d l e s and the limbs except those above the humeri of the forelimbs and below the t i b i a e and f i b u l a e of the hind limbs.  Individual v a r i a t i o n s i n ash content, the  age  of the chicks and the dosages of parathormone used are indicated i n the tables of r e s u l t s .  A l l i n j e c t i o n s were  subcutaneous. Only two pigeons were a v a i l a b l e . One served as a control and the other received i n t r a p e r i t o n e a l i n j e c t i o n s of parathormone as indicated In the tables of r e s u l t s . b i r d s were the same age.  Both birds were k i l l e d by gas.  The  16 The muscles taken were those of the breast only.  The same  bones were taken as f o r the chick but with the breast bone included.  The cleaning method was as f o r the chicks and  mice. Parathormone ( L i l l y ) was used as a source of parathyroid hormone.  This i s an active extract of beef para-  thyroids and contains twenty units per cc.  One unit of  t h i s extract obtained by G o l l i p s method i s defined as  one-  hundredth the amount required to r a i s e 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 r e l a t i v e intolerance of  other animals to the hormone, average, dosages given were approximately  ten times that required i n dogs when calculated  on a u n i t s per gm. basis. 10 gms. each, frogs 25 gms., and pigeons about 150  Goldfish weighed mice 20 gms.,  approximately chicks 100  gms.  gms.  T o t a l mineral content was determined by ashing as follows:  the fresh tissue was placed i n an oven at 95 - 100 C e  f o r not l e s s than 48 hours to extract a l l moisture.  The  tissues were then placed i n s i l i c a crucibles previously weighed to 4 plaees on the chalnomatic balance.  The  cruc-  i b l e s containing the tissue were cooled f o r 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 i n l e s s than 8 hours.  The cruc-  i b l e s were removed from the muffle furnace and when s u f f i c i e n t l y 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 i n the muffle furnace f o r one or two more hours. Cooling and weighing of ash were exactly as f o r dry tissue. A l l crucibles were re-weighed before each set of determinations. A l l animals were k i l l e d and determinations carried out twenty-four hours after the i n j e c t i o n s designated i n the tables.  With the exception of g o l d f i s h skeleta, a l l  bones were crushed i n a mortar before drying.  18 -  RESULTS  GOLDFISH Effect of Temperature  The e f f e c t of moderate variations i n temperature shown i n Table I .  are  There i s no s i g n i f i c a n t 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 e f f e c t on the t o t a l mineral content of bones or musoles.  Massive doses given at high temperatures  produce no p a r t i c u l a r change i n mineral content.  (table  HI)  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 i n April.  There i s an increase i n bone ash with no change In  muscle ash.  The difference i n t o t a l mineral content of  bones from males and females (Table IV) i s also quite marked, being higher i n females. i n muscles.  There i s no s i g n i f i c a n t difference  Parathormone i n j e c t i o n s as indicated bring  about an increase i n bone ash with no change i n muscles. MICE Table Y shows that parathormone increases the ash content of bones while i t seems to have no effect on muscle. The s l i g h t increase i n muscle ash shown i n Table IV i s probably i n s i g n i f i c a n t .  A l l mice were males.  F i g . 1 shows  these changes better. '  I Y/A  Fig. 1  I Control ^ Units Parathormone  The effect of parathormone on the t o t a l mineral content of bones and muscles of micei  - 20 CHICKS The v a r i a t i o n s i n bone and muscle ash content from one i n d i v i d u a l to another are s l i g h t as shown i n Table VI • The i n i t i a l e f f e c t of parathormone i s to increase the ash content of bones by about 2$.  