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Plasma calcium regulation associated with induced hypocalcemia and hypercalcemia Mensen, Esther Doris 1958

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PLASMA CALCIUM REGULATION ASSOCIATED WITH INDUCED HYPOCALCEMIA AND HYPERCALCEMIA by ESTHER DORIS MENSEN B.A., University of B r i t i s h Columbia, 1956 A'. THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department of Physiology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1958j ABSTRACT The plasma calcium l e v e l i s one of the most precisely regulated constants of the i n t e r n a l environment, and the large reservoir of calcium i n the skeleton i s primarily responsible for this homeostasis. The experiments presented i n t h i s thesis were designed to study quantitatively the regulation of plasma calcium. Acute hypocalcemia was induced by continuous intravenous EDTA infusion (a calcium chelating agent) at a known rate, and hypercalcemia was induced by intravenous calcium gluconate infusion. The rate used i n most cases was 10 mg. calcium per kg. for one hour. Both mobilization and storage of calcium appeared to depend on an equilibrium with a l a b i l e calcium storage pool i n bone. The rate of storage or mobilization was shown to be proportional to the amount of blood coming i n contact with this l a b i l e pool i n bone (bone blood flow), and the plasma/bone difference i n C a + + a c t i v i t y . Bone blood flow was measured using the Pick Pri n c i p l e f o r calcium storage, and i t was calculated to be 6.46 - 0.60$ of the cardiac output (14 dogs). The e x t r a c e l l u l a r f l u i d calcium was also estimated and found to be 15.73 - 0.72 mg/kg (14 dogs), corresponding to an ex t r a c e l l u l a r f l u i d volume of approximately 20$ of body weight. Less than 5% of the injected calcium was excreted i n the urine. The l a b i l e calcium storage pool i n bone was estimated from the changes i n the bone-blood equilibrium a f t e r calcium was injected, and was found to be 2 - 5 times greater than the ext r a c e l l u l a r calcium. The net loss of calcium from the plasma af t e r calcium i n j e c t i o n , which i s assumed to equal the rate at which calcium i s used f o r bone mineralization less calcium released by resorption, was estimated as 1 - 2 mg. Ca/kg/hr. or 0.15 - 0.35$ of the t o t a l bone calcium per day. The methods described provide a means of assessing quantitatively the factors involved i n acute regulation of the plasma calcium l e v e l . 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 th a t 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 g a i n s h a l l not be allowed without my w r i t t e n permission. Department of Physiology The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date April 15, 19^8 ACKNOWLEDGMENTS The author expresses special thanks to Dr. D. H. Copp, Professor of Physiology, f o r his constant encouragement and guidance and fo r sponsorship of thi s thesis; to Dr. E. C. Black and Mr. A. J . Honour f o r t h e i r interest; to Mrs. N. Wilson f o r her technical assistance; to Mr. K. Henze f o r the diagrams; to Miss P. Head f o r the typing of the manuscript; and to the Physiology s t a f f f o r t h e i r interest and help i n the experimental work over this period. i TABLE OF CONTENTS page I« Introduction A. General scope of material 1 B. The importance of the skeleton i n the ; homeostasis of calcium 1 C. Structure of bone 2 D. Growth of bone 3 E. Transport of calcium 6 F. Calcium balance 7 G. Homeostatic balance of plasma calcium 8 H. The kidneys i n calcium homeostasis 10 I . Plan for experiments 10 I I . Materials and Methods A. A n a l y t i c a l 12 1. Plasma calcium determination with EDTA t i t r a t i o n 12 2. Urine calcium determination 13 B . Solutions used 14 1. For plasma calcium determinations 14 C. Infusion solutions 15 1. EDTA 15 2. Calcium gluconate 15 3. Parathyroid extract 16 i i page D. Infusion apparatus 16 1. Continuous delivery machine 16 2. Kymograph infusion machine 17 E. Blood sampling apparatus 17 E. Animal procedures 17 G-. Experimental procedures 18 1. Calcium gluconate infusions 18 2. EDTA infusions 19 3. Calcium gluconate and EDTA infusions during maintenance dose of parathyroid 19 extract 4. Calcium gluconate infusion a f t e r stat dose of parathyroid extract 20 5. Measuring arterio-venous differences during calcium gluconate and EDTA infusions 20 I I I . Results 21 A. Calcium gluconate and EDTA infusions 21 1. In the normal animal 21 2. In the thyroparathyroidectomized dog 22 3. After nephrectomy 23 4. After l i g a t i o n of the ureters 23 5. During a maintenance dose of parathyroid extract i n a parathyroidectomized dog 24 B . Urine calcium excretion a f t e r calcium gluconate infusion 24 i i i Page C. Arterio-venous, marrow differences during calcium gluconate and EDTA infusions 25 D. Methods used f o r calculations 26 IV. Discussion 27 A. Acute calcium storage by bone 27 B. Calcium storage compared with bone clearance of Ca-45 28 C. Calcium storage compared with calcium mobilization 29 1. EDTA infusion before calcium gluconate infusion 30 2. Calcium gluconate infusion before EDTA infusion 30 D. Changes i n bone-blood equilibrium l e v e l a f t e r EDTA and calcium gluconate infusions 32 E. Calcium storage pool i n bone and rate at which calcium i s used for bone mineralization 33 P. Effect of slow calcium infusion 34 1. In a normal dog 34 2. In a thyroparathyroidectomized dog 34 G. Calcium excretion i n the urine 35 H. E f f e c t of ure t e r a l l i g a t i o n and nephrectomy on removal of injected calcium from the blood stream 36 I. E f f e c t of - parathyroid extract on calcium storage, calcium mobilization, and bone-blood equilibrium l e v e l 37 V. Summary and Conclusions 38 VI Appendix 40 A. Rate of calcium storage i n bone following intravenous infusion (Discussion) 40 Method of calculation 1. Spe c i f i c calcium storage and mobilization 42 2. Bone blood flow 44 3. The e x t r a c e l l u l a r calcium space 44 4. Example of calculations 46 B. Estimation of l a b i l e calcium storage pool i n bone (Discussion) and method of calcul a t i n g the net loss of plasma calcium af t e r calcium infusion (Discussion) 49 Example of calculations 1. Estimation of l a b i l e calcium storage pool i n bone 51 2. Estimation of net loss of calcium from the plasma 52 VII Figures VIII Tables IX Acknowledgments X Bibliography 1. I. INTRODUCTION A. General scope of problem. The homeostasis of the calcium concentration i n blood i s very important physiologically i n order to maintain normal function of a l l tissues of the body, especially nerves and muscles. This thesis i s concerned with acute restoration of plasma calcium to equilibrium when there i s rapid addition to or removal of calcium from the blood stream. This i s achieved by intravenous injections of calcium gluconate, or of a calcium chelating agent, EDTA. The role of the skeleton i n thi s acute regulation of blood calcium has been studied. Also the rates of restoration of blood calcium to equilibrium have been estimated. B. The Importance of the Skeleton i n the Homeostasis of  Calcium. The body contains approximately 15 g. of calcium per kg. body weight. There i s about \ g. i n the entire extra-c e l l u l a r compartment, and 111 g» i n soft tissue ( i n t r a c e l l u l a r ) i n a 70 kg. man.