Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The effects of severe phosphorous deficiency on calcium metabolismin the rat Suiker, Alice Petronella 1958

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1958_A8 S8 E3.pdf [ 2.92MB ]
Metadata
JSON: 831-1.0106170.json
JSON-LD: 831-1.0106170-ld.json
RDF/XML (Pretty): 831-1.0106170-rdf.xml
RDF/JSON: 831-1.0106170-rdf.json
Turtle: 831-1.0106170-turtle.txt
N-Triples: 831-1.0106170-rdf-ntriples.txt
Original Record: 831-1.0106170-source.json
Full Text
831-1.0106170-fulltext.txt
Citation
831-1.0106170.ris

Full Text

THE EFFECTS OF SEVERE PHOSPHORUS DEFICIENCY ON CALCIUM METABOLISM IN THE RAT by ALICE PETRONELLA SUIKER B.A., Brigham Young U n i v e r s i t y , Utah, 1951 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS1 i n the Department of Physiology We accept t h i s t h e s i s as conforming to thej required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1958 i v ABSTRACT Young Wistar female r a t s weaned at 25 days were placed on a control d i e t , or a d i e t extremely d e f i c i e n t i n phosphorus but adequate i n a l l other respects. After f i v e weeks on the d i e t they were injected with 10 microcuries of high s p e c i f i c a c t i v i t y radiocalcium. The animals were k i l l e d at varying periods after i n j e c t i o n and samples.of bone, teeth and soft tissues were taken for chemical, radio-isotope, and h i s t o l o g i c a l analysis. The phosphorus deficient animals showed a marked demineralization of the skeleton, lower radiocalcium uptake by bone and higher radiocalcium uptake by the teeth. The accretion and resorption rate of bone i n the phosphorus deficient animals was markedly reduced. The resorption rate, however, was higher than the accretion rate and accounted f o r the reduced mineralization of the bone. The femur of the r a c h i t i c animal had an exchangeable calcium portion of 13% as compared to A--8% i n the control animal. The teeth of the phosphorus d e f i c i e n t animal showed a reduced accretion and a t t r i t i o n rate, and a s t a t i s t i c a l l y evident difference i n the chemical calcium and phosphorus content. The accretion rate was higher than the a t t r i t i o n rate, so that the teeth remained well mineralized. The depression of the accretion rate was not as marked as that observed i n the bone, therefore, the marked demineralization of the r a c h i t i c animals' bones was not evident i n the teeth. The serum plasma levels f o r calcium and phosphorus were 9.43 mg.% and 2.86 mg.% i n the r a c h i t i c animal and 9.91 mg.$ and 7.24 mg.% i n the V control animal. The disappearance of plasma radiocalcium was not as rapid i n the r a c h i t i c animals. Starvation of phosphorus deficient animals resulted i n a lower plasma calcium and raised plasma phosphorus l e v e l similar to that observed i n parathyroidectomized animals. The so f t tissue calcium concentration i n the r a c h i t i c animals as compared to the control animals was higher for a l l soft tissues examined with exception of the kidney and blood plasma where there was no s i g n i f i c a n t difference. The amount of calcium i n the various muscle compartments was calculated. The r a c h i t i c animal had a higher i n t r a - c e l l u l a r calcium concentration and the same e x t r a - c e l l u l a r concentration when compared with the control animal. There was no difference i n the phosphorus concentration of the control and r a c h i t i c animals' soft tissues. H i s t o l o g i c a l studies of the femur of the r a c h i t i c and control animal showed that the r a c h i t i c femur had a wider epiphyseal cartilage which was not uniform i n width. The bone trabeculae showed wide i r r e g u l a r seams of unca l c i f i e d osteoid matrix. H i s t o l o g i c a l and calcium analysis of the kidneys of the phosphorus deficient animals showed no evidence of calcium deposits or nephrocalcinosis. H i s t o l o g i c a l studies of the parathyroid glands of r a c h i t i c animals showed a decrease, i n the volume of the gl a i d s , i n the size of the nuclear surface, and i n the amount of cytoplasm present, when compared to the glands of the control animals. This study of calcium k i n e t i c s i n the phosphorus deficient animal coupled with the h i s t o l o g i c a l findings shows the p o s s i b i l i t y that phosphorus v i deficiency i n rats produces a hypoparathyroid condition as a homeostatic mechanism to conserve phosphorus f o r the s o f t tissues. In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed, without my w r i t t e n permission. Department of '^HAy^uo-^>^  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver B, Canada. ACKNOWLEDGEMENTS The author would l i k e t o extend her appreciation to Dr. D.H. Copp f o r h i s prof i t a b l e discussions, and invaluable c r i t i c i s m ; to Dr. E.C. Black for h i s counsel and encouragement; to Dr. Ramamurti for his aid i n the h i s t o l o g i c a l .analysis; to Mr. B. Twaites f o r the h i s t o l o g i c a l preparations; to Mr. K. Henze for the preparation of the graphs; and t o the Canadian Dental Research Board whose grant made t h i s .work possible. . i i i TABLE OF CONTENTS Page I INTRODUCTION 1 1. Review of literature 1 2. Purpose of research . 9 II MATERIALS AND METHODS 10 I I I RESULTS 13 1. Observations 13 2. Plasma 13 3. Soft tissues 14 4. Excretion 15 5. Bones and teeth 15 6. Histological 16 IV DISCUSSION 18 1. Effect of phosphorus deficiency on bone metabolism 18 2. Effect of phosphorus deficiency on metabolism i n teeth.. 20 3. Soft tissue changes i n phosphorus deficiency Mt- 21 A. Skin 21 B. Muscle 22 C. Kidney 23 4. The parathyroid gland i n phosphorus deficiency 24 V SUMMARY 27 VI CONCLUSIONS 29 VII APPENDIX 30 VIII TABLES 35 IX FIGURES 42 X BIBLIOGRAPHY 53 INTRODUCTION Bone has provided absorbing problems for physiologists for many hundreds of years. The point of interest has varied from time to time but the basic problem i n the past has been the property of bone as a supporting structure. Bone possesses physiological as well as structural advantages, i t i s not a dead tissue but a store house of labile mineral which aids i n the homeostatic regulation of blood calcium and phosphorus levels, and as a mineral supply for the actively metabolizing tissues of the body. Since bone contains 99% of the body calcium and 80 to 85% of the body phosphorus, i t i s evident that for development of well mineralized bone, calcium and phosphorus are nutritional needs. Calcium deficiency in wild animals and birds i s a rare phenomenon but lack of phosphorus i s a hazard to both plant and animal growth. The impaired animal growth i n phosphorus deficiency stimulated the interest of the effect of low phosphorus diet on the development of rickets. The earliest reference to the effect of phosphorus on bones was made by Wegner (71). This investigator, working i n Virchow's laboratory, stated that elementary phosphorus increased bone formation i n growing animals and birds. Kassowitz (43) claimed elementary phosphorus stimulated repair of rachitic bones while Kissel (44) reported no bone changes from feeding phosphorus. Phemister (55) confirmed Wegner's and Kassowitz*s observation that phosphorus was productive of bone formation. Sherman and Pappenheimer (64) reported the addition of alkaline potassium phosphate to 2. a rachitogenic d i e t high i n calcium and low i n phosphorus prevented ri c k e t s i n rats when 0.4$ phosphate was added, and an equivalent amount of calcium withdrawn from the d i e t . Hess et a l . (38) and others have confirmed the observation of Sherman and Papperiheimer. McCollum et a l . (49) showed that the addition of 2$ cod l i v e r o i l to rachitogenic d i e t , high i n calcium and low i n phosphorus, was curative of r i c k e t s i n ra t s . Phemister et a l . (56) also reported phosphorus or phosphorus plus cod l i v e r o i l produced s i m i l a r r e s u l t s i n bone development. McCollum et a l . (50) found that starvation of r a t s , made r a c h i t i c on a high calcium low phosphorus diet induced a cure of the r i c k e t s at the expense of other tissues of the body. Similar results were reported by Cavin (14) on r a c h i t i c rats which were fasted f o r two days. The serum phosphorus i n the r a c h i t i c starved animals rose to 16 mg.$ and the serum calcium l e v e l dropped to 6 mg.$. Sherman and Pappenheimer (64) and McCollum et a l . (50) produced similar r a c h i t i c conditions i n rats. They f e l t that the r i c k e t s resulted from the low phosphorus high calcium content of the diet coupled with a lack of some organic substance contained i n cod l i v e r o i l . However, the diets used by a l l the investigators were def i c i e n t i n other respects. Osborne et a l . (54), Steenbock and Black (66) and Brown et a l . (11) a l l t r i e d to reduce the phosphorus i n the rachitogenic diets but were unsuccessful i n producing a phosphorus l e v e l s u f f i c i e n t l y low to produce the symptoms of severe phosphorus deficiency. Schneider and Steenbock (61) described a diet which contained 0.04$ phosphorus. Jones (41) prepared a 3. diet based on the use of p u r i f i e d beef blood f i b r i n as the protein. The phosphorus content was 0.02$. Day and McCollum (25) reported the effect of a diet extremely low i n phosphorus (0.0.7$) but n u t r i t i o n a l l y adequate i n a l l other respects. The animals on t h i s d i e t Showed the characteristic changes of r i c k e t s (upon gross and h i s t o l o g i c a l examination). They found a normal phosphorus concentration i n soft tissues, lowered concentration i n the plasma, and marked loss of phosphorus from the bones. They concluded that the content of phosphorus i n soft tissues remains remarkably constant, while bone phosphorus i s able to undergo appreciable changes i n amount without manifestations of i n j u r y to the body. Coleman '. et a l . (21) using a d i e t with a phosphorus content of 0.005 to 0.015 mg.