With prolonged administration  the effeot i s diminished u n t i l on the fourth day a f t e r increasing d a i l y dosages the bone ash i s decreased about 2$ below normal.  Muscle i s unaffected.  There i s no difference  i n ash content between 12 day o l d chicks and 16 day o l d chicks.  F i g . 2 shows the s l i g h t up and down e f f e c t pro-  duced i n bones by parathormone i n j e c t i o n s .  Muscles show no  s i g n i f i c a n t change from the normal as shown i n F i g . 2. J Control  Fig. Z  The e f f e c t of parathormone on the t o t a l mineral content of bones and muscles of growing chicks.  -  21  -  PIGEONS Parathormone evidently softens the bones of pigeons although the survey was not extensive enough to permit conclusions to be drawn. the time.  Only two pigeons were available at  These were from the same hatching.  A noticeable  increase was noted i n the muscle as well as i n 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 parathormone.  The photograph  ( F i g . 3) i l l u s t r a t e s t h i s .  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 b i r d . Bones on r i g h t are from control b i r d of same age.  - 22 Prior to k i l l i n g 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 i n the ash content but, i n 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 o f parathormone.  Stewart and  P e r c i v a l (192?) found that parathormone r a i s e s the serum calcium more i n cats than In rabbits but l e s s than In dogs. Tholldte (1928) found the same species difference the a c t i o n of parathormone.  regarding  He found that repeated doses  did not show a summation of e f f e c t i n cats and rabbits as has been reported to occur i n dogs.  J a f f e et a l (1932) gave  further evidence that r a b b i t s are r e l a t i v e l y tolerant to parathormone.  He found the same with guinea pigs but the  resistance of these animals i s not so strong as In r a b b i t s . Burrows (1938) found that parathyroid extract retards growth i n young r a t s , the r e l a t i v e retardation being proport i o n a l to the d a l l y dosages.  Over three u n i t s per day r e -  sulted i n a r a p i d l o s s i n weight and death In a few days. P a r t i a l l y parathyroidectomized r a t s tolerated the dosages better;  Microscopically Burrows found that e a r l y i n j e c t i o n s  brought about a resorption of bone, a decrease i n osteoblastsand t h e i r _change to fibrous tissue - and an increase i n osteoc l a s t s leading to o s t e i t i s f i b r o s a .  Continued i n j e c t i o n s ,  however, lead to a reversal of these processes, producing h y p e r c a l c i f i c a t i o n of bone leading ultimately t o "marble bone" disease. normal,  S t i l l further i n j e c t i o n s cause a return to  m the present s e r i e s of experiments only an Increase  - 34 i n the t o t a l 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 l o g i c a l explanation f o r the increase In  each case i s the time f a c t o r .  Each series of determinations  shown i n the tables represents the ash content twenty-four hours a f t e r the animal had received the designated i n j e c t i o n , m  a l l p r o b a b i l i t y the period when the ash content was low -  had been bipassed since parathormone i s known to act quite rapidly. The various degrees of s u s c e p t i b i l i t y to the hormone can probably be attributed to class differences since the dosages were approximately s i m i l a r with regard to body weight i n each case.  Since species differences i n 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 p o s s i b i l i t y 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 a c t i o n of the hormone.  Cox and Imboden (1936)  produced soft, thick, knobbed bones, e a s i l y l i a b l e to fracture by i n j e c t i n g parathormone into r a t s and keeping the animals on a high phosphate d i e t .  But i n spite of t h i s the bones  had a normal ash content. Goldfish d i d not respond to the hormone even i n extremely large doses.  