(l5) The skeleton contains 99$ of the body's calcium, and i s primarily involved i n the homeostatic regulation of the serum calcium. The kidneys and i n t e s t i n a l t r a c t are important i n normal calcium balance, but are of minor 2. importance i n comparison to the large calcium reservoir of bone, i n restoring equilibrium during rapid removal or addition of calcium to the e x t r a c e l l u l a r f l u i d . C. The Structure of Bone. Bone s a l t i s a compound of calcium, phosphate, and hydroxyl ions, and water, with a c h a r a c t e r i s t i c apatite pattern. The crystals are described as f l a t tablets or rods a few hundred angstroms i n length and breadth and only a few unit c e l l s thick (30 - 70 A 0 ) . The findings most commonly reported, from electron microscopy, are that the crystals are rod- or needle-shaped. It has also been suggested by Pinean and Engstrom ( I O ) , that the tablets appear to be aggregations of rods. In intact bone, these c r y s t a l s are found to be closely associated with collagen i n an organized fashion, with the long axes of the crystals oriented i n the long axes of the collagen f i b r e s . Because of the minute size of the c r y s t a l s , the s p e c i f i c surface area of bone mineral i s so large, that surface phenomena dominate the chemical behaviour of the bone mineral (2.2.). The s t r u c t u r a l units of bone are the osteone ( f o r lamellar or "hard" bone), and the trabecula (for soft and cancellous type). There i s a minute artery and vein within the osteone. The trabeculae are completely surrounded by e x t r a c e l l u l a r f l u i d of the highly vascular marrow. In both 3. types, throughout the tissues, there are spaces ( c a n a l i c u l i and lacunae) through which tissue f l u i d s flow. 04-) Bone c e l l s , or osteoblasts, produce a matrix of collagen and polysaccharide, along which are deposited the hone s a l t s i n a normal environment. Bone mineral i s a quite impure hydroxy apatite, having as i t s p r i n c i p a l impurities, carbonate, 6$; c i t r a t e , 17°; sodium, 0.7$; and magnesium, 0.7$ with traces of f l u o r i d e . The impurities i n bone are there as a passive physicochemical consequence of the presence of these ions i n the f l u i d s i n which the crystals form. (ZZ.) D. The Growth of Bone. During growth of bone, a newly formed matrix i s added to the surface just under the layer of osteoblasts. When t h i s c a l c i f i e s , a new layer of matrix i s l a i d upon the older, pushing i t back farther from the s i t e of most active exchange with the e x t r a c e l l u l a r f l u i d s . However, these layers are s t i l l a store-house that could become available i f exposed. Bone i s l i v i n g tissue, and therefore i s constantly being destroyed, and newly formed. These processes occur over and above the pure exchange reactions between atoms of the crystals and the e x t r a c e l l u l a r f l u i d s . (i5) The bone mineral participates i n e l e c t r o l y t e metabolism throughout the l i f e t i m e of the animal because of the constant "turnover". 4 . Neuman c l a s s i f i e s the osteone maturation and r e a c t i v i t y i n three i o n i c processes: (1) d i f f u s i o n i n t o the h y d r a t i o n s h e l l (2) ion-exchange or ion-displacement a t the c r y s t a l s u r f a c e (3) thermal r e c r y s t a l l i z a t i o n w i t h i n the c e l l . These are modified under the i n f l u e n c e of p h y s i o l o g i c a l c o n d i t i o n s . Because the serum i s supersaturated w i t h r e s p e c t to calcium and phosphate, the tendency i s f o r a l l bone s t r u c t u r e g r a d u a l l y to a t t a i n maximal m i n e r a l i z a t i o n . Thus the c r y s t a l s grow slo w l y , to the complete or near complete e x c l u s i o n of water, and the more f u l l y m i n e r a l i z e d the s t r u c t u r e , the more r e s t r i c t e d are the c i r c u l a t i o n , d i f f u s i o n , and exchange o f i o n s . From t h i s i t f o l l o w s that the age o f the bone i s the primary determinant of i t s chemical r e a c t i v i t y . T h i s a p p l i e s to any bone s t r u c t u r e , such as t r a b e c u l a , a Haversian system, an i n t e r s t i t i a l o r s u b p e r i o s t e a l l a m e l l a . Young bone i s more v a s c u l a r and has a h i g h e r water content, so that t h i s bone remains i n e q u i l i b r i u m w i t h the body f l u i d s . The c r y s t a l s are s m a l l and e x h i b i t r a p i d s u r f a c e exchange, r e c r y s t a l l i z a t i o n and i n t r a c r y s t a l l i n e exchange. As the water content f a l l s to i t s minimal v a l u e , w i t h advancing age, there r e s u l t s an ever i n c r e a s i n g p r o p o r t i o n of the s k e l e t o n that i s o l d , f u l l y m i n e r a l i z e d , and n o n - r e a c t i v e . However, a l l the osteones i n the a d u l t s k e l e t o n are not i n e r t . 