$, vitamin A 25 U.S.P. units/gm., vitamin D 8 U.S.P. units/gm. and optimum i n a l l other respects, developed severe r i c k e t s i n growing rata. Roentgenograms showed a marked reduction of mineral i n the skeleton of the d e f i c i e n t animals and a delay i n the appearance of o s s i f i c a t i o n centers i n the vertebrae. H i s t o l o g i c a l studies of the t i b i a and metacarpal bone, caudal vertebra and the costochondral junction showed severe r i c k e t s i n the phosphorus deficient animals. Increase of the Ca'/P r a t i o from 28:1 to 78:1 did not appear to i n t e n s i f y the severity of the r i c k e t s . Definite changes were observed with increasing age and time on low phosphorus diet. The gross deformities of the bones i n r i c k e t s were attributed to the p o s s i b i l i t y of the f a i l u r e of the intercoluranar matrix and osteoid matrix to c a l c i f y . Remodeling resorption f a i l e d to take place and the volume of osteoid was increased. Cramer and Steenbock (24) varied the calcium content of a low phosphorus diet and showed that the negative calcium balance due to a low phosphorus 4. diet can be changed to a positive one i f the calcium content of the diet i s raised over 1%. This change i s not evident i n the absence of vitamin D. Much has been reported on the bone changes i n phosphorus deficient rickets, however poor correlation has been found between the mineral content of bone and that of teeth when animals are subjected to phosphorus deprivation. Gies and Perlzweig (34) reported chemical aberrations of the teeth due to phosphorus deficiency. However, his observations are of limited value because of the use of non-purified diets. Karshan (42) found a 50% decrease i n bone mineral of rats on low phosphorus ration but no corresponding change i n the teeth. These findings were confirmed by Gaunt and Irving (33), and F o l l i s et a l . (30). I t seems incongruous that i n young animals, only the bone should exhibit such a marked degree of hypocalcification especially i n view of histologic demonstrations of the effects of phosphorus deficiency on teeth as shown by Becks and Ryder (9), Rosebury and Karshaw (59), Weinmann and Schour (72) and Coleman et a l . (20). These workers found disturbances i n the calcification and ossification of the dentin of incisors and molars of rachitic animals. The formation and calcification of the dentin was severely retarded, the pump chambers of the incisors and molars were enlarged and the zone of predentin abnormally wide. Enamel formation and calcification were normal, however, the enamel organ showed cystic degeneration. Although disturbances i n gross and histological structure and i n mineral balances were noted i n rachitic animals, i t was d i f f i c u l t to study 5. quantitatively the metabolic changes taking place u n t i l the advent of radio-isotopes. Conn and Greenberg (19) studied the effect of vitamin D on phosphorus metabolism, using They found the uptake of P32 by the bone was increased as much as 25-3C$ i n the rats which received vitamin D. In another study of P32 d i s t r i b u t i o n i n rats raised on a low phosphorus diet Gaunt et a l . (32) found a d e f i n i t e l y increased retention of radio-phosphorus i n those animals raised on a phosphorus d e f i c i e n t d i e t . The major part of the P^2 was taken up by the muscle. These results were confirmed by Claassen and Wostmann (16, 17, 18). Feaster et a l . (29) compared the turnover of P-^ 2 i n soft tissue and bone of^growing rats maintained on diets that were lacking i n vitamin D and low i n phosphorus but otherwise adequate. The rate on low phosphorus diet deposited more P32 i n t h e i r soft tissues than the two groups fed adequate phosphorus diets, but less i n the femur, as indicated by P32 uptake and radioautographs. Total phosphorus values indicated depletion of t h i s element i n the femurs but not i n the soft tissues. Radioau tographs showed only a s l i g h t deposition of P32 i n the proximal end of the femur of the phosphorus deficient animals as compared with the femurs of the rats receiving adequate phosphorus. Myers (51) found a very high uptake of P32 i n the bones of rats recovering from r i c k e t s . Carlsson (12) found that i n vitamin D deficient r a c h i t i c rats there was a marked decrease f C a ^ r a t i o between femur n c i s o r s . The dministra-tion of vitami D increased t h i s r a t o . Copp t a l . (23) studied he ur over of radioc l ium i n r c h t i c r ts at varying n t e r v a l s a f t e r6 » intravenous injection of high specific activity radiocalcium. They used a diet extremely low i n phosphorus (o.oo8-0.015$) but adequate i n a l l other respects. After a maximum uptake i n the skeleton 1 hour after administra-tion of radiocalcium there was a rapid loss of radiocalcium, so that the percentage of the dose retained by the deficient skeleton decreased by about 50$ i n one day. The slope of the curve then gradually decreased, and about 4 days after the administration of radiocalcium the curve f e l l only slightly. According to these authors, the f i r s t stage suggests the presence of a very labile calcium fraction. The young control animals on the other hand had a maximum uptake i n one hour after injection but lost only approximately 5% of their Ca^5 uptake i n 16 days. Radioautographs of bones from animals injected with Ca45 were very similar. The radiocalcium was found only i n areas i n which bone salt was present, and none was found i n the uncalcified osteoid matrix. Lindquist (48) observed a rapid removal of Ca45 from the femurs of vitamin D deficient rachitic rats. Metabolic studies with Ga^ 5 and P^2 have shown that the uptake by bones varies considerably with type, area and age of the tissues. Differences according to bone type were well established by Rogers and Weidmann (57). Variations i n the absorption of isotopes i n the different areas of bone shaft were clearly demonstrated by Amprino (l) and Engfeldt et al.(28) using radioautographic techniques, and also by Rogers et a l . (58) who measured uptake directly. The age of bone greatly influences the rate of isotope incorporation. The uptake of isotope i s greater i n the developing bones of the young than i n the mature bones of the adult animal as was demonstrated by Leblond et a l . (45) and Hansard and Crowder (37). 7. These variations have been attributed to local structural differences in the bone tissue, such as differences i n micro-structure (Amprino ( l ) ) , crystal size (Hevesy et a l . (39)) and in the degree of calcification (Hevesy et a l . (39) and Amprino (1)). I t was suggested by Amprino (l) and Engfeldt et a l . (28) that the turnover rate of radioisotopes was proportion-a l to the relative amount of recently formed but not yet f u l l y calcified bone. The radiographic findings were later confirmed by radioactivity measurements i n the bone-powder samples taken from the various regions of the shaft bone of the rabbit by Rogers et a l . (58). Isotope incorporation i s comparatively rapid immediately after the administration of the isotope, and slows down markedly within a few hours. Hevesy et a l . (39) explained the i n i t i a l phase as an ion-exchange reaction and the subsequent slower phase as the biological recrystalization. I t was thought that the rate of reaction i n the i n i t i a l phase decreased with the exhaustion of the easily exchangeable fraction of bone salt c r y s t a l l i t e s , and that the second phase was entirely governed by the rate of recrystaliz-ation. Neuman and Neuman (53) defined recrystalization more e x p l i c i t l y as an ionic-diffusion through the solid crystal. One of the main reasons for regarding the i n i t i a l uptake as an ion-exchange absorption was that the reaction was too fast to be accounted for by the new bone formation and could indicate only, that an equilibrium was quickly established between the exchangeable ions of the surface area of the bone and the blood minerals. According to this theory, bone growth i s of minor importance for the redistribution of administered radiocalcium and radiophosphorus i n the skeleton. 8. Carlsson (12), on the other hand, demonstrated that eighteen hours after the administration of the isotope to growing raits: the d i s t r i b u t i o n of radiocalcium was closely related to the growth and the c a l c i f i c a t i o n of the different parts of the skeleton. Lindquist (48) made simi l a r observations. ' Carlsson (13) also showed that the uptake i n the i n c i s o r teeth was an i r r e v e r s i b l e process and expressed the view that even i n the skeleton, exchange processes were of minor importance. Bauer (4) explained the decrease i n turnover rates of bone s a l t by the rapid f a l l of isotope concentration i n the blood. Since bone and blood minerals are i n dynamic equilibrium, bone s a l t s formed i n the early phase of the reaction, when the tracer concentration i s s t i l l high, contain more isotope than those formed i n the l a t e r stages. Neuman and Hodge (52) have stated that " chemical exchange reactions between bone and the ions i t yi e l d s to solution have made i t impossible to use isotopes as an index of the amount of calcium and phosphorus being l a i d down i n bone." Bauer et a l . (5) do not agree with t h i s point of view, and consider that " i t i s indeed possible by means of tracer technique to d i f f e r e n t i a t e between various processes occurring i n the c a l c i f i e d t i s s u e , and also i n part to measure them quantitatively." Bauer defined the exchange reactions as reversible processes while accretion (incorporation of mineral i n the c a l c i f i c a t i o n of osteoid tissue) and resorption (actual breakdown of c a l c i f i e d bone tissue) should be regarded as two i r r e v e r s i b l e processes working more or less independently of each other at different s i t e s . As a rule there i s a d i s t i n c t i n t e r v a l of time between accretion and resorption. Assuming Bauer's (4) assumptions to be correct, i t i s the purpose of t h i s research to calculate the k i n e t i c s of calcium metabolism with Ca^5 i n bones, teeth, and soft tissues of rats on a control and r a c h i t i c producing phosphorus deficient d i e t , u t i l i z i n g the Bauer et a l . (5) method f o r measuring calcium metabolism. The changes produced i n these experiments should be d i f f e r e n t i a t e d from r i c k e t s as defined by Lindquist (48) who stated, "Most authors seem, however, to agree that r i c k e t s i s a condition of vitamin D deficiency r e a d i l y recognized by a decrease i n the inorganic phosphorus i n the serum and by certain s k e l e t a l changes." The r i c k e t s i n t h i s research i s due to a severe phosphorus deficient d i e t with an adequate amount of vitamin D. 10. MATERIALS AND METHODS EXPERIMENT I Two series of female Wister rats average weight 55 grams were r e s t r i c t e d to one of the experimental diets described i n Table I . In the f i r s t series there were 15 control and 15 phosphorus deficient animals; i n the second series there were 20 control and 20 phosphorus d e f i c i e n t animals. The animals were maintained on the d i e t f o r f i v e weeks at which time the animals were injected i n t r a - p e r i t o n e a l l y with 0.25 ml. of isoto n i c saline solution containing about 10 microscuries of radiocalcium of high s p e c i f i c a c t i v i t y (2 Mc/mg. stable Ca.). At time of i n j e c t i o n the urinary bladder was emptied. The animals were k i l l e d i n groups (3 per group series 1 and 4 per group series 2), at 1, 3, 6, 12, and 24 hours after i n j e c t i o n . Care was taken to make the groups as simi l a r as possible with regard to body weight. Immediately after i n j e c t i o n , each of the animals intended to survive f o r s i x hours or more were placed i n i n d i v i d u a l cages provided with an arrangement for separation of urine and faeces. The animals at time of death were given ether anaesthesia. Samples of blood, skin, gut, l i v e r , muscle, kidney, femur, t i b i a plus f i b u l a , t a i l , i n c i s o r s , molars, and remaining carcase were taken. Muscle, s k i n , l i v e r , and kidney samples were weighed as soon as they were removed. Urine and faeces from the 6, 12, and 24 hour animals were collected. 11. The samples were dried and then dry ashed i n a muffle at 1000° F. f o r removal of a l l organic material. N i t r i c acid was then added and the samples were brought into solution, wet ashed and made up to a known volume. Plasma calcium was analyzed by a modification of Lehman (46). Calcium i n the bone, teeth and soft tissues, were measured by the Comar (22) modification of the Clark-Collip method. Phosphorus was measured by the Sumner (68) modification of Fiske and Subbarow. Plasma radiocalcium was determined on 0.2 ml. samples of plasma dried i n stainless s t e e l planchets. Other radiocalcium samples were prepared using polyethylene tubes and stainless s t e e l planchets as described by Comar (22). Samples were counted with a t h i n end window Geiger counter and corrections f o r s e l f absorption were made. Samples of kidney, femur, and block sections of trachea (containing thyroid plus parathyroid), from r a c h i t i c and control animals were placed i n formal alcohol f o r h i s t o l o g i c a l studies. The thyroids plus parathyroids for h i s t o l o g i c a l studies were s e r i a l sectioned and analyzed for function as described by Engfeldt ( 2 7 ) . EXPERIMENT I I 42 Female Wistar rats average weight 50 grams were r e s t r i c t e d to the same diet as experiment I . 21 rats received the phosphorus d e f i c i e n t diet and 21 rats received the control d i e t . At the end of four weeks, 14 deficient and 14 control rats were given an intra-peritoneal i n j e c t i o n of radiocalcium as i n experiment I . 7 rats from each group were k i l l e d at 8 and 24 hours after i n j e c t i o n . Samples of blood, femur, t i b i a plus f i b u l a , i n c i s o r s and 12. molars were taken. The remaining animals were maintained on t h e i r diets t i l l the end of f i v e weeks at which time they received 10 micro-curies by intra-peritoneal i n j e c t i o n . These animals were k i l l e d 24 hours after i n j e c t i o n . Samples of blood, femur, t i b i a plus f i b u l a , i n c i s o r s and molars wereotaken. Radiocalcium and chemical calcium were determined as i n experi-ment I. 13. RESuIffS  Observations Pig.I showa that the control animals gained weight rapidly on the control diet (Table i ) . Rats on the phosphorus deficient diet had a slower rate of growth, which at three weeks levelled off and l i t t l e or no gain i n weight was noted from the third to f i f t h week. The control animals showed no v i s i b l e signs of deficiency. The phosphorus deficient animals developed a greasy coat after one week on the diet. This greasy coat could have been due, perhaps, to the marked hoarding and digging complex which developed i n these animals about this time. These animals would drag their food from the food dishes through the feeding tunnel into their cages. After three weeks on the phosphorus deficient diet, a marked weakness i n the legs of the animals was noted. The red keratinization about the eyes, noted by other authors, was not seen i n these animals. At the time of death, the control animal was eating approximately 10 grams of food per day compared with 4 grams for the phosphorus deficient animal... At autopsy, the bones of- the phosphorus deficient animals were soft and pliable with a club-shaped epiphysis and an abrupt junction with the diaphysis characteristic of rickets. Plasma Table I I I shows the normal calcium and low phosphorus plasma values, 45 characteristic of phosphorus deficient rickets. Ca a c t i v i t y values of the plasma were higher for the r a c h i t i c animal (Table I I I , Figures 2 a & b and 4 a & b). 45 This i s to be expected i n view of the small uptake of Ca by the phosphorus deficient animal (Table I I ) . When phosphorus deficient animals were fasted for 24 hours, the plasma calcium level f e l l to 7.5 mg.$ and the plasma phosphorus level rose to 8.5 mg.#. There was no significant change i n the blood plasma levels of the control fasted animal. -*"""' 14. Soft Tissues No difference i n the chemical phosphorus content of the soft tissues was noted with the exception of the low blood plasma level i n the experimental animals. There was, however, a higher chemicalAContent i n a l l soft tissues of the rachitic animal, with the exception of the kidney and blood plasma which showed no difference. The soft tissue values are found i n Table IV. One hour after injection of Ca4'' approximately 11$ of the dose was present i n the skin of the animal. The radiocalcium disappeared very rapidly from the skin during the f i r s t s i x hours and then declined" more gradually. The rate of 45 disappearance of Ca^' from the skin of the rach i t i c and control animal was not significantly different (Fig. 3 a & b). The control animal skin specific a c t i v i t y curve coincided very nearly with the plasma specific a c t i v i t y curve of Fig. 4 a & b) but the phosphorus deficient animal skin specific a c t i v i t y curve showed values which were markedly lower. This was also true for l i v e r . One hour after injection of radiocalcium, muscle contained approximately 10$ of the dose i n the control animal and 8$ i n the rach i t i c animal; 24 hours after injection approximately 2$ remained i n the muscle of the control animal and 3.8$ i n the ra c h i t i c animal. Radiocalcium was removed from the muscle at a much 45 slower rate than was the plasma Ca . The rate of removal of radiocalcium was higher i n the control animal than i n the rach i t i c animal (Fig. 3 a & b). The specific a c t i v i t y curves for muscle i n Fig. 4 a & b do not drop as rapidly as the plasma curves. The calculated extra-cellular f l u i d calcium content of muscle of 8$ showed no difference between the experimental and control animals. The difference i n t o t a l calcium content i n mg.$ between the control and the rachitic animal appeared to be due to the proposed i n t r a - c e l l u l a r content, the control animal having an i n t r a - c e l l u l a r calcium content of 3*18 mg.$ and the rachitic 15. animal 5.06 mg.$ (Table IV). Using Bauer's kinetic equation #5 as demonstrated i n soft tissue calculations, the muscle calcium was calculated to move from the assumed extra-cellular pool into the i n t r a - c e l l u l a r pool at a rate of 0.23 mg/hr. for both control and rachitic animals. Excretion 45 Ca was excreted more^raprdly from the r a c h i t i c animal,.as demonstrated by the one to twenty-four hour values for, the carcass shown i n Table I I . and urine and faeces values Table IIA. Approximately 5$ of the injected dose of radiocalcium was found i n the gut plus faeces after 24 hours. The radiocalcium appeared to be excreted into the gut i n the digestive juices and then p a r t i a l l y reabsorbed. After fi v e weeks on a phosphorus deficient diet, the rachitic animal lost 0.33 mg/hr. of calcium i n i t s urine as compared to 0.015 mg/hr. i n the control animal (Table IIA, Fig. 7). Bones and Teeth The control animals had a much higher bone mineral content than the rachitic animals. This difference was not as marked i n the teeth. The amount of mineral i n the rac h i t i c animals' teeth showed a small but significantly lower content. 45 The teeth i n the rachitic animal, i n contrast to the bone, had a higher Ca uptake, when compared with the control animal. Table I I . Figs. 5 a & b and 6 a & b represent observed values for radiocalcium 45 45 i n femur and incisors of the control and rac h i t i c animals. The Ca A and Ca E curves represent calculated values. These values were calculated using the A and E values from Bauer's equations 4b and 5, and substituting these values into equations 1 and 2. I t may be noted that the sum of Ca 4^ A plus Ca 4^ E equals 45 Ca observed for a l l values from 6 to_24 hours. For values from 1 to 6 hours, 16. the radiocalcium for the exchangeable portion was not i n equilibrium with the radiocalcium of the blood plasma and, therefore, i f equation 2 i s used for calculation of the exchangeable radiocalcium i n bone, a.value which i s much too high i s obtained. 