The m o r t a l i t y rate was not abnormal  - 35 Thus mammalian, parathyroid extract Is completely non toxic i n these animals.  We might expect such negative r e s u l t s In  f i s h In the l i g h t of the f a c t that parathyroid glands are not generally recognized i n any class of animals below amphibia.  Evidently parathormone does not have any pharma-  c o l o g i c a l nor s p e c i f i c action on bone or kidney. I t Is i n t e r e s t i n g to f i n d a d e f i n i t e sex difference i n frogs with regard to bone mineral content.  This d i f f e r -  ence was recorded i n the spring, at the height of the mating season.  I t would be i n t e r e s t i n g 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 e a r l i e r investigators (Waggener, 1930.) and Zhenevskaya, (1948).  In order of  responsiveness frogs rank second to mice i n these I n v e s t i gations and the mode of response i s the same i n both. Mice received equivalent doses of parathormone to those given frogs.  Their s u s c e p t i b i l i t y i s s l i g h t l y greater  using the Increase In mineral content as the c r i t e r i o n . This might be expected since the parathyroids have been d e f i n i t e l y established as e s s e n t i a l to l i f e i n mammals.  In  amphibians the functional s i g n i f i c a n c e of these organs i s problematical.  On the other hand the difference i n response  might be a temperature  effect.  The m o r t a l i t y r a t e as a  r e s u l t of parathormone i n j e c t i o n s i s rather high In mice, seven of the experimental animals dying two to three hours a f t e r r e c e i v i n g the i n j e c t i o n s .  Autopsy revealed l e s i o n s  - 26 i n the intestine which were not the r e s u l t of i n j u r y from injection.  I t has been pointed out that f i s h can withstand  much larger doses with no apparent 111 e f f e c t s .  There were  no deaths among the amphibians during the experimental period.  The r e s u l t s obtained with mice are i n f u l l agreement  with the investigations c a r r i e d out by Silberberg and S i l b e r berg (1941).  These investigators found that parathormone  caused an increased c a l c i f i c a t i o n , degeneration and o s s i f i cation of the epiphyseal cartilage i n 5 weeks o l d mice. But i n these same mice there was no stimulation of the p r o l i f e r ation of the epiphyseal cartilage at a time when under normal conditions the p r o l i f e r a t i o n of t h i s 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 o s t e i t i s f i b r o s a could be produced even a f t e r prolonged administration of the hormone, although  this  condition was produced i n r a t s and guinea pigs subsequent to administering equivalent doses. The r e s u l t s obtained i n chicks are i n t e r e s t i n g since other Investigators have f a i l e d to obtain any p o s i t i v e r e sponse i n fowls.  In our experiments chicks are d e f i n i t e l y  more tolerant to the a c t i o n of parathormone than the other higher vertebrates studied but the r e s u l t s Indicate that they are not e n t i r e l y immune.  S p i s n i (1946) observed that  parathormone had a p a r t i a l i n h i b i t o r y e f f e c t on the growth of young rabbits but found i t to have no e f f e c t on the growth  2? of young chicks.  Rabbits, as has been pointed out, are  among the most r e s i s t a n t animals themselves.  Avery et a l  (1940) found no s i g n i f i c a n t 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 r e -  s u l t s In t e s t i n g 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$. more of the order of 51*3$.  The author f i n d s i t to be The discrepancy l i e s i n the  method of cleaning, at least i n part.  The method employed  here insured that the bones were stripped completely of a l l adhering tissue as well as t h e i r contained marrow.  