5-There i s a continuous p a t t e r n of Haversian r e m o d e l l i n g by which e r o s i o n c a v i t i e s are c o n t i n u a l l y forming, and new Haversian systems developing. Thus, there r e s u l t s a constant supply o f a c t i v e exchangeable bone m i n e r a l throughout the l i f e of the animal. (2.2.) Three mechanisms f o r the process of c a l c i f i c a t i o n have been suggested by Neuman. "(1) That the c o l l a g e n f i b r e s of bone s p e c i f i c a l l y possess the chemical property of i n d u c i n g the p r o d u c t i o n of c r y s t a l n u c l e i perhaps through the presence i n the molecule of a phosphorylated amino a c i d . (2) That i n c a r t i l a g e , the p r o t e i n i s rendered " a c t i v e " by the enzymatic t r a n s f e r of a pyrophosphate group from ATP to the p r o t e i n . (3) That the a c t i v e n u c l e a t i o n centre i n c a r t i l a g e i n v o l v e s a complex between c o l l a g e n and the mucopolysaccharide, c h o n d r o i t i n s u l phate." The f i r s t d e a l s with m i n e r a l i z a t i o n i n forming bone, and the l a s t two with the m i n e r a l i z a t i o n of c a r t i l a g e . (2.1) McLean d e f i n e s r e s o r p t i o n as "the p u t t i n g i n t o s o l u t i o n of a complicated s t r u c t u r e i n such a f a s h i o n t h a t i t disappears, i t s end-products e n t e r i n g the blood stream. R e s o r p t i o n a l s o progresses inward from the s u r f a c e s of bone; i t never a r i s e s w i t h i n the deeper l a y e r s of the s t r u c t u r e . " One s m a l l f r a c t i o n of b o n e i s a l r e a d y i n f l u i d 6. form, so i t may be e a s i l y removed. The remainder i s i n s o l i d form and i n s o l u b l e i n aqueous f l u i d s , so t h a t i t must be rendered s o l u b l e i n water before i t can be t r a n s f e r r e d to the body f l u i d s . K o e l l i k e r i n 1873 O^5) suggested that the o s t e o c l a s t s eroded bone by chemical means, but d i d not s p e c i f y the nature of the chemical a c t i o n . An assumption i s now made t h a t the bone s a l t may be d i s s o l v e d whenever another substance having a s t r o n g e r a f f i n i t y f o r calcium i s i n a s o l u t i o n i n contact w i t h bone. I t i s assumed that there i s c o n t i n u a l l y a p p l i e d to the s u r f a c e of bone, a s o l u t i o n that w i l l depolymerize mucopolysaccharides, d i g e s t c o l l a g e n and h o l d calcium i n a f i r m combination. Such a mechanism would r e q u i r e only c e r t a i n enzyme systems and an organic substance to combine wi t h calcium. ( 2 . 0 ) E. Transport of Calcium. The t r a n s p o r t of calcium to and from bone i s accomplished by the blood stream, e x t r a c e l l u l a r f l u i d , and lymph. Ca-45 s t u d i e s have demonstrated the enormous s u r f a c e area of bone on which a c t i v e i o n i c exchange processes can occur, and an immediate t r a n s f e r between bone and blood can take place because of t h i s a c t i v e exchange. In t h i s type of r e a c t i o n the ions p a r t i c i p a t i n g i n the exchange are present on the s u r f a c e s of the bone s a l t c r y s t a l s . ( 2, ) 7. I t i s w e l l e s t a b l i s h e d , that most of the i n j e c t e d calcium i s c a r r i e d by the blood to the bone, but the extent to which organs or s e c r e t i o n s may i n f l u e n c e the removal of calcium from the c i r c u l a t i o n i s p o o r l y understood. (13) F. Calcium Balance. The calcium content of the serum i s dependent on the balance between how much enters and how much l e a v e s the blood stream. Calcium enters by two sources; from the i n t e s t i n e by a b s o r p t i o n , and from the s k e l e t o n by r e s o r p t i o n . Calcium leaves the plasma by e x c r e t i o n i n the u r i n e , i n the f e c e s (being d e r i v e d from i n t e s t i n a l j u i c e c a l c ium which may remain unabsorbed) or i t may be deposited i n bone s a l t . ( 7 ) The normal t o t a l serum calcium l e v e l i s 10 mgm.$, or 2.5 mM/liter. Some of t h i s i s bound to p r o t e i n , and the r e s t , 6.5 mg. per cent (1.62 mM/l) i s u l t r a - f i l t r a b l e and f r e e l y d i f f u s i b l e a c r o s s the normal c a p i l l a r y membrane. Of t h i s d i f f u s i b l e calcium, a smal l f r a c t i o n i s i n the form of s o l u b l e complex i o n s . The i o n i z e d calcium i s estimated a t 1.33 mM/l ( F i g . 1A) The r e l a t i o n s h i p of calcium to plasma p r o t e i n has been i n c o r p o r a t e d i n a f o r m u l a t i o n that d e s c r i b e s the plasma as a s o l u t i o n o f a p a r t i a l l y i o n i z e d e l e c t r o l y t e , c a l c i u m p r o t e i n a t e , the i o n i z e d and u n i o n i z e d f r a c t i o n s being i n 8. e q u i l i b r i u m with each other. Or. Homeostatic R e g u l a t i o n of Plasma Calcium. The calcium i n s o l u t i o n i n the plasma i s i n constant exchange w i t h the calcium o f e x t r a c e l l u l a r f l u i d of the bones. T h i s exchange i s present a t a l l l e v e l s o f calcium i o n c o n c e n t r a t i o n i n the f l u i d s o f the body. The p a r a t h y r o i d glands f u n c t i o n to maintain the constancy o f the calcium c o n c e n t r a t i o n i n the plasma at 10 mg.fo (2.5 m M / l i t e r ) . Experiments of Copp _et a l show that turnover of calcium i n blood and bone may occur independently o f p a r a t h y r o i d a c t i v i t y . So, whatever may be the tr u e chemical and p h y s i c a l nature o f the s k e l e t o n , some of the calcium present i n the s k e l e t a l t i s s u e s i s a v a i l a b l e f o r m o b i l i z a t i o n i n time of need, and some o f the s k e l e t a l t i s s u e i s a v a i l a b l e to s t o r e excess calcium. The p a r a t h y r o i d s are a dominant f a c t o r i n the a b i l i t y o f bones to provide calcium to the e x t r a c e l l u l a r f l u i d s when necessary. Mclean makes a d i s t i n c t i o n between : (1) A mechanism a c t i n g i n one d i r e c t i o n o n l y , i . e . m o b i l i z a t i o n of calcium from the bones under the i n f l u e n c e o f the p a r a t h y r o i d glands. (2) The t r a n s f e r o f calcium between blood and bone, i n both d i r e c t i o n s , independent of p a r a t h y r o i d f u n c t i o n . 9 . T h i s i s thought of as a d u a l mechanism, one p a r t a c t i n g by d i f f u s i o n e q u i l i b r i u m between the plasma and the l a b i l e f r a c t i o n o f the bone m i n e r a l . The p a r a t h y r o i d hormone causes d e s t r u c t i o n of both the m i n e r a l and o r g a n i c components of bone. Ac c o r d i n g to h i s i n t e r p r e t a t i o n , the l a b i l e f r a c t i o n o f the calcium of the m i n e r a l i s e a s i l y a c c e s s i b l e to i o n i c exchange with the f l u i d s of the body. On the other hand, the s t a b l e f r a c t i o n of calcium does not d i s s o l v e r e a d i l y and r e q u i r e s the a c t i o n o f the p a r a t h y r o i d hormone f o r i t s l i b e r a t i o n . (2.0) There has a l s o been found a r e l a t i o n s h i p between c i t r a t e and c alcium metabolism, which i n d i c a t e s that the metabolic r e a c t i o n s which i n f l u e n c e the c a l c i u m of the body can a f f e c t the accumulation o f c i t r a t e i n the t i s s u e s . The bone i s r e l a t i v e l y r i c h i n c i t r a t e which cannot be e l u t e d from the bone by water. T h i s c i t r a t e i s thought to be h e l d on the s u r f a c e of the bone c r y s t a l s by v i r t u e o f i t s property of complexing with c a l c i u m . In v i t r o s t u d i e s have shown the presence of c i t r o g e n a s e and a c o n i t a s e a c t i v i t y i n bone t i s s u e , and i t has been suggested that c i t r a t e accumulation i n the s k e l e t o n could r e s u l t from metabolic a c t i v i t y of bone c e l l s . I f serum i s supersaturated, some c e l l u l a r mechanism must be p o s t u l a t e d by which t h i s supersaturated s t a t e can be maintained. (12.) 