24 hours after injection of radiocalcium, 42$ of the Ca^ i n the phosphorus deficient rat's femur was i n the exchangeable portion, while only 16$ was present i n the exchangeable portion of the control animal. The teeth of the phosphorus deficient animals had a higher uptake of radiocalcium than did the control animals. Since there was not much difference i n the accretion rate between the two groups of experimental animals and the specific a c t i v i t y of the rachitic animals' plasma was much higher, this was to be expected. Tables V and VI show the accretion, resorption and exchangeable portions of the experimental animals. The values for accretion were s l i g h t l y higher i n the experimental animals i n experiment I I . This may have been due to the fact that these animals were smaller than those in- experiment I. The phosphorus deficient as compared to the control animal had a markedly lower bone accretion and resorption rate. The resorption rate i n the rac h i t i c animal was higher than the accretion rate and accounted for the marked demineralization of the skeleton of the phosphorus deficient animal. The accretion and a t t r i t i o n rates i n the teeth of the rachitic animals was si g n i f i c a n t l y lower than that of the teeth of the control animals. Histological Studies Histological studies of the femur showed that the epiphyseal cartilage of the phosphorus deficient rats was markedly wider than that of the control rats, i t was not uniform i n width but broad i n the centre and narrow toward the periphery. The major increase i n width was i n the zone of maturing cartilage. There was a decreased c a l c i f i c a t i o n i n the area of calcifying cartilage. The bone trabeculae 17. showed wide irregular seams of uncalcified osteoid matrix. There was l i t t l e evidence of osteoclastic a c t i v i t y and i t appeared as i f the uncalcified osteoid matrix acted as an insulator against osteoclastic a c t i v i t y . 3y normal histological stains (H. & E.), the glomeruli and tubules of the phosphorus deficient animals' kidney showed no abnormality. Calcium deposits of nephro-calcinosis were not found. Chemical analysis showed no difference i n the calcium concentration of the r a c h i t i c animals kidneys as compared to the control animals. Histological studies of the rachitic animals parathyroid glands showed a decrease i n the volume of the glands, i n the size of the nuclear surface, and the amount of cytoplasm present when compared to the control animals glands. 18 DISCUSSION Day and McCollum (25) observed marked bone resorption and negative balance i n r a t s reared on a d i e t d e f i c i e n t i n phosphorus. They f e l t that the bone resorption was necessary to provide phosphorus f o r the essential needs of the soft tissues. So severe was the deficiency that the animals died after 8 - 1 0 weeks from collapse of the softened r i b cage and respiratory f a i l u r e . Copp et a l . (28) found l i t t l e radiocalcium remained i n the r a c h i t i c animals 8 days after i n j e c t i o n of Ca45. They concluded that t h i s may be due to the i n a b i l i t y of the r a c h i t i c animals to form new bone s a l t . This conclusion was confirmed by radioautographs which showed that radiocalcium was only found i n areas of new bone s a l t formation and none was found i n the un c a l c i f i e d osteoid of r a c h i t i c bone. They found that the i n i t i a l uptake by the bones of both normal and r a c h i t i c animals was very rapid. However, while the isotope appeared to remain fi x e d i n the skeleton of the normal animals, i t was rapidly l o s t from the bones of the animals with low phos-phorus r i c k e t s . They concluded that ion exchange was undoubtedly an important factor i n the uptake of radiocalcium by the skeleton, and i s perhaps the only s i g n i f i c a n t one i n the adult animal but hot i n the young animal. The very rapid i n i t i a l uptake i n t h e i r young animals, with f i x a t i o n and a very slow l o s s , argued against an exchange process and argued f o r the p o s s i b i l i t y that the f i x a t i o n of the radiocalcium was probably due to i t s incorporation into new bone s a l t . Their r a c h i t i c r a t s on the other hand, had an i n i t i a l rapid uptake with no f i x a t i o n and a rapid loss from the 19. skeleton suggesting a very l a b i l e calcium f r a c t i o n i n these animals. They calculated the l a b i l e calcium f r a c t i o n i n these animals to be 13% of the bone s a l t present. Carlsson (12) found that r a c h i t i c rats bones continued to grow but f a i l e d to c a l c i f y . Bauer et a l . (7) i n studies on human r i c k e t s with radio-phosphorus found that i n children with vitamin D deficiency r i c k e t s the accretion rate of bone salt was lower than i n normal children of the same age. Bauer et a l . (8) found i n a case of osteoporosis studied by means of radio-phosphorus that the accretion rate was about one t h i r d that of a normal subject. In the experiment described i n t h i s thesis, i t was found that rats subjected to a d i e t extremely low i n phosphorus had an accretion rate i n the femur approximately one-third that of the control animal and i n the carcass an accretion rate approximately two-fifths that of the control animal. The exchangeable portion of the r a c h i t i c femur was approximately 13% while that i n the control femur i t was 4 - 8$. Bones from experiment I I had a higher accretion rate than those from experiment I. This could be attributed to the fact that these animals were younger. Bauer et a l . (5) stated that the accretion and resorption rates are d i r e c t l y proportional to the increase i n weight of the body as a whole. The rate of accretion diminished with age. The resorption rate of the r a c h i t i c animal was only one h a l f of the resorption rate of the control animal. Even though the resorption rate i n the r a c h i t i c animal was lower than i n the control animal i t was s t i l l higher than the accretion rate of the r a c h i t i c animal and, therefore, accounted for the marked demineralization of the 20. skeleton. Bauer (4) gave a value of 0.1O mg/hr. for the accretion rate of normal i n c i s o r s . Bauer et a l . (5) gave a value of 0.08 mg/hr. f o r the accretion rate of the i n c i s o r s . The i n c i s o r average value from t h i s experiment for the four i n c i s o r s of the r a t was 0.084 mg/hr. The control t i b i a plus f i b u l a value of accretion and resorption obtained i n t h i s experiment corresponded very clo s e l y to the values obtained by Bauer et a l . (5, 6). Their values were dif f e r e n t i n that they are f o r both t i b i a s and f i b u l a was not included. In the introduction i t was noted that many authors have found a de f i n i t e difference i n the h i s t o l o g i c a l structure of r a c h i t i c teeth but that none had noted a d e f i n i t e difference i n chemical content. This could be due to the f a c t that the difference between the accretion rate of r a c h i t i c and control animals was not markedly lower and that the a t t r i t i o n -rate i n the r a c h i t i c animal was lower. Therefore, the bones w i l l show marked changes i n t h e i r chemical structure long before a s t a t i s t i c a l difference i n the teeth can be noted. Becks and Ryder (9) noted that a r a c h i t i c diet produced marked pathological changes i n the dentin i f the diet was maintained during the development of the teeth. These changes increased i n extent and.severity as the.period of the d i e t was prolonged. Weiraann and Schour (72) found that rats on a low phosphorus diet showed no difference i n enamel formation and c a l c i f i c a t i o n . They found, however, that the dentin formation and c a l c i f i c a t i o n was retarded and d i s -turbed. The enamel seemed immune to the metabolic disturbances of r i c k e t s . 21. This difference i n behavior of enamel from that of other hard tissues such as dentin, cementun and bone, may be caused by the chemical difference of the matrix of these tissues. Leicester (4-7) described the protein of enamel as a type of keratin, r e l a t i v e l y low i n the sulfur. The adult enamel matrix contains a muco-polysaccharide i n addition to the keratin. The protein of dentin i s collagen. Zipkin and Piez (73) found that sound enamel contained 98.7 mg.$ c i t r i c acid while dentin contained 888.0 mg.% c i t r i c acid. Atkinson (2) showed that enamel and not dentin could act as a semi-permiable membrane r e s i s t i n g the passage of large molecular but not of small ions. Sodium chloride took 10 to 30 days to penetrate through enamel. These chemical differences may account for the difference i n the reaction of dentin and enamel to ricket-producing die t s . Therefore, dentin, with a matrix s i m i l a r to bone and a high c i t r i c acid content, seems to react i n a manner si m i l a r to bone when the animal i s fed a r a c h i t i c diet. The s p e c i f i c a c t i v i t y curves f o r skin showed a marked difference between the r a c h i t i c animal and the control animal. The control animal showed a s p e c i f i c a c t i v i t y curve which followed very clo s e l y the blood plasma curve. Skin s p e c i f i c a c t i v i t y of the r a c h i t i c animal was higher than the blood plasma at f i r s t and then f e l l w ell below the blood plasma spec i f i c a c t i v i t y curve. The r a c h i t i c animal had a higher t o t a l tissue chemical calcium concentration. One could postulate that the higher calcium l e v e l and lower s p e c i f i c a c t i v i t y values of the r a c h i t i c animal could be due to a larger, very slowly exchangeable portion i n the skin of the animal. This slowly exchangeable or non-exchangeable portion could correspond to the calcium concentration that Belanger (10) found i n the hair immediately adjacent to the root sheath below the keratogenous zone. 22. Eichelberger and McLean (26) observed a muscle concentration of 3.26 mg.$ calcium i n dogs. The i n t r a - c e l l u l a r concentration was 3.79 mg.$. The extra-cellular concentration was estimated to 6.52 mg.