We  would expect a higher ash content under these conditions. . Off a l l the animals studied pigeons were probably most responsive to the parathormone.  The bone ash  the  content  does not show t h i s but f i g * 3 d e f i n i t e l y Indicates a softeni n g of osseold t i s s u e *  The increase In ash content of the  bones of the experimental  animal was  due to the f a c t that  the beetles used In cleaning them were able to enter the marrow c a v i t y through the epiphysis which had been eroded. The bones of the control animal, because of t h e i r r e l a t i v e hardness prevented the beetles from entering the marrow cavity.  This condition combined with the f a c t that the  fibrous material had been eaten would tent to r a i s e the ash content per gram of dry t i s s u e .  - 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 i n the  muscle was the result of the drop i n the blood.  The same  author tried the effect of parathormone on muscle calcium and found the results variable, pression of muscle calcium occur.  but i n no case did a deThis i s i n agreement  with the results of the present investigation.  In no case  was there a significant change i n 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 i s not l i k e l y 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 i n the same species there i s a much greater individual variation 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 i s seldom less than three times the lowest.  This makes i t  d i f f i c u l t to demonstrate the small changes brought about by such an agent as parathormone. Burns (1933) found the same variations.  I t i s not l i k e l y that such a procedure as ashing  would detect small changes r e s u l t i n g from parathormone administration unless done In rather large quantities. Dennis and Gorley (1932) attribute these wide v a r i a t i o n s to the contamination of the tissue with blood and lymph. Few investigators have studied the e f f e c t of parathormone on the ash content of t i s s u e s .  P i s a (1934) report-  ed no change i n the t o t a l ash content of r a t s under parathormone administration.  Frugoni (1934) reported an  increased percentage of body ash In thyroid  or parathy-  roide eternized r a t s . The present experiments have been the f i r s t to t e s t the e f f e c t of parathormone on amphibia and f i s h . respond but f i s h are completely immune.  Amphibians  The theory o f  Waggener (1930) that the parathyroids developed with the land habitat should be more f u l l y investigated.  I t presents  an Interesting problem as to how mineral metabolism Is regulated i n aquatic forms.  There Is the p o s s i b i l i t y that  vitamin D i s the sole f a c t o r .  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 g o l d f i s h a l l animals showed an effeot of parathormone but the e f f e c t varied with the species. The pigeons probably are most reactive but the use of only two b i r d s does not permit d e f i n i t e conclusions to be drawn.  Pigeons were the only ones to show a pronounced  e f f e c t on muscle tissue with a r i s e i n t o t a l mineral content. A l l animals with the exception of chicks and g o l d f i s h show a r i s e In percentage bone ash.  Goldfish d i d not r e -  spond and chicks showed an I n i t i a l r i s e followed by a depression. Mice showed the greatest increase In bone ash.  Frogs  were l e s s 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 s l i g h t l y . variations i n ash content are only s l i g h t .  Individual  A new method i s  described f o r cleaning bones of adhering tissue f o r t h i s type of work. Variation i n temperature  does not a f f e c t the ash  content of g o l d f i s h . Parathyroid mechanism, whether i t acts d i r e c t l y 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, E f f e c t of parathyroid preparations on the blood calcium of the fowl. Poultry S c i . , 19: 321 - 325, 1940. Bard, P h i l i p , Macleod s physiology In Modern Medicine. 