10. H. The Kidneys i n Calcium Homeostasis. Normally more than 99$ of the f i l t e r e d calcium i s reabsorbed by the r e n a l t u b u l e s . In experiments of Poulos, where calcium c h l o r i d e and calcium gluconate i n f u s i o n s were c a r r i e d out, t u b u l a r r e a b s o r p t i o n i n c r e a s e d n e a r l y i n p r o p o r t i o n to the i n c r e a s e d r a t e of d e l i v e r y of calcium i n t o the glomerular f i l t r a t e , and no t u b u l a r r e a b s o r p t i v e maximum was observed. (2.4) Wolf performed experiments where dogs r e c e i v e d steady intravenous i n f u s i o n s of calcium c h l o r i d e or c a l c i u m gluconate f o r f i v e hours. There was no e f f e c t i v e t h r e s h o l d f o r r e t e n t i o n of calcium, and plasma c o n c e n t r a t i o n s of t h i s i o n are apparently not r e g u l a t e d e x t e n s i v e l y by r e n a l functions' (2.k) I . P l a n f o r Experiments. 1. The p h y s i o l o g i c a l s i g n i f i c a n c e of processes governing the r a p i d interchange between the bone c r y s t a l s and the c i r c u l a t i n g f l u i d s . The extent to which the a v a i l a b l e s k e l e t o n m i n e r a l can p a r t i c i p a t e i n b u f f e r i n g a g a i n s t s h i f t s i n the calcium composition of the e x t r a c e l l u l a r f l u i d s i s p o o r l y understood. The importance of the s k e l e t o n i n the homeostatic r e g u l a t i o n of the c a l c i u m - i o n c o n c e n t r a t i o n i n the blood was i n v e s t i g a t e d . T h i s was accomplished by e s t i m a t i n g acute storage of calcium or acute m o b i l i z a t i o n of calcium by bone, a f t e r intravenous i n j e c t i o n s of c alcium gluconate or EDTA. These i n j e c t i o n s were performed under normal c o n d i t i o n s , and i n dogs f o l l o w i n g 11. removal o f the t h y r o i d and p a r a t h y r o i d glands. Intravenous i n j e c t i o n s were gi v e n f o l l o w i n g removal of the kidneys, or f o l l o w i n g l i g a t i o n of the u r e t e r s to compare the removal of i n j e c t e d calcium w i t h t h a t of the normal animal. In o t h e r experiments the e x c r e t i o n of calcium i n the u r i n e was measured d u r i n g c a l c i u m gluconate i n f u s i o n s . The e f f e c t of c a l c i u m i n f u s i o n s i n r a i s i n g the bone-blood calcium e q u i l i b r i u m or i n c r e a s i n g s k e l e t a l a c t i v i t y was a l s o s t u d i e d . A r t e r i a l , venous, and marrow samples were simultaneously taken d u r i n g calcium gluconate or EDTA i n f u s i o n s to demonstrate the d i f f e r e n c e i n plasma calcium c o n c e n t r a t i o n . Methods f o r c a l c u l a t i n g the f o l l o w i n g are o u t l i n e d ( i n the appendix). (1) the rate of acute calcium storage i n bone. (2) the r a t e of m o b i l i z a t i o n by bone. (3) bone-blood flow. (4) the amount of calcium i n v a r i o u s e x t r a c e l l u l a r f l u i d compartments. (5) the volume of v a r i o u s e x t r a c e l l u l a r f l u i d compartments. ( 6 ) the amount of a v a i l a b l e calcium i n bone. (7) the r a t e a t which calcium i s used f o r bone m i n e r a l i z a t i o n . 12. IIIJ MATERIALS AND METHODSf A. A n a l y t i c a l . 1. Plasma calcium determination with EDTA t i t r a t i o n . The plasma calcium was determined by a rapid t i t r a t i o n with EDTA. This determination can be done within ten to f i f t e e n minutes a f t e r the blood sample i s taken from the animal. The method i s a modification of Lehmann's. (18) 0.2 ml. of plasma i s diluted with 5 ml. of d i s t i l l e d water. The pH i s adjusted to 12 with concentrated NaOH, and ammonium purpurate.. i s used as an indicator. The photometric t i t r a t i o n method described by Campbell (—) and Pales ( S> ) was adapted so that a single K l e t t colorimeter could be used. Because there i s only a small difference i n the maximum l i g h t absorption of the ammonium purpurate as compared to calcium purpurate, the end point must be determined photometrically, and a monochromatic l i g h t source i s ess e n t i a l . A second order interference f i l t e r with transmission at 500 Lambda i s usedto obtain t h i s . ( k> ) To determine serum or plasma calcium the following procedure was employed: (1) 0.2 cc. plasma i s placed i n a 10 cc. K l e t t tube. (2) 5 ml. d i s t i l l e d water i s added. (3) the pH i s adjusted to 12 with concentrated NaOH (1-2 drops). (4) one drop of caprylic alcohol i s added to prevent foaming while mixing. 13. (5) enough ammonium purpurate i s added so the reading f o r calcium purpurate i s about 200 on the Kle t t colorimeter ( 2-5 drops). (6) the mixture i s mixed up by a jet of a i r from a polyethylene tube attached to an aquarium aerator. (7) the t i t r a t i o n i s done with 0.01$ sodium versenate from a 2 ml. burette. As d i l u t e EDTA i s added, complexing with calcium, the calcium purpurate i s changed to ammonium purpurate and the l i g h t absorption rapidly decreases. When a l l of the calcium i s complexed with EDTA, any further decrease i n l i g h t absorption i s due to simple d i l u t i o n of the ammonium purpurate, which occurs very slowly as more versene i s added. The l i g h t absorption i s plotted against the mis. of 0.01$ versene added. The end point i s the point of intersection of the steepest slope and the d i l u t i o n curve (Fig.j|B). Table I shows t y p i c a l values f o r a standard. The t i t r a t i o n i s reproducible within * 0.05 mg.$ to 0.1 mg.%. 2. Urine calcium determination. ( 5 ) Take urine samples into small Erlenmeyer f l a s k s . Add one to two cc. of concentrated n i t r i c acid and b o i l u n t i l the solution i s colourless. Dissolve precipitate by adding 2 - 3 cc. of water. Add: 1 cc. ammonium oxalate; 1 drop methyl red; 2 cc. 20$ acetic acid; 20$ ammonium hydroxide u n t i l the colour 14. i s salmon pink. Leave overnight. Then: centrifuge; remove supernatant; wash i n 2$ ammonium hydroxide; recentrifuge; remove supernatant. Add 2 cc. 20$ s u l f u r i c acid and heat on hot plate to 90°C. Tit r a t e with 0.0126 N KMnO^ u n t i l the colour-is pink. Standard: Take 2 cc. of 10 mg.