%. They found, however, that for t h i s estimate of e x t r a - c e l l u l a r f l u i d calcium concentration, the amount of f l u i d needed i n the e x t r a - c e l l u l a r pool to account for the calculated amount of calcium present was disproportionately high. They concluded, therefore, that the excess calcium present must be combined with the connective tissue f i b r e s of the muscle. G i l b e r t and Wallace (35), working on frog muscle, separated the calcium of muscle into f i v e compart-ments. These compartments are shown i n the s o f t tissue calculations. They found that by t h i r t y minutes the e x t r a - c e l l u l a r muscle radiocalcium was i n equilibrium with the surrounding medium and the i n t r a - c e l l u l a r radiocalcium reached equilibrium i n 5 hours. They concluded that there was an electrochemical gradient much l i k e that f o r sodium, pushing calcium into the c e l l . They postulated that there was a calcium pump, a c t i v e l y removing calcium from the muscle c e l l . They found that, as part of the i n t r a - c e l l u l a r calcium, there appeared to be a non-exchangeable or very slowly exchangeable portion. Using Bauer's equation 5 i t was possible to calculate the movement of calcium from the e x t r a - c e l l u l a r to the i n t r a -c e l l u l a r pool. The rate of movement of calcium was calculated to be 0.23 mg/hr. and was not s i g n i f i c a n t l y different i n experimental compared to the control. The concentrationAcalcium i n the e x t r a - c e l l u l a r muscle f l u i d was 8 mg.% f o r both groups of experimental animals. The calcium i n the assumed i n t r a - c e l l u l a r pool was markedly dif f e r e n t . The control animals had' 3.18 mg.% and the r a c h i t i c animals had 5.06 mg.% i n t h e i r i n t r a - c e l l u l a r f l u i d . 23. The marked difference i n the disappearance rate of radiocalcium from the muscle as compared t o the other soft tissues could be due to the large i n t r a - c e l l u l a r calcium pool i n muscle, and to the kinetic tmovements of calcium i n and out of t h i s pool. The higher i n t r a - c e l l u l a r muscle calcium concentration of the r a c h i t i c animal may be due to a l a r g e r , slowly exchangeable portion i n the i n t r a - c e l l u l a r pool. This higher i n t r a -c e l l u l a r calcium concentration could explain the slower release of muscle radiocalcium from the r a c h i t i c animal. The i n t r a - c e l l u l a r pools i n the other soft tissues examined were small i n comparison to the size of t h e i r e x t r a - c e l l u l a r pools and, therefore, the rates of movement of calcium could not be determined with any degree of accuracy. Raised i o n i c calcium l e v e l s tend to i n h i b i t voluntary and involuntary muscle and the nervous tissue. Freeman and McLean (31) found anorexia, lassitude and lack of muscle tone i n puppies on a phosphorus deficient diet. Day and McCollum (25) noted the development of muscle weakness i n the legs of t h e i r r a t s and the same weakness was noted i n these experiments. I t i s possible that t h i s weakness was due to an accumulation of calcium i n the i n t r a - c e l l u l a r muscle pool of the animal. Schneider and Steenbock (62) found that animals raised on a phosphorus deficient diet f o r 20 weeks developed urinary c a l c u l i which were p r a c t i c a l l y pure calcium c i t r a t e . The vitamin D content of t h e i r diet was 380. I.U./lOOOm. of diet. Sager and Spargo (60) found urinary c a l c u l i i n t h e i r animals fed a low phosphorus d i e t . The rats which developed urinary c a l c u l i were also protein depleted. Coleman et a l . (21) and Copp et a l . (23) did not f i n d urinary c a l c u l i i n th e i r phosphorus deficient animals. Chemical analysis 24. of the kidneys i n t h i s experiment showed no difference i n the chemical calcium or phosphorus content of the experimental animals. H i s t o l o g i c a l examination did not reveal any pathological changes i n the kidneys of r a c h i t i c animals. Day and McCollum (25) noted that the phosphorus deficient animal seemed to adjust to the phosphorus deficient d i e t by what appeared to be homeostatic mechnisras. There appeared to be an increased absorption of calcium from the gut i n order to t r y to conserve the phosphorus excreted i n the i n t e s t i n a l j u i c e s . There was a marked conservation of phosphorus by the kidney and nearly a l l of the calcium absorbed from the i n t e s t i n a l t r a c t was excreted by the kidney even though the calcium l e v e l did not r i s e above 10 mg.%. F o l l i s et a l . (30) examined the parathyroids of the phosphorus deficient animal and, even though the c e l l s i n the parathyroids of the control animal appeared larger than those i n the r a c h i t i c animals, they concluded that there was no difference between the glands, since the control animal was a larger animal. Engfeldt (27) found that animals on a high calcium, low phosphorus diet developed hypoplastic parathyroid glands while animals on a high phosphorus, low calcium d i e t developed hyperplastic glands. Engfeldt (27) based his diagnosis of a c t i v i t y of the parathyroid gland on measurement of c e l l volumes by s e r i a l sections, the amount of mitosis taking place i n the gland, and the r e l a t i v e cytoplasm to nuclear volumes H i s t o l o g i c a l studies on the parathyroid glands of the phosphorus deficient animals showed findings similar to those found by Engfeldt (27). 25. Jones found that parathyroidectomized rats fed a phosphorus diet had a' calcium level of 10 mg.% and had a phosphorus level of 2 mg.%, characteristic of rickets. He concluded that the phosphorus deficient diet completely changed the blood picture from one of parathyroid deficiency to one of rickets. McCollum et a l . (49) and Cavin (14) reported that starvation of phosphorus deficient animals lowered the blood calcium level and raised the blood phosphorus l e v e l , to levels comparable to that of a parathyroidectomized animal. Phosphorus deficient animals developed i n this laboratory were subjected to a 24 hour fast, the blood plasma calcium and phosphorus levels of these animals were similar to those observed by Cavin (14) and McCollum et a l . (49). T aim age et a l . (69) found that i n the normal animal parathyroid hormone appears to aid i n controlling the renal threshold for phosphate excretion, thereby maintaining a normal phosphate level. Loss of the hormone altered the kidney mechanism so that essentially no phosphate was excreted u n t i l the serum level reached or surpassed 13 mg.$. Calcium excretion increased considerably after parathyroidectomy and then decreased as the serum level f e l l to 6 mg.%. Therefore, there also appeared to be a lowering of the renal threshold to calcium i n the absence of parathyroid hormone. Soerk and Silber (67) showed an inhibitory influence of para-thyroid hormone upon tubular reabsorption of phosphate i n the parathyroid-ectomized animal. From this evidence and the results obtained i n this experiment i t would appear that rats on a phosphorus deficient diet develop hypoplastic parathyroid glands which may serve as a homeostatic mechanism for 26. conservation of phosphorus. This brings up the question as to whether ric k e t s due to calcium deficiency are due to another mechanism or not. I f the parathyroid hormone raises the renal threshold f o r calcium and i s stimulated by a low plasma calcium l e v e l , one would expect to f i n d that the body homeostatic mechanisms would t r y to maintain the blood calcium l e v e l i n a calcium deficient animal by stimulating the parathyroid glands. Engfeldt (27) found t h i s to be the case when he examined parathyroid glands from animals on a calcium and calcium d e f i c i e n t d i e t . The p o s s i b i l i t y that there might be a difference i n the r i c k e t s produced by these two dif f e r e n t types of r a c h i t i c diets was made by Shaw (62) i n his statement, "The morphological c r i t e r i a of r i c k e t s are found i n the bony shaft and at the junction of the shaft and the c a r t i l a g e . Two separate processes are involved: f i r s t , the f a i l u r e of the epiphyseal cartilage c e l l s to complete the sequences of p r o l i f e r a t i o n , maturation, and degeneration; and, second, the f a i l u r e of the osteoid matrices to c a l c i f y . Apparently, the changes are closely s i m i l a r whether produced by vitamin D deficiency i n the species that require i t , or by calcium or phosphorus deficiency, or by imbalanced calcium to phosphorus r a t i o . One possible exception i s i n the r a t , where hypertropic, u n c a l c i f i e d cartilage and osteoid appear to be much more prominent i n diets with high calcium to phosphorus r a t i o than i n those with low calcium to phosphorus r a t i o " . 2-7. \ Summary 1. Young Wistar female rats raised on a phosphorus deficient diet for f i v e weeks, developed severe r i c k e t s . No deficiency was noted i n the control animals. 2. The serum plasma levels for calcium and phosphorus were 9.43 tng.% and 2.86 mg.$ i n the r a c h i t i c animal and 9.91 mg.$ and 7.24 mg./S i n the control animal. 3. The r a c h i t i c animal showed marked demineralization of the skeleton, lower radiocalcium uptake by the bone, and higher radiocalcium uptake by the teeth when compared with the control animal. 4. The disappearance of plasma radiocalcium was not as rapid i n the r a c h i t i c animals because of the low accretion rate i n these animals. 5. The accretion and resorption rate of bone i n the phosphorus de f i c i e n t animals was markedly reduced. The resorption rate, however, was higher than the accretion rate and accounted for the reduced mineralization of the bone. 6. The femur of the r a c h i t i c animal had an exchangeable calcium portion of 13% as compared to 4-8/£ i n the control animal. 24 hours after i n j e c t i o n of radiocalcium, 4-2% of the radiocalcium present i n the femur of the r a c h i t i c animal was i n the exchangeable portion, as compared to 16% i n the control animal. 