9th ed. C.V. Mosby Co., 1941. t  Burns, G. M.  The calcium'content of muscle. Biochem. J . , 27: 22 - 32, 1933.  Burrows, R.B. variations produced i n bones of growing r a t s by parathyroid extracts. Am. J . Anat., 62: 23? - 290, 1938. Chanutin, A., and S. Ludewig, The e f f e c t of r e n a l I n s u f f i c iency on the response of serum calcuim a f t e r administration of parathyroid hormone i n the r a t . Am. J> P h y s i o l . , 129: 242 - 243;, 1940. C o l l i p , J . B.,The extraction of a parathyroid hormone which w i l l prevent or control parathyroid tetany and which regulates the l e v e l of blood c a l oium. J . b l o l . Chem., 65: 395 - 438, 1925. Cook, J.W., and E.I.Robertson, The e f f e c t of minerals and vitamin D on the percentage bone-ash of young chicks. Poultry S c i . , 19: 385 - 388, 1940. Cox, W; M., and M. Imboden The r o l e of calcium and phosphorous i n 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 excessive calcium ingestion content of tissues with and application- of u l t r a - v i o l e t chem., 66: 609 - 617, 1925.  effect of on the ealeuim without the l i g h t . J". b l o l .  Fong - Yen Hsu and Chtao I s a l The calcium content of the s k e l e t a l muscles"after thyrdparathyroidectomy and parathormone i n j e c t i o n s . Chinese J . P h y s i o l . , 4_: 423; - 29, 1930 (abstract only). Frugonl, Piero  The t o t a l content of calcium and phosphorous of r a t s a f t e r thyroparathyroidectomy and the e f f e c t of an excess of parathormone. Arch. Sol.Med., 58: 237 - 252, 1934 (abstract o n l y ) .  - 32 J a f f a , H, L., Bodansky, A., and J . E . B l a i r The e f f e c t s of parathormone and ammonuim chloride on the hones of r a b b i t s , 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., E f f e c t of parathyroid hormone and o f tuberc l o s i s on the serum and tissue calcuim of guinea pigs* Bioehem. J . , 29: 8095 - 2100, 1935. Logan, M.A.  The early e f f e c t s of parathyroid hormone on the blood and u r i n e . 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. "~~ P i s a , Manlio  The "total calcium and phosphorous i n r a t s treated with small doses of parathormone. Arch. S c i . Med., 58: 629-632, 1934 (abstract only) •  Pugsley, X. I . , C o l l l p , J.B.,, Selye, H;, and D. L. Thomson. . Observations, concerning, the mechanism, of parathyroid hormone a c t i o n . B r i t . J.Exp. Path., 15: 335 -336,1934. Pugsley, L. I . , and Hans Selye The h i s t o l o g i c a l changes i n the bone responsible f o r the action of parathyroid hormone on the calcium metabolism of the r a t . J . Physiol.,,79: 113 - 117,1933. * Ross, W.F., and T.R.Wood The p a r t i a l p u r i f i c a t i o n and some observations oh the nature of the parathyroid hormone. J . b i o l . Chem., 146: 49-58, 1942. ^ Schereschewsky, Helene Uber den bau des epithelgewebes i n der pharyngeal klemenregion der knochenfische-. C.R. (Doklady) Acad. S o l . URSS, N.S., 29: 165 - 168,.1940. : V Silberberg, M., and R. SUberberg E f f e c t s of hormones on the skeleton of mice, guinea pigs, arid r a t s . Endocrinology, 89: 4175 - 482, 1941.  - 33 -  S p i s n i , Dino  Experimental studies on hyperparathyroidism provoked i n chickens. Boll.Soo. I t a l . B i o l . , 21: 71 -75, 1946 (abstract only)  Stewart, C. P., and G. H. P e r c i v a l 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 i n d i f f e r e n t kinds of animals. Krankheitsforseh, 6: 397 - 492, 1928. (abstract o n l y ) .  1/  Tweedy, W.R.,  and W.W. Campbell The e f f e c t of parathyr o i d extract upon the d i s t r i b u t i o n , retention and exoretion of l a b e l l e d 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 i n s u f f i c i e n c y on the action of parathyroid hormone i n the dog. Amer. J . P h y s i o l . , 115: 514 - 519, 1936.  Waggoner, R.A.,An experimental study of the parathyroids i n the anura. J . exp. Z o o l . . 57: 13-55,1930.  