$ calcium standard into centrifuge tube. Add 1 cc. ammonium oxalate; 1 drop methyl red; 2 cc. 20$ acetic acid. Proceed as above. B. Solutions used: 1. For plasma calcium determination with EDTA t i t r a t i o n . (a) Ammonium purpurate (0.1$). 400 ml. of d i s t i l l e d water are boiled to 200 ml. to remove the oxygen, which oxydizes ammonium purpurate"*", especially i n l i g h t . 200 mg. of ammonium purpurate are added and the solution i s immediately covered with mineral o i l , and the flask stored i n a cardboard container i n the r e f r i g e r a t o r . Only 3 or 4 ml. of the solution are required per day f o r plasma calcium determinations. (b) Disodium Versenate. A stock solution of 0.1$ sodium versenate i s prepared 2 from regular sodium versenate. The 0.1$ solution i s diluted to 0.01$ f o r plasma calcium determinations. Calcium indicator reagent, procured from Hagan Corporation, the Buromin Company of Calgon Inc., P.O, Box 1346, Pittsburgh,Pa.,U.S.A p 'Supplied by Beresworth Chemical Company,Framingham,Mass.,U.S.A. 15. C. Infusion Solutions. 1. EDTA (about 6$). Disodium ethylene-diamine-tetraeetate (EDTA) is. a calcium chelating compound. Prom the physiological point of view, the important c h a r a c t e r i s t i c of a chelate i s that i t s a b i l i t y to ionize i n solution i s very low. (2.0) EDTA forms a soluble, non-toxic and very stable complex with calcium at the pH of blood, and removes calcium as far as b i o l o g i c a l a c t i v i t y i s concerned, as e f f e c t i v e l y as i f i t were actually extracted from blood. (23) The reaction i s immediate and quantitative. When Ca-EDTA was infused intravenously at the same rate as the o r i g i n a l EDTA, no effect was observed on t i t r a b l e calcium l e v e l i n plasma, i n blood pressure, or i n the general condition of the animal. (4> ) A stock solution f o r infusions of approximately 6.0$ i s prepared, with the pH adjusted to 7.4$ with NaOH. When standardized, one ml. of this solution should chelate 5-7 mg. of calcium. This solution i s diluted with 5$ glucose i n water (Abbott) when infused into an animal. With the rate of the infusion pump calibrated, the rate of calcium chelation can be controlled to equal 10 mg/kg/hr. or any other rate required. 2. Calcium gluconate. Some of the calcium gluconate used was obtained i n 10 cc. ampoules (No.239, Parke Davis and Co. Ltd.) of 10$ calcium gluconate. They were found on analysis to contain 16. 8-10 mg, calcium per ml. This was diluted with 5$ glucose i n water to the calculated concentration for infusion. (10 mg./kg/hr). Other calcium gluconate solutions were prepared from calcium gluconate powder (about 8$ calcium). This powder was dissolved i n warm water and warm 5$ glucose i n water to give a concentration of 5$ glucose and 5-7$ calcium. This mixture was heated u n t i l the powder dissolved. Thus one ml. of solution would contain 5-7 mg. calcium. This was again diluted to give the appropriate concentration f o r infusion. A l l of the calcium gluconate infusion solutions were analyzed by the EDTA t i t r a t i o n method fo r t h e i r exact calcium content. 3. Parathyroid Extract. Parathormone was the material used (Parathyroid extract), supplied through the courtesy of E . L i l l y and Co. Ltd., and was from Lot #2126 - 678089, expiration date Aug. 1, 1958, and Lot #7148 - 687712, expiration date Aug. 1, 1958. The extract was prepared from beef glands by the method of C o l l i p . The parathyroid extract was diluted with 5$ glucose i n water to give the appropriate concentration. D. Infusion Apparatus. 1. The f i r s t type of infusion pump used gives a continuous delivery (Figure £ ) . I t i s a "Motor Driven Compensator", produced by the American Instrument Company. A syringe i s driven by a constant rate motor, the rate can be 17. adjusted by the selection of several gears, and d i f f e r e n t sizes of syringes. 2. The second type does not give a continuous flow although the mis. delivered per hour can be varied to a much greater extent (10-200G ml./hr). A rubber tube i s attached to a piece of f l a t metal which l i e s p a r a l l e l to a cam shaft. The cams on the shaft are set at d i f f e r e n t angles so that each compresses the tube i n succession, i n such a manner that the solution i s sucked up and pushed through the tubing (Figure 3 ) . E. Blood Sampling Apparatus. 2 ml. blood samples were taken with dry or moist heparinized 5 ml. syringes, so that no c l o t t i n g or d i l u t i o n of plasma would occur. The blood was delivered from the syringes into dry heparinized hematocrit tubes through pieces of dry polyethylene tubing. The tubes were centrifuged f o r 10-15 minutes and the plasma removed with a Pasteur pipette. F. Animal Procedures. Most of the animals used were adult mongrel dogs, fasted overnight before use. They were anaesthetized and maintained under anaesthetic with nembutal during the experiment. Bogs used f o r s u r v i v a l experiments were given an i n j e c t i o n of 1 ml. of p e n i c i l l i n i n o i l (300,000 units) intramuscularly at the conclusion of the operation. Antiseptic technique was 18. attempted d u r i n g experiments. Complete b i l a t e r a l thyroparathyroidectomy was performed, and these animals were giv e n a maintenance dose of -kgr. d e s s i c a t e d t h y r o i d every o t h e r day. Blood samples were obtained from the j u g u l a r v e i n and a r t e r i a l samples from the femoral a r t e r y o r c a r o t i c a r t e r y . Most of the i n f u s i o n s were c a r r i e d out through the l a r g e s u p e r f i c i a l v e i n s of the f r o n t and hind l e g s . I n f u s i o n s o f 5% glucose i n water were performed when no other i n f u s i o n was c a r r i e d on, to keep the animal w e l l hydrated. Nephrectomy and u r e t e r a l l i g a t i o n was done u s i n g the r e t r o p e r i t o n e a l approach. The i n c i s i o n was made j u s t i n f r o n t of the a n t e r i o r s u p e r i o r i l i a c spine and extended to a p o i n t below the base of the l a s t r i b . The animals were kept warm by bl a n k e t s and heat lamps. G. Experimental Procedures. 