7. The teeth of the phosphorus deficient animal showed a reduced accretion and a t t r i t i o n rate, and a s t a t i s t i c a l l y evident difference i n the chemical calcium and phosphorus content. The accretion rate was higher than the a t t r i t i o n rate, so that the teeth remained w e l l mineralized. The depression of the accretion rate was not as marked as that observed i n the bone and, therefore, the marked demineralization of the r a c h i t i c animals bones was not evident i n the teeth. 8. Starvation of the phosphorus deficient animals resulted i n a lower plasma calcium and raised plasma phosphorus similar to that observed i n parathyroidectomized animals. 9. The soft tissue calcium concentration i n the r a c h i t i c animal as compared to the control animal was higher for a l l soft tissues examined with the exception of the kidney and blood plasma where there was no s i g n i f i c a n t difference;, There was no difference i n the phosphorus concentration of control and r a c h i t i c animals soft tissues. 10. The amount of calcium i n the various muscle compartments was calculated. The e x t r a - c e l l u l a r compartment contained 16.6 mg/Kg. body weight for both the c o n t r o l and r a c h i t i c animals, and 8.0 mg.% i n the e x t r a - c e l l u l a r f l u i d pool. The i n t r a - c e l l u l a r calcium concentration was 5.06 mg.% f o r the r a c h i t i c animal and 3.18 mg.% for the control animal. 11. The rate of calcium movement from the e x t r a - c e l l u l a r to i n t r a - c e l l u l a r pool of muscle was calculated to be 0.23 mg./hr. 12. Radiocalcium disappeared rapidly from the soft tissues of the experimental and control animal. The disappearance of radiocalcium from the soft tissues coincided with the disappearance from the blood plasma for a l l soft tissues examined with the exception of muscle, where the rate of disappearance was much slower. 13. H i s t o l o g i c a l studies of the femur of r a c h i t i c and control animals showed that the r a c h i t i c femur had a wider epiphyseal cartilage which was not uniform i n width. The bone trabeculae showed wide ir r e g u l a r seams of u n c a l c i f i e d osteoid matrix. 14. By normal h i s t o l o g i c a l stains (H. & E.), the glomeruli and tubules of the r a c h i t i c animals kidneys showed no abnormality. H i s t o l o g i c a l and chemical analysis of the kidneys of these animals showed no evidence of calcium deposits or nephrocalcinosis. 15. H i s t o l o g i c a l studies of the parathyroid glands of r a c h i t i c animals showed a decrease, i n the volume of the glands, i n the si z e of the nuclear surface and i n the amount of cytoplasm present, when compared to the glands of the control animals. CONCLUSION This study of calcium k i n e t i c s i n the phosphorus deficient animal coupled with the h i s t o l o g i c a l findings shows the p o s s i b i l i t y that phosphorus deficiency i n r a t s produces a hypoparathyroid condition as a homeostatic. mechanism to conserve phosphorus for the soft tissues. CALCULATION OF RESULTS. Calcium can be taken up by the skeleton and be removed again by two different mechanisms: 1, physical exchange reactions between the bone sa l t and the surrounding f l u i d j 2, accretion (formation) and resorption (dissolution) of bone s a l t . By d e f i n i t i o n the exchange reactions are reversible processes and do not r e s u l t i n any net change i n bone salt mass. Accretion and resorption, on the other hand, can be regarded as two i r r e v e r s i b l e processes working more or less independently of each other at different s i t e s . As a rule there i s a d i s t i n c t i n t e r v a l of time between accretion and resorption. The following p r i n c i p l e s and calculations are described by Bauer et a l . (6). The amount of an isotope such as radiocalcium present i n a c a l c i f i e d tissue can be written as follows: 4 5 c a o b s * 4 5 ° a E + 4 5 c a A * 4 5 c a R ( 1 ) 45ca0kS i s the t o t a l amount of radiocalcium present i n the c a l c i f i e d tissues: 45cag i s the amount of radiocalcium present i n the exchangeable f r a c t i o n of the bone s a l t : 45ca^ i s the amount of radiocalcium incorporated into the non-exchangeable portion of bone salt through accretion: 4 5 Ca^ i s the amount of radiocalcium that has been removed from bone s a l t "through resorption: Assuming that the s p e c i f i c a c t i v i t y , (radiocalcium/stable calcium) of the exchangeable bone calcium f r a c t i o n rapidly attains the same value as the 31. s p e c i f i c a c t i v i t y of the plasma calcium, and that these two s p e c i f i c a c t i v i t i e s can then be taken as equal, we get 4 5 C a E » E x S (2) E, Amount of chemical calcium i n the exchangeable f r a c t i o n of the bone s a l t j S, Specific a c t i v i t y of the plasma calcium. Since i t must be assumed that 45ca and 40ca are deposited i n the bone sa l t i n the same proportions as those simultaneously occurraLng i n the blood, we get 45caA - A x T x % (3) A, Rate of calcium accretion, T, Interval of time between administration of radiocalcium and observation, Sj{ Average s p e c i f i c a c t i v i t y of serum calcium during t h i s time i n t e r v a l . Combination of equations (1), (2), (3), gives; 4 5 c a o b s " E x S + A x T x S M - *5caR (4) At s u f f i c i e n t l y short intervals radiocalcium resorbed can be disregarded. From observations made at two d i f f e r e n t i n t e r v a l s of time and T 2 we get 45cao bs 1 = E x S i + A x T i x SM 1 (4a) 4 5 C a o b s 2 - E x S 2 + A x T 2 x S M 2 (4b) From these two equations we obtain, 4 5 C a o b s 1 - A x T i x S H ! $ S± : " _ (5) 4 5 c a o b s 2 - A x T 2 x S M 2 f S 2 32. E i s assumed to be the same f o r the short i n t e r v a l involved. After A has been calculated from equation (5), E can be calculated by introducing the calculated A value into equation (4a). Equation (5) can be used for ca l c u l a t i n g the accretion rate of the whole skeleton. In t h i s c a l c u l a t i o n the whole bocty i s treated i n the same manner as a c a l c i f i e d tissue, the ^Caobs i s equal to 100% minus the percentage of dose l o s t v i a the kidneys and the g a s t r o i n t e s t i n a l t r a c t , and the E value i s the amount of calcium i n the exchangeable f r a c t i o n of bone, teeth and the calcium present i n the soft tissues. The accretion rate (A) of the bones and teeth were calculated from the calcium results by introducing into equation (5) the 6 and 24, 6 and 12, and the 12 and 24 hour radiocalcium observed values from Table I I and the S and SJI values from Table I I I . I f e a r l i e r values than those at 6 hours are used for the ca l c u l a t i o n of E, the c r i t e r i o n of equation (2) i s not met, and res u l t s which are too low w i l l be obtained. Sample calculation 6 and 24 hour t i b i a plus f i b u l a values from Table I I . ( phosphorus deficient animals) S and Sj4 vales from Table I I I . (phosphorus d e f i c i e n t animals) 1.69 m 2.45 - 3.33 x 6 x A 0.95 * 2.72 - 1.82 x 24 x A A - 0.041 mg./hr. Substituting for E we obtain 1.69E = 2.45 - 3.33 x 6 x 0.041 E = 0.96 mg. Using equations (2 and 3) calculated values for 45Ca,ft. and ^Qag 6 hr. 4 5 C a A = 0.041 x 6 x 3.33 a 0.82 4 5 C a E = 0.96 x 1.69 - 1.62 2.44 E x p . 4 5 C a o b s = 2.45 12 hr. 45ca A - 0.041 x 12 x 2.48 - 1.19 4 5 C a E = 0.96 x 1.42 - 1.36 24 nr. 2.55 • " ' - 2.57 4 5 C a A - 0.041 x 24 x 1.82 - 1.79 4 5 C a E = 0.96 x 0.95 - 0.91 2.70 " -?- 2.72 Resorption and a t t r i t i o n rate c a l c u l a t i o n experiment I I . The gain i n bone and teeth calcium from 4 to 5 weeks i s due to the difference i n the two processes accretion and resorption. Values for accretion were calculated as per equation (5) and the chemical calcium measured, i n the animals k i l l e d at four weeks. The same analysis was done for the f i v e week animals. The gain i n calcium weight i s due to calcium accreted minus calcium resorbed. Knowing the rate of accretion, the period of time involved and the actual amount of calcium gained over t h i s period of time, the rate of calcium resorption was calculated. Soft tissue calculations. The muscle radiocalcium disappearance curve appeared to have two major components one which was r a p i d l y exchangeable and another slowly exchangeable portion. The slower exchangeable portion was calculated 34. using equation (5) of the bone k i n e t i c s . The slowly exchangeable portion was assumed to be the movement of calcium from e x t r a - c e l l u l a r to i n t r a -c e l l u l a r f l u i d . The rapid exchangeable portion was assumed to be the exchangeable calcium present outside the c e l l . The movement of radiocalcium into the c e l l gave a uniform ca l c u l a t i o n from 1 to 6, 3 to 6, and 1 to 3 hours, using values for muscle radiocalcium found i n F i g . 3 as the radio-calcium observed values, and substituting these values into equation (5) a value f o r A of 0.23 mg./hr., movement of calcium from the ext r a - c e l l u l a r to i n t r a - c e l l u l a r f l u i d was obtained f o r both r a c h i t i c and control animals. Substituting for E, 1.50 mg. calcium was calculated to be present i n the rapid l y exchangeable e x t r a - c e l l u l a r portion of the r a c h i t i c animal and 2.50 mg. of calcium i n t h i s portion of the control animal. Skeleton (65) values of muscle 45$, and skin 18$, of body weight were used. Muscle wt. » 75$ water and skin wt. * 65$ water. Walser (70) values of muscle extra-cellular water 18.7$ and skin e x t r a - c e l l u l a r water 66.4$ were used. G i l b e r t and Wallace. (35) found that calcium i n muscle was distr i b u t e d i n f i v e areas, 10$ on the c e l l surface, 17$ connective t i s s u e , 12$ extra-c e l l u l a r f l u i d . These portions they assumed to be e x t r a - c e l l u l a r , therefore 26$ of the extra-cellular portion was on the c e l l surface, 44$ connective tissue and 30$ ext r a - c e l l u l a r f l u i d . These values were used i n Table IV. They also found 24$ i n t r a - c e l l u l a r f l u i d and 37$ non exchangeable or very slowly exchangeable which they assumed to be i n t r a - c e l l u l a r . 