i / Zhenevskaya, R.P., Restoration of cal culm content i n 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 o f moderate v a r i a t i o n s  i n temperature  on the p e r c e n t ash composition o f bones and muscles o f goldfish.  Temp.°C.  No,Fish  No. Deternu  Muscle #Ash Ave- Range rage  13  20  8  5.72  5.305.99  16  20  4  5.78  5.695.92  go  20  5.62  5.415.72  Bone % Ash Average  Np Bet jrm.  ' range"  61.65 SO .3053.0  62 .53 51.7552.97  60.88 59.5163.73  TABLE I I The effect of average doses of parathormone on the ash content of bones and muscles of g o l d f i s h . Dosages increased 1 unit per day. F i s h at 20°C. I o. F i s h  Muscle # Ash  I O. I eterm. 1  Average  Ave- Range rage 1 Unit Inj.  10  5.56  5;47 5; 68  Control  10  5.67  2 Units In;).  a  5.52  62.18  61.21 62.99  5.675.68  2  61.41  60.8661.96  2  5.345.71  3  61.62  61.44 61.93  3  3  62.40  61.96 62.90  3  3  61*09  60.6261.66  3  60.86  59;43 62.43  3  9  5.87  5.626.13  4 Units InJ.  9  5.65  5.555.74  5 Units Inj.  Control  10 Units Inj.  10  10  9  5  5.57  5.65:  5.58  5.59  Range  3  Control  Control  No. Determ.  lone # Ash  5.515.60  3  5.595.68  3  61.64  61.3362.11/  3  5.465 .66  3  61.29  60.3562 . £ 6  3  1  58.75  1  iil  TABLE I I I E f f e c t of large doses of parathormone and extreme temperatures on bone and muscle ash content of goldfish, Dosages 10 u n i t s per day f o r 3 days.  No.Fish  Muscle % Ash Ave- Bange rage  15° C Control  So. Determ.  ;2one fo Ash Average  No. Determ.  Range  10  5.59  5.515.67  60.61  60.1261.29  10  6.47  6.186.81  61.49  60.9762.32  30 Unit s 10  5.56  5.405.67  62.20  61.5962.48  30°C Control  10  5.46  5.235.73  61.57  61.1262.11  10  5.65  5.555.73  61.97  61.4562.78  30 Unit 3 10  5.37  5.525.84  61.46  61.2061.77  InJ. 10 Unit*  Inj. • 10 Unitb  TABLE J\T E f f e c t o f parathormone on the hone and muscle ash content of frogs. Dosages Increased 5 u n i t s per day. Seasonal and sex differences i n ash content. Muscle fo Ash  No* Frogs  Average Group I (February) Control  6  4.44  ro. )eterm.  Range  4.37-4.59  InJ. . 6 Units  2  12 U n i t s  2  Average  No; Determ.  Range  5 7.29-52.64 '  56.90  56.35>7.63  3.77-  4  4.35  4.32 4.12- 4.164.58 4.51  4  64.83 58.37 64.43 - 67.61 65.14 69.14  4.10  3.364.51  4  68.62,  4.12  4  Males. Fern- I a l e s Females ales.  r  2  >  60.93  Males Fem- Scales Females ales Group I I (April). Gontrol  Bone $ Ash  :  67; 69 70.05 *  4  4  4  -  V  -  TABLE V  E f f e c t o f parathormone on the bone and muscle ash content o f mice. Dosages i n c r e a s e d 2 u n i t s per day f o r 3 days.  No.Mice  ro.  Muscle %> Ash , ieterm. Average Range  Control  8  Intf. . 2 Units  6 Units  6  Bone fo Ash Average Range  4.46  4.36 4.53  5  53.74 -  4.58  4.524.61  3  59.25  4;674.98  3  59.77 .  4.79  Mo.  )eterm;  52.7Q54.50 57.88-  4  6i.45<  3  59*56 59;88  3  TABLE VI E f f e c t of parathormone on the bone and muscle ash content of chicks. Dosages increased 5 u n i t s per day. To.Chieks  Muscle % Ash  No; Determ.  Average Range IS days old •Control  1  4.46  *Contro; L  1  4.72  4.535 .04  13 days old Control.  4.76  4.'574.98  Inj. (20 Units) a  4.65  15 days old Control  8  Inj. (.30 uni-ds) a  4.38 4.78  Bone % Ash  NO.  : Jeter ml. Average Range  3  51.10  51.0251.18  2  51.54  49.9253.26  2  51.30  51.1151.67  4.454.82  53.47  52.7154.32  4.80  4.554.93  51.28  51.1351.42  4.57  4.314.89  52.71  51.2953.72  16 days bid. Control  4.57  t.531.6G  51.38  50.3752.22  mj, (35 U n i t s ) !  £.75  U535.05  49.38  18.76i9.70  Kept separate to determine i n d i v i d u a l v a r i a t i o n s .  - vii -  TABLE VII  E f f e c t of parathormone on the bone and muscle ash content of pigeons. Dosages increased 10 units per day.  No.Birds  Muscle % Ash Average -Range  WTi Determ. I  Bone % Ash Average  Range  Contrc1  4.60  4.464.72  49.31  48.49 49*88  (180 q m t s f o r S days) 1  5.68  5.475.91  51.89  51.1852.78  TJbT  Deterktt  3  

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