1. Calcium gluconate i n f u s i o n s . Calcium gluconate was i n f u s e d i n t r a v e n o u s l y a t a continuous r a t e o f 10 mg/kg/hr, u s u a l l y f o r a p e r i o d o f one hour, although d u r a t i o n o f i n f u s i o n s range from \ to 2\ hours. C o n t r o l samples were taken before the i n f u s i o n , d u r i n g the i n f u s i o n , and f o r s e v e r a l hours a f t e r the i n f u s i o n stopped ( u n t i l the plasma c a l c i u m returned to normal, o r to some new l e v e l ) . T h i s procedure was c a r r i e d out on the normal dogs, 19. thyroparathyroidectomized dogs, nephrectomized dogs, and dogs i n which the ureters were l i g a t e d . During a l l of the experiments, blood samples were taken f o r calcium determination. During several experiments, urine samples were taken f o r calcium analysis. Stat (instantaneous) doses of calcium gluconate from 3-25 mg/kg were injected intravenously i n thyroparathyroid-ectomized dogs. Also, long infusions of 9-12 hours at 2-4 mg. calcium/kg/hr were given to control and thyro-parathyroidectomized animals. 2. EDTA infusions. EDTA was injected intravenously at a continuous rate, so that 10 mg. calcium/kg/hr would be chelated, and thus removed from the plasma. The infusion continued -^-1 hr. Blood samples were taken before the infusion, during the infusion, and several hours a f t e r the infusion. 3. Calcium gluconate and EDTA infusions during maintenance dose of parathyroid extract. Immediately a f t e r b i l a t e r a l thyroparathyroidectomy, parathyroid extract was infused at 0.1 units/kg/hr. This appeared to be the maintenance dose of parathormone f o r the majority of the dogs used, and maintained the blood l e v e l f o r at least 10 hours. EDTA was infused for one h a l f hour at a rate to chelate 5 mg. calcium/kg/hr. Time for recovery was allowed, then 5 mg. calcium/kg/hr. were infused over \ hour to 20. r e p l a c e t h a t calcium removed with EDTA. The maintenance dose of parathormone was simultaneously proceeding w i t h the EDTA and calcium gluconate i n f u s i o n s . The EDTA and calcium gluconate i n f u s i o n s were repeated on the same dog, as above, with a d d i t i o n a l parathormone i n the EDTA and calcium gluconate s o l u t i o n s . 4. Calcium gluconate i n f u s i o n a f t e r a s t a t dose o f parathormone. A thyroparathyroidectomized dog was g i v e n a s t a t dose of parathormone (10 u n i t s / k g . ) . The plasma calcium rose and l e v e l l e d o f f . At t h i s p o i n t c a l c i u m gluconate was g i v e n c o n t i n u o u s l y a t a r a t e o f 10 mg./kg./hr. f o r one hour. 5. Measuring a r t e r i o - v e n o u s d i f f e r e n c e o f plasma c a l c i u m d u r i n g calcium gluconate and EDTA i n f u s i o n s . A r t e r i a l , venous, and marrow blood samples were taken simultaneously a t the end of both calcium gluconate and EDTA i n f u s i o n s . The a r t e r i a l sample was taken from the femoral a r t e r y or the c a r o t i d a r t e r y ; venous sample from the j u g u l a r or femoral v e i n ; and the marrow blood sample from the femurs. 21. i n i 'RESULTS A. Calcium gluconate and EDTA infusions. 1. In the normal animal. (a) In over 200 control determinations i n 62 dogs, the mean control l e v e l of plasma calcium was found to be 10.08 mg.% (standard deviation - 0.27 mg.%; standard error 0.03 mg.^).1 By the end of the calcium gluconate infusion, the plasma calcium had ri s e n to 12-15 mg.$. After the infusion was stopped, the plasma calcium returned to the control l e v e l or to some new l e v e l , above the control l e v e l . (Fig. 4 & 5). An i r r e g u l a r i t y i n the descending curve as shown i n Fig.5 occurred i n most of the animals. The o v e r a l l rapid f a l l or loss of calcium from the c i r c u l a t i o n i s due to the disappearance of calcium from the a r t e r i a l plasma into the bone. Thus circulatory mixing i s complicated by the fact that the injected calcium does not remain within the blood system. The ir r e g u l a r r i s e s i n the descending curve might be explained by the return of calcium to the central c i r c u l a t i o n from extra-ske l e t a l areas of the body, such as the v i s c e r a . ( 17 ) Fig . 4- shows the r a p i d return of the blood calcium to the control l e v e l . In F i g . 5 the plasma calcium returned to a new l e v e l , representing a r i s e i n the equilibrium between bone S t a t i s t i c a l analysis by Dr. L.W.E. Flather i n summer of 1957. 22. and blood. (b) Comparison o f calcium gluconate and EDTA i n f u s i o n curves with the i n f u s i o n sequence ( P i g . 6 & 7). The blood calcium returned to the c o n t r o l l e v e l a f t e r both i n f u s i o n s i n P i g . 6. In F i g . 7 the bone blood e q u i l i b r i u m has been lowered a f t e r EDTA i n f u s i o n , presumably-due to the i n i t i a l i n f u s i o n of calcium gluconate. (c) Calcium gluconate was i n f u s e d a t a r a t e of 4 mg./kg./hr. f o r 9.5 hours, i n t o a normal dog. The plasma calcium l e v e l rose above the c o n t r o l l e v e l , then l e v e l l e d o f f f o r s e v e r a l hours. The plasma calcium l e v e l f e l l promptly to a l i t t l e below c o n t r o l a f t e r the i n f u s i o n was stopped. ( F i g . 8). The calcium excreted i n the u r i n e was 5.26$ o f the c a l c i u m i n f u s e d i n t r a v e n o u s l y . The u r i n e was c o l l e c t e d by c a n u l a t i o n o f one u r e t e r . 2. E f f e c t o f calcium gluconate i n f u s i o n s i n thyroparathyroidectomized dogs. Calcium gluconate was i n f u s e d a t 10 mg./kg./hr. f o r d i f f e r e n t time i n t e r v a l s . ( F i g . 9). The e q u i l i b r i u m l e v e l rose i n p r o p o r t i o n to the t o t a l amount of ca l c i u m i n f u s e d . The f i r s t i n f u s i o n was a t o t a l o f 5 mg./kg; the second 10 mg./kg; and the t h i r d a t o t a l of 25 mg./kg. (see a l s o Table X ) . In one parathyroidectomized dog the plasma c a l c i u m 23. l e v e l returned rapidly to the pre-injection l e v e l a f t e r calcium infusion. (Pig. 10). In a l l other dogs under the same conditions, the plasma l e v e l came back to a new and higher l e v e l , and then f e l l at a very gradual rate. Calcium gluconate was also given i n single or stat doses of 5-20 mg./kg./hr. The plasma calcium l e v e l rose sharply a f t e r the i n j e c t i o n and then f e l l to a new equilibrium l e v e l (Pig. 11). Calcium infusions at slow rates of 1-3 mg./kg./hr. were done on thyroparathyroidectomized dogs (Fig. 12). The plasma calcium rose immediately a f t e r the calcium infusion started. The r i s e was not l i n e a r , and the plasma