35. TABLE I Composition of the Experimental (Phosphorus-Deficient) Diet* Purified beef blood fibrin 20.0$ #Sucrose 60.0$ Hydrogenated cottonseed o i l (Crisco +) 10.0$ Pish liver o i l (400 U.S.P. units vitamin D and 1,000 U.S.P. units vitamin A per gram) 2.0$ Sodium Chloride 1.0$ Potassium chloride 1.5$ Magnesium sulfate.7H20 6.3$ Ferric citrate 0©1$ Calcium carbonate 1.0$ Vitamin mixture (l) 1.0$ Trace element mixture (2) 1.0$ * In the control diet the 1.0 per cent sodium chloride i s replaced with 2,4 per cent disodium phosphate. (l) Vitamin mixture(per 100 Gm. of diet) (2) Trace element mixture(per 100 Gm. diet) Thiamine hydrochloride 1.0 mg. CuS04.5H20 13.0 mg. Riboflavin 1.0 mg. MnS04 4.0 mg. Pyridoxine 0.5 mg. ZnS04.7H20 2.0 mg. P—aminobenzoic acid 1.0 mg. CaCl2.6H20 1.0 mg. Nicotinic acid 5.0 mg. KI 008 mg. Calcium pantothenate 5.0 mg. Sucrose 979o2 mg. 1.0 Gm. I-inositol 20.0 mg. Choline chloride 100.0 mg. Folic acid 1.0 mg. Ascorbic acid 100.0 mg. Sucrose 765.5 mg. 1.0 Gm. + Registered trademark # Phosphorus deficient diet sucrose = 62.1$ Control diet sucrose =60.7$ TABLE I I Radiocalcium and Chemical Average-Values for Hard Tissues from Experiment I iChemical Dose Ca45 Group Ca/mg. P/mg. 1 hr. 3 hr. 6 hr. 12 hr. 24 hr. Femur P def. 9ol7± 0.28 4.75± 0.14 3.07± 0.16 2.80+ 0.14 2.74± 0.10 2.81+ 0.08 2.91± 0.09 Control 5 0 . 8 5 ± 1.23 25.68+ Oi58 3.05± 0.09 3.54± 0.04 3.59+ 0.18 3.69± 0.12 3.81± 0.16 Tibia + P def. 10.57+ 0.35 5.25+ 0.15 2.45± 0.12 2.42± 0.21 2.45± 0 .36 2.57± 0.10 2.72± 0.08 Fibula Control 46.17+ 1.00 24.32+ 0«65 2.68+ 0.11 3.00+ 0.08 3*29+ 0.04 3.40± 0.03 3.62± 0.16 Whole P def. 9.88± 0.57 5 . 3 3 ± 0.19 2.38± 0.07 3 . 3 3 ± 0.13 3.25± 0.46 2.92± 0.07 2.61± 0.22 T a i l Control 63 . 79± 1.92 34.66+ 1.03 2.63± 0.35 3.71± 0.19 4.64± 0.10 3.87± 0.09 3.53± 0.10 Incisors P def. 37.68+ 0.93 21.01+ 0.82 1.07± 0.08 1 . 2 6 ± 0.11 1.58± 0.18 2.16± 0.11 2.87± 0.17 Control 48.95± 1.13 27.64± 0.96 1.06± 0.08 1.58± 0.08 1.90± 0.06 1.98± 0.14 2.18± 0.04 Molars P def. 3 0 o 5 0 ± 0o42 16.98± 0.17 0 . 5 3 ± 0 .06 0.58± 0.04 0.58± 0.04 0.77± 0.06 0.68± 0.02 Control 34.74± 0.63 ;, 19 . 75± 0o40 0.48+ 0.04 0.48± 0.01 0.50± 0.01 0.55± 0.03 0.47± 0.02 Whole Carcass P def. Control, 396.5 1404.8 280.4 830.7 99.9 ± 1.6 99.0 ± 1.8 96.5 ± 2.8 99.0 + 1.0 92,3 ± 1.8 97.2 ± 3.0 84.9 ± 2.1 98.3 ± 2.6 85.6 ± 2.9 97.1 ± 2.2 Average Total Recovery = 97*0 ± 1.1$ TABLE II A Urine Gut Chemical ea45 $ Dose i' Ca Clearance Group Ca/mg. P/mg. 1 hr. 3 br. 6 hr. 12 hr. 24 hr. i , mg/hr. Plasma cc/mii0l2 P def. 7.91 ± 0.82 0.17± 0.02 5.46 ± 1.01 9.58 ± 0.59 11.81 ± 0.28 14.55 ± 2.13 0.33 2.57 Control 0.34 ± 0.08 5.98 ± 0.42 0.14 ± 0.02 0.25 ± 0.06 0.11 ± 0.07 0.39 ± 0.12 0.015 0.079 9 def. 12.66 + 0.93 37.46 ± 1.21 6.18 ± , 0.12 5.74 ± 0.66 3.90 ± 0.53 4.72 ± 0.50 4.82 ± 0.56 -0 4 Control 34.00 ± 1.35 52.32 ± 1.63 7.92 ± 0.27 6.74 ± 0.36 5.34 ± 0.24 5.60 ± 0.35 4.67 ± 0.41 Faeces P def o Control 9.82 ± 0.86 19.04 + 1.44 0.75 ± 0.08 4.95 ± 0.82 38. P. def. $» Control S1 P. def. Sm Control Sm TABLE III Plasma Levels Ca4^ 1 hr«- 3 hr. 6 hr. 12 hr. 24 hr. 5.03 ± 0.25 6.97 6.39 2.55 ± 0.30 1.69 ± 0.24 1.42 ± 0.04 4.59 3.72 3.33 2.46 2.48 1.65 0.95 ± 0.04 5.54 ± 1.60 ± 0.98 ± 0.66 ± 0.33 ± 0.06 0.19 0.05 0.03 0.06 1.82 1.08 S« = % Dose Ca 4 5 / mg. Sm SB Average -specific activity during interval Chemical Calcium and Phosphorus Concentrations i n Plasma Ca mg. $ P. mg. P. def. Control. 9.43 ± 0.34 9.91 ± 0.15 2.86 ± 0.33 7.24 ± 0.21 Muscle P. def* Control TABLE IV Calcium and Phosphorus Concentrations of Soft Tissues ->2 Phosphorus g./lOG g. g./total organ 2.25 ± 0.09 2.22 ± 0.04 0.92 1.50 Calcium mg./lOOg. mg./total mg./Ex.^ mg* c e l l 4 mg* conn* 4 mg«$ Ex. 4 mg.$ i n t r a tissue c e l l , f l u i d c e l l 6.77 ± 0.25 5.64 ± 0.44 organ 2.76 3.81 ce l l u l a r surface 1.50 0.39 2.50 0.65 0.65 1.10 8.0 8.0 5.06 3.18.: Skin P. def. Control Liver P. def. Control 0.94 ± 0.02 1.00 ± 0.03 2.74 ± 0.12 2.72 ± 0.87 0.15 ± 0.27 ,..:0.012 0.019 10.05 ± 0.36 8.91 ± 0.41 8.51:± 0.58 5.81 + 0.42 1.64 2.40 6.38 0.41 1.37 2.12 7.58 4.74 V J J Kidney P. def. 2.80 ± 0.032 18.4 ± 0.20 0.07 0.75 Control 2.77 ± 0.05 18.6 ± 6.28 0.05 1.08 1. Control animal average weight = 150.0 g. P. def. animal average weight = 90.8 g. 2. Walser (70) $ water $ extracellular muscle 75 18.7 skin 6 5 66.4 3. Calculated using Bauer equation (5) 4. Calculated Gilbert (35) c e l l surface = 26$ exchangeable Ca conn, tissue = 44$ " " extra c e l l . = 30 $ " " 40. TABLE V #Accretion Rate and Exchangeable Portions of Hard Tissues i n Experiment I Femur P. def. Control mg. Ca A E $ E 9.17 ± 0.28 0.041 1.14 12.4 50.85 ± 1.23 0.124 1.86 3.65 Tibia + Fibula P. def. Control Incisors P. def. Control 10.57 ± 0.35 0.041 0.95 9.0 46.16+1.00 0.120 1.55 3.35 37.68 ± 0.93 0.061 0.24 0.64 48.59 ± 1.13 0.077 0.70 1.44 Molars P. def. Control 30.50 ± 0.42 0.008 0.35 1.17 34.74 ± 0.63 0.013 0.39 1.12 Whole Carcass less gut P. def. Control 383.9 1370.8 1.14 33.0 8.5 2.93 53.3 * 3.8 .* Includes soft tissues . # Accretion and exchange values are the average values from the 6 & 12, 6 & 24, and 12 and 24 Hour calculations. In a l l cases A & E values for the period of time calculated were almost exactly the same. TABLE VI Rate of Accretion, Resorption, Attrition and Exchangeable Portions of Experiment II mg. Ca start Exp. mg. Ca 4 wks mg. Ca 5 wks A mg/hr R mg/hr E mg. Femur P. def. 11.17 ± 0.27 7.72 ± 0.29 7.38 ± 0.21 0.056 0.058 1.05 Control 57.78 ± 1.05 43.77 ± 0.76 0.161 0.125 5.08 Tibia + Fibula P. def. Control 10.95 ± 0.15 8.78 ± 0.50 55.71 ± 0.67 8.64 ± 0.24 39.66 ± 0.44 0.054 0.151 0.055 0.116 0.99 2.65 Incisors P. def. 18.84 ± 0.22 51.67 ± 0.74 35.26 ±* 0.55 0.062 0.055* 0.81 Control "-'. 35.21 0.99 40.21 0.41 0.092 0.062 0.12 Molars P. def. 20.55 ± 0.52 25.57 ± 0.55-27.54 ± 0.46 0.011 0.001 0.20 Control 29.34 ± 0.78 52.11 ± 0.55 0.018 0.001 0.45 Whole Carcass less Cut P. def. Control -1.51 5.86 1.57 5.00 E * The animals gnawed on the wire cages causing the possibility that the mg. Ca present may be low and therefore the attrition rate may be higher than the true value. 42. Body Weight Gain. Weaned to Diet -25 Days. gms 50 H Weeks on Diet. Gain i n body weight on control and phosphorus deficient diet 43. F i g . 2 Mean radiocalcium s p e c i f i c a c t i v i t y of blood plasma. 44. % dose Time in Hours i F i g . 3a Disappearance of radiocalcium from soft tissues of the control r a t . 45. % dose Low P Rats. O 4 8 12 16 20 24 T i m e i n H o u r s . F i g . 3b Disappearance of radiocalcium from the soft tissues of the phosphorus deficient animal. 46. %Dose mg. Co 5 n Ca - Specific Activity Soft Tissues Control Rats 0 Muscle — Plasma. 1 yver Skin Time in Hours. F i g . 4a Soft tissue radiocalcium s p e c i f i c a c t i v i t y curves of the control animal. 47. %Dose mg. Ca -i x Ca — Specific Activity Soft Tissues Phosphorus. Deficient Rats. 8 16 Time in Hours Liver T 1 r F i g . 4b Soft tissue radiocalcium s p e c i f i c a c t i v i t y curves of the phosphorus deficient animals. 48. Femur. • (Control) ! I % Dose Ca 4 6 j O -i 1 1 1 1 1 1 0 8 16 24 Time in Hours. F i g . 5a Role of accretion (A) and exchange reactions (E) i n the uptake and removal of radiocalcium i n the control femur. The curves labeled Ca45(E) and Ca45(A) were calculated according to equations (2) and (3) respectively of the appendix. 49. Femur. ( Phosphoros Deficient) % Dose Co O 8 16 24 Time in Hours. Fig. 5b Role of accretion (A) and exchange (E) reactions i n the uptake and removal of radiocalcium i n the phosphorus deficient femur. The curves labeled Ca45(E) and Ca45(A) were calculated according to equations (2) and (3) respectively, of the appendix. 50. Incisors. ( Control ) Co 0 OBS. -f— 0 8 16 Time in Hours. —i • 24 Fig. 6a Role of accretion (A) and exchange (E) reactions i n the uptake of radiocalcium in.the control incisors. The curves labeled Ca45(E) and Ca45(A) were calculated according to equations (2) and (3) respectively of the appendix. . . 51. Incisors. ( Phosphoros Deficient ) % Dose Ca 3.0 - i 45 2.0 -.0 Ca 45 0BS. -A c a 4 5 A. .0 1.0 - 0 / / / I I A - i 1 1 r 8 16 Ca 45 -X E. 24 Time in Hours. F i g . 6b Role of accretion (A) and exchange (E) reactions i n the uptake of radiocalcium in.the phosphorus deficient i n c i s o r s . The curves labeled Ca45(E) and Ca45(A) were calculated according to equations (2) and (3) respectively.of the appendix. 52. Phosphorus Deficient. Control. Gut 16 mg./doy Diet Faeces 10 mg./doy Plasma 9.43 m g . % I t Urine 0.33 m g / h r . Bone 9 Gut 4 0 m g . / d a y Diet 1.51 mg/hr. > 1.57 mg/hr Faeces 19 mg. /day Plasma 9.91 m g . % I f Urine 0.015 m g / l i r Bone 9 3 . 8 6 mg/h r . » 3 .00 mg/hr . F i g . 7 Calcium balance i n the control and r a c h i t i c animal. \ 53. BIBLIOGRAPHY 1. Amprino, R.; Autoradiographic analysis of the distribution of labelled Ca and P i n bones. Experientia, 8: 20, 1952. 2. Atkinson, H.F. j An investigation into the permeability of human enamel usihg osmotic methods. Br i t . Dent; J., 84$ 113, 1948. 3. Bauer, G.C.H.j The importance of bone growth as a factor i n the redistribution of bone salt. I_ Redistribution of radio-active calcium in the skeleton of rats. J. Bone J t . Surg., 36A: 375, 1954. 4. J Bone salt metabolism in bone growth and bone repair studied i n rats by means of Ca45, p32, and Na 2 2. Acta. Orthop. Scand., 23: 247, 1954. 5. Bauer, G.C.H., Carlsson, A., and Lindquist, B.j Evaluation of accretion, resorption and exchange reactions, i n the skeleton. Kgl. Fysiograf. Sa l l s k . i Lund Forhandl., 25: 1, 1955. 6. ; A comparative study on the metabolism of Ba^O a n d Ca45 in rats. Biochem J., 63: 535, 1956. 7. 3 Bone salt metabolism i n human rickets studied with radioactive phosphorus. Metabolism, 5s 573, 1956. 8. j Accretion rate of bone salt i n osteoporosis studied by means of p52. Acta Med. Scand., 158: 139, 1957. 9. Becks, H., and Ryder, W.B.; Experimental rickets and calcification of dentin. A.M.A. Arch. Path. 12: 358, 1931. 10. Belanger, L.F.j The entry of Ca45 into the skin and other soft tissues of the rat: An autoradiographic and spodographic study. J» Histochem. Cytochem., 5: 65, 1957. 