calcium f e l l immediately a f t e r the infusion was stopped. The time index (abcissa) i n Fig.12. was shortened, and the rates of disappearance of calcium from the blood stream during and a f t e r the infusion were calculated from the r e s u l t i n g curve. (Fig. 13) 3. When calcium gluconate was infused a f t e r nephrectomy, the plasma calcium rose and then f e l l to a new l e v e l above control, which was maintained for a few hours. There was no immediate gradual f a l l i n the new calcium l e v e l as soon as an equilibrium was reached. The mobilization curve was s i m i l a r to those obtained i n a normal dog. (Fig.14) 4. Immediately a f t e r ureteral l i g a t i o n , and i n some cases both thyroparathyroidectomy and u r e t e r a l l i g a t i o n , calcium gluconate and EDTA were infused. (Fig. 15) The curves were 24. s i m i l a r to those obtained i n a normal dog. Thus, disappearance of i n j e c t e d calcium from the blood stream i n t h i s experiment cannot be a t t r i b u t e d to e x c r e t i o n i n the u r i n e . 5. Calcium gluconate and EDTA i n f u s i o n s o f one h a l f hour d u r a t i o n each were c a r r i e d out d u r i n g a maintenance dose o f p a r a t h y r o i d e x t r a c t (0.1 u n i t s / k g . / h r . i n experiment p r e s e n t e d ) . The plasma calcium d i d not r e t u r n to normal a f t e r the EDTA i n f u s i o n . Thus the maintenance dose o f p a r a t h y r o i d e x t r a c t i s enough to maintain the blood c a l c i u m a t the normal l e v e l when the p a r a t h y r o i d s are removed, but i s not enough to b r i n g the blood calcium back to the normal l e v e l a f t e r removal of calcium w i t h EDTA. The amount of calcium t h a t was removed from the blood stream d u r i n g the one h a l f hour i n f u s i o n of EDTA was r e p l a c e d by a one h a l f hour i n f u s i o n of calcium gluconate. T h i s r e s t o r e d the plasma calcium l e v e l to normal. A d d i t i o n a l p a r a t h y r o i d e x t r a c t i n the EDTA s o l u t i o n r e s t o r e d the plasma calcium c l o s e r to normal. ( P i g . 16) B. Urine calcium e x c r e t i o n . The method f o r a n a l y s i s was not a c c u r a t e i n the m a j o r i t y of experiments. Urine was c o l l e c t e d by c a t h e t e r i z a t i o n i n s e v e r a l experiments, and s m a l l p o r t i o n s of u r i n e were s p i l t while a s h i n g . More accurate u r i n e a n a l y s i s was accomplished when the u r i n e was c o l l e c t e d by c a n u l a t i o n o f one u r e t e r . The a s h i n g method was a l s o improved. But adequate allowances 25. were made f o r these sources of error, and the determination i s expressed as an approximation. The accurate analyses are indicated i n the tables (accurate). Tables II and III present urine calcium excretion i n the normal dog and i n the parathyroidectomized dog. Since not much more than 5% of the injected calcium was excreted i n the urine, very l i t t l e disappearance of injected calcium from the blood stream could be attributed to excretion i n the urine. C. "Results showing the difference between a r t e r i a l , venous  and marrow blood samples during calcium gluconate and  EDTA infusion. (Fig. 17) Prior to the calcium infusion, the plasma calcium was 5 mg.$. The plasma calcium was i n equilibrium with the calcium on the surface of the bone c r y s t a l s , and the e x t r a c e l l u l a r f l u i d around i t . The plasma calcium was suddenly raised by calcium infusion, so that i t was no longer i n equilibrium with the l a b i l e bone calcium. Eventually t h i s equilibrium was re-established at a higher l e v e l , due to storage of calcium i n the bone storage pool. Thus at the end of the infusion, the carotid artery blood sample (assumed to be mixed a r t e r i a l sample) had more calcium than the marrow sample, and also more than the femoral vein blood sample, which had just returned from the bone. In other experiments the difference i n plasma calcium between the femoral artery, femoral vein, and jugular vein was measured, during calcium gluconate and EDTA infusions. (Fig.18) 2 6 . F i v e dogs showed s i m i l a r r e s u l t s . During EDTA i n f u s i o n s the femoral v e i n sample had the highest plasma calcium because i t had j u s t returned from the l o n g bones of the l e g where calcium m o b i l i z a t i o n was t a k i n g p l a c e . The j u g u l a r v e i n sample was lower s i n c e the blood was coming c h i e f l y from the b r a i n . The a r t e r i a l sample was the lowest f o r i t contained a mixture of blood from a l l p a r t s of the body. The reverse was true d u r i n g c a l c i u m gluconate i n f u s i o n s . D. Methods f o r c a l c u l a t i n g c a l c ium storage and m o b i l i z a t i o n  by bone; bone blood flow; e x t r a c e l l u l a r f l u i d spaces; calcium storage p o o l i n bone; and r a t e at which c a l c i u m  i s used f o r bone m i n e r a l i z a t i o n , are found i n the appendix. The summary of the r e s u l t s i s presented i n the d i s c u s s i o n . 27. IV.1''DISCUSSION A. Acute Storage by Bone. When calcium i s injected intravenously, i t disappears rapidly from the blood, moving into bone and e x t r a c e l l u l a r space. The assumption i s made that the blood passing through the bone i n contact with bone mineral i s restored to i t s pre-in j e c t i o n l e v e l during one passage. (Fig. 19) On the basis of this assumption, the rate at which calcium i s stored i n bone, and the bone blood flow may be estimated by the methods given i n the appendix, p. 4/0 . By these methods, the calcium storage may be calculated i n mg./min. f o r various plasma calcium l e v e l s . The storage rate was found to be d i r e c t l y proportional to the increase i n plasma calcium above the control l e v e l , f o r when each storage rate i s divided by the increase i n the plasma calcium l e v e l , a constant i s obtained. This constant i s expressed i n mg. Ca/min./mg.$ r i s e i n the plasma calcium. (See Appendix for d e t a i l s on calculations.) The f i r s t part of the curve i n Fig . 23 shows a r i s e during the infusion, and then a rapid f a l l at the end of the infusion to the previous l e v e l , or a new equilibrium l e v e l . This represents the rapid movement of the injected calcium into i n t e r s t i t i a l space and bone. The rate of this movement i s d i r e c t l y proportional to the bone blood flow measured as $ of cardiac output. The bone blood flow ranged from 3-8$ of the cardiac output i n 14 adult dogs. 80$ ranged between 5-8$ of 28. the c a r d i a c output.:/..