11. Brown, H.B., Shohl, A.T., Chapman, E.E., Rose, C.S. and Saiirwein, E.M.; Rickets i n rats. X l l l The effect of various levels and ratios of calcium to phosphorus i n the diet upon the production of rickets. J. Biol. Chem., 98: 207, 1923. 12. Carlsson, A.j' Metabolism of radiocalcium i n relation to calcium intake i n young rats. Acta pharm. tox., Kbh., 7s Suppl. 1. 1951. 13. J On the mechanism of skeletal turnover of lime salts. Acta physiol. scand., 26: 200, 1952. 14. Cavin, A.W. j The effect of fasting on the inorganic salts i n the blood serum of normal and rachitic rats. J. B i o l . Chem., 59s 237, 1924. :5,4. 15. Clark, E.P., and Collip, J.B.j A study of the Tisdall method for determination of blood serum calcium with suggested modification. J. Biol. Chem., 63: 461, 1925. n 16. Claassen, V., and Wostmann, B.S.J.j The uptake of injected radioactive phosphorus i n the skeleton of the growing white rate. I The uptake of intravenously injected P32 by rachitic and control animals during the f i r s t 24 hours. Biochim. biophys. acta, 12: 432, 1953. 17. J Uptake of P32 i n the femur of growing rachitic and normal rats as compared with the uptake i n the total skeleton. Biochim. biophys. acta. 10: 625, 1953. 18. j Uptake of injected radioactive phosphorus in the skeleton of the growing white rat. I l l Metabolism of skeletal P^2 i n experiments of longer duration, performed with rachitic and control animals. Biochim. biophys. acta, 13: 48, 1954. 19. Cohn, W.E., and Greeriberg, D.M.j Studies i n mineral metabolism with the aid of a r t i f i c i a l radio-isotopes. I l l The influence of vitamin D on the phosphorus metabolism of rachitic rats. J. Bi o l . Chem., 130: 625, 1939. 20. Coleman, R.D., Becks, H., Copp, D.H., and Fransen, A.M.; Skeletal changes i n severe phosphorus deficiency in the rat. I I Skull, teeth and mandibular joint. Oral Surg., 6: 756, 1953. 21. Coleman, R.D., Becks, H., Kohl, F. Van N., and Copp, D.H.j Skeletal changes in severe phosphorus deficiency of the rat. I_ Tibia, metacarpal bone, costochondral junction, caudal vertebra. A.M.A. Arch. Path., 50: 209, 1950. 22. Comar, C.L.-, Hansard, S.L., Hood, S.L., Plumlee. M.P., and Barrentine, B.F.j Use of Ca45 i n biological studies. Nucleonics, 8: 19, 1951. 23. Copp, D.H., Hamilton, J.G., Jones, D.C., Thompson, D.M., and Cramer, C.F.j The effect of age and low phosphorus rickets on calcification and the deposition of certain radioactive metals i n bone. Trans. Macy Conf. on Metabolic Interrelations, 3: 226, 1951. 24. Cramer, J.W., Steenbock, H.j Calcium metabolism and growth i n the rat on a low phosphorus diet as affected by vitamin D and increased i n calcium intake. Arch. Biochem., 63: 9, 1956. 25. Day, H.G., McCollum, E.V.j Mineral metabolism, growth, and symptomatology of rats on a diet extremely deficient i n phosphorus. J. Biol. Chem., 130: 269, 1939. 26. Eichelberger, L., McLean, F.C.j The distribution of calcium and magnesium between the c e l l s and the extra cellular fluids of skeletal muscle and l i v e r i n dogs. J. Biol. Chem., 142: 467, 1942. 55. 27. Engfeldt, B. •. Studies on parathyroidal function i n relation to hormonal influences and dietetic conditions. Acta endocr. Kbh., 5* Suppl. 6, 1950. " II 28. Engfeldt, B., Engstrom, A., and Zetterstrom, R.; Renewal of phosphate i n bone minerals. II Radioautographic studies of the renewal of phosphate i n different structures of bone. Biochim. biophys. acta, 8s 375, 1952. 29. Feaster, J.P., Shirley, R.L., McCall, J.T., and Davis, G.K.j P32 distribution and excretion i n rats fed vitamin D.free and low phosphorus diets. J. Nutrit., 51: 381, 1953. 30. F o l l i s , R.B. J r . , Day, H.G., and McCollum, E.V.; Histological studies of the tissues of rats fed a diet extremely low i n phosphorus. J. Nutrit., 20; 181, 1940. 31. Freeman, S., and McLean, F.C.j Experimental Rickets. Blood and tissue changes i n puppies receiving a diet very low i n phosphorus with and without vitamin D. A.M.A. Arch. Path., 32: 387, 1941. 32. Gaunt, W.E., G r i f f i t h , H.D., and Irving, J.T.: The assimilation of radioactive phosphorus following phosphorus deficiency i n rats. J. Physiol., 100: 372, 1942. 33. Gaunt, W.E., and Irving, J.T.j The influence of dietary calcium and phosphorus upon tooth formation. J. Physiol., 99t 18, 1940. 34. Gies, W.J.', and Perlzweig, W.A. j The influence i n dogs and rats on diets deficient i n calcium or phosphorus. J. A l l i e d Dent. Soc, 11: 80, 1916. 35. Gilbert, D.L., and Wallace, O.F.j Calcium equilibrium i n muscle. J. Gen. Physiol., 40: 393, 1957. 36. Hammet, F.S.j Studies of thyroid apparatus. XLV The role of the thyroid and parathyroid glands in the chemical differentiation of bone during growth. J. Biol. Chem., 7 2 : 527, 1927. 37. Hansard, S.L., and Crowder, H.M.j The physiological behavior of calcium i n the rat. J. Nutrit., 62: 325, 1957. 38. Hess, A.F., Unger, L.J., and Pappenheimer, A.M.) Experimental rickets i n rats. J. Biol. Chem., 50: 77, 1922. 39. Hevesy, G.C., Levi, H.B., and Rebbe, O.H.j Rate of rejuvenation of the skeleton. Biochem. J., 34: 532, 1940. 40. Jones, J.H.j The influence of the removal of the parathyroid glands on the development of rickets i n rats. J. B i o l . Chem., 106: 701, 1934. 41. j A study of rachitogenic diets composed of purified food materials. J. Nutrit., 17: 601, 1939. 56. 42. Karshan, M.; Calcification of teeth and bones on rachitic and non-rachitic diets. J. Dent. Res., 13: 301, 1933. 43. Kassowitz, M. $ Die phosphorusbehandlung der Rachitis. Zeitsch. fur Kind. Med. Bd. 7: S36, 1884. 44. Kissel, A. Ueber die pathologische-anatomischen veranderungen i n den Knochen wachsend^r Thiere unter dem Einfluss minimalen phosphordosen. Virchows Arch, fur pathologische Anatomie, Bd. 144* S94, 1896. 45. Leblond, CP., Wilkinson, G.W., Belanger, L.F., and Robichon, J.; Radio-autographic visualization of bone formation i n the rat. . Am. J. Anat. 86: 289, 1950. 46. Lehman, J.j A photoelectric micro method for direct t i t r a t i o n of calcium i n serum with ethylenediamine tetra acetate. Scand. J. Clin, and Lab. Invest., 5: 203, 1953. 47. Leicester, H.M.j The biochemistry of teeth. Annual Rev. Biochem., 22: 341, 1953. 48. Lindquist, B.j Effect of vitamin D on the metabolism of radiocalcium i n rachitic rats. Acta paediat., Upps., 41: Suppl. 86. 1952., 49. McCollum, E.V., Simmonds, N., Shipley, P.G., and Park, E.A.$ Studies on experimental rickets. J. B i o l . Chem., 51: 41, 1922. 50. Studies of experimental rickets. Johns Hopkins Hospital B u l l . , 33: 31, 1922. 51. Myers, H.M.j The comparative turnover rate of p32 i n normal and deficient rats teeth and bones during recovery from phosphorus deficiency. J. Dent. Res., 34: 225, 1955. 52. Neuman, W.F., and Hodge, H.C. $ Role of the skeleton i n pharmacological and toxicological responses, i n V.A. D r i l l , Pharmocology i n Medicine, McGraw H i l l , New York, 1954. 53. Neuman, W.F., and Neuman, M.W. $ The nature of the mineral phase of bone. Chem. Rev., 53: 1, 1953. 54. Osborne, T.B., Mendal, L.B., and Park, E.A.J Experimental production of rickets with diets of purified food substances. Proc. Soc. Exp. Biol., 21: 87, 1923. 55. Phemister, D.B.j The effect of'phosphorus on growing, normal, and diseased bones. J. Am. Med. Assoc., 70: 1737. 1918. 56. Phemister, D.B., Miller, E.M., and Bonar, B.E.j The effect of phosphorus in rickets.. J. Am. Med. Assoc., 76: 850, 1921. 57. 57. Rogers, H.J., and Weidmann, S.M.j Metabolism of alveolar bone. B r i t . Dent. J . , 90s 7, 1951.. 58. Rogers, H.J., Weidmann, S.M., and Jones, H.Q.; Studies on the s k e l e t a l tissues. I l l The rate of exchange of the. inorganic phosphate i n different bones and parts of bones i n various species of mammal. Biochem. J . , 54s 37, 1953. 59. Rosebury, T., and Karshan, M.j Pathological changes i n teeth of rats produced by synthetic diets. J . Dent. Res., 11: 137, 1931. 60. Sager, R.H., and Spargo, B.j The effects of low phosphorus ration on calcium metabolism i n the r a t with the production of calcium c i t r a t e urinary c a l c u l i . Metabolism, 4: 519, 1955. 61. Schneider, H., and Steenbock, H.j A low phosphorus d i e t and the response of rats to vitamin D. J . B i o l . Chem., 128: 159, 1939. 62. j Low phosphorus diets and urinary l i t h i a s i s . J . B i o l . Chem., 128: p l x x x v i i , 1939. 63. Shaw, J.H.j Effect of n u t r i t i o n a l factors on bone and teeth. Ann. N. York Acad. Sc., 60: 733, 1955. 64. Sherman, H.C, and Pappenheimer, A.M.; A d i e t i c production of ri c k e t s i n rats and i t s prevention by inorganic s a l t s . Proc. Soc. Exp. B i o l . , 18: 193, 1921. 65. Skeleton, H.j The storage of water by various tissues of the body. Arch. Int. Med. 40: 140, 1927. 66. Steenbock, H., and Black, A.j Fat soluble vitamins. XXIII The induction of growth promoting and c a l c i f y i n g properties i n fats and. t h e i r unsaponifiable constituents by exposure to l i g h t . J . B i o l . Chem., 64: 263, 1925. 67. Stoerk, H.C, and S i l b e r , R.H.j Parathormone i n renal reabsorption of phosphate. Laborat. Invest., 5: 213, 1956. 68. Sumner, J.R.j A modification of the Fiske and Subborow method f o r phosphate analysis. Science, 100$ 413, 1944. 69. Talmage, R.V., Kraintz, F.W., and Buchanan, G.D. $ Effect of parathyroid extract and phosphate s a l t s on renal calcium and phosphate excretion after parathyroidectomy. Proc. Soc. Exp. B i o l . , 88:- 600, 1955. 70. Walser, M., and Bodenlos, L.J.j Composition of skin as compared with muscle. Am. J . P h y s i o l . , 178: 91, 1954. it 71. Wegner, G.; Der Einfluss des Phosphorus auf den Organismus. Arch, fur pathologische Anatomie und Physiologie., Bd 55: S l l , 1872. 58. 72. Weinmann, J.P., and Schour, I . j I_ Experimental studies i n c a l c i f i c a t i o n . I l l The ef f e c t of parathyroid hormone on the alveolar bone and teeth of the normal and r a c h i t i c r a t . Am. J . Path., 21: 857, 1945. 73. Zipkin, I . , and Piez, K.A.J The c i t r i c acid content of human teeth. J . Dent. Res., 29: 498, 1952.. 74. Zucker, T.F., H a l l , L., and Young, M.j Growth and c a l c i f i c a t i o n on a di e t deficient i n phosphate but otherwise adequate. J. N u t r i t . , 22: 139, 1941. i 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0106170/manifest

Comment

Related Items