(Table IV) The second p a r t of the curve which shows a more grad u a l s l o p e , represents the f a l l o f the new e q u i l i b r i u m l e v e l between the blood and the l a b i l e p o r t i o n of bone. The r a t e of t h i s f a l l depends on the r a t e o f net removal of calcium. ( a c c r e t i o n or i r r e v e r s i b l e uptake i n bone m i n e r a l i z a t i o n , l e s s bone r e s o r p t i o n ) . The i n j e c t e d calcium i s c h i e f l y s t o r e d i n bone, s i n c e only a r e l a t i v e l y s m a l l p r o p o r t i o n (5% or l e s s ) i s excreted i n u r i n e . T h i s i n c r e a s e i n s t o r e d calcium may i n c r e a s e the e q u i l i b r i u m l e v e l . Two kinds of storage curves are shown i n the normal and parathyroidectomized dogs. ( F i g s . 4 and 5) The plasma c a l c i u m l e v e l r e t u r n s to the normal c o n t r o l l e v e l i n some dogs, and i n others i t r e t u r n s to a new and h i g h e r l e v e l . T h i s d i f f e r e n c e may depend on the r a t e at which c a l c i u m i s l e a v i n g the plasma to be used f o r bone m i n e r a l i z a t i o n . The d i f f e r e n c e may a l s o depend on the s i z e of the calcium storage p o o l i n bone. T h i s has been expressed as a r a t i o o f bone storage c a l c i u m / e x t r a c e l l u l a r f l u i d c alcium. I f the calcium i n the bone storage p o o l i s l a r g e enough i n comparison to the e x t r a c e l l u l a r f l u i d calcium, a r i s e i n the e q u i l i b r i u m between the two, due to storage of calcium, might not be n o t i c e a b l e . B. Calcium storage compared w i t h bone clearance' of Ca-45. When r a d i o s a l c i u m i s i n j e c t e d i n t r a v e n o u s l y , i t 29. disappears rapidly from the blood, moving into i n t e r s t i t i a l space and bone. The ki n e t i c s of the blood disappearance curves have been analyzed for ra t s , rabbits, c a t t l e and humans. Armstrong found that approximately half the blood calcium i n the rat exchanged with calcium i n the i n t e r s t i t i a l f l u i d per minute, so that the two may be considered a single compartment. There i s also very rapid movement of Ca-45 into bone, the l i m i t i n g factor being bone blood flow. Prederickson, Honour and Copp determined the i n i t i a l bone clearance of Ca-45 from blood i n the rat and obtained a value of 5-8$ of the cardiac output. Similar values have been obtained with Sr-90 and P-32. (11) • ' . C. Storage vs. Mobilization. When calcium i s removed from the blood stream by EDTA infusion at 10 mg./kg./hr. a mirror image of the storage curve i s obtained, and i n most animals the s p e c i f i c mobilization equals the s p e c i f i c storage as defined i n the appendix. ( Tables IV and V) This indicates an equilibrium between the blood calcium and a l a b i l e calcium pool i n bone, which can be approached from either the hypocalcemia or hypercalcemia side. It suggests the presence of a l a b i l e reservoir i n bone available f o r calcium storage, or calcium release i n times of calcium stress. The rate at which mobilization or storage occurs i s proportional to the amount of blood coming i n contact with the bone mineral or 30. calcium reservoir ( i . e . bone blood flow), and the difference i n Ca++ a c t i v i t y between the blood and bone pool. The bone blood flow was calculated by using both._calcium storage curves and calcium mobilization curves. .The. results obtained were almost i d e n t i c a l . By calcium storage calculations BONE BLOOD FLOW = 6.46 - *0.60 fo of cardiac output. By calcium mobilization calculations BONE BLOOD FLOW = 6.37 i * 0 . 6 l % of cardiac output * standard error 1. EDTA infusion p r i o r to calcium gluconate infusion (Fig. 6) I f the EDTA infusion i s carried out f i r s t and followed by a calcium infusion, the rate of calcium mobilization i s not s i g n i f i c a n t l y d i f f e r e n t from the rate of calcium storage. (Table VI) When the s p e c i f i c sirorage and s p e c i f i c mobilization was expressed graphically, the sirorage and mobilization tended to f a l l along the same l i n e running through the o r i g i n . (Fig.2 0 ) . Using Dog 7-45 as~an example, the results indicate the presence of an equilibrium between the l a b i l e f r a c t i o n of bone and the blood. Whether calcium i s removed from the blood stream with EDTA or added by calcium infusion, the equilibrium i s regained at the same rate. (Table VII). 2. Calcium gluconate infusion p r i o r to EDTA infusion.(fig. 7-) There i s evidence that hypercalcemia depresses 31. p a r a t h y r o i d gland f u n c t i o n . ( 8 ) This i n t u r n w i l l lower the calcium e q u i l i b r i u m between bone and blood. I n v e r s e l y , a low blood calcium i s thought to s t i m u l a t e p a r a t h y r o i d f u n c t i o n , thus r a i s i n g the e q u i l i b r i u m . During these experiments, hypercalcemia was produced f i r s t and seemed to have some d e f i n i t e e f f e c t on the m o b i l i z a t i o n r a t e of calcium, and a l s o on the bone-blood e q u i l i b r i u m l e v e l . Table V I I I shows c a l c u l a t i o n s of c a l c i u m storage and ca l c i u m m o b i l i z a t i o n when calcium gluconate was i n f u s e d before EDTA. I f t h i s t a b l e i s compared wi t h Table VI, no s i g n i f i c a n t d i f f e r e n c e between the two can be seen. When m o b i l i z a t i o n and storage i s p l o t t e d a g a i n s t the d i f f e r e n c e i n calcium l e v e l , the slopes are s i m i l a r , but i n c e r t a i n animals there i s a marked d i f f e r e n c e i n the i n t e r c e p t . ( P i g . 21^22) The slope of the l i n e r epresents the bone blood flow, which should be constant. Most of the graphs i n P i g . 21 show the storage l i n e p a s s i n g through the o r i g i n . The m o b i l i z a t i o n l i n e may cut the x - a x i s , g i v i n g a negative m o b i l i z a t i o n r a t e , a t 0 mg.$ de p r e s s i o n i n the plasma calcium. T h i s negative m o b i l i z a t i o n i n d i c a t e s some upset i n the normal plasma calcium balance. The r a t e o f t h i s negative m o b i l i z a t i o n would depend on the r a t e o f net removal of calcium, which i s the a c c r e t i o n o r i r r e v e r s i b l e uptake i n bone m i n e r a l i z a t i o n l e s s bone r e s o r p t i o n . 32. D. Changes i n the Bone-Blood Calcium E q u i l i b r i u m . Changes i n the bone-blood calcium e q u i l i b r i u m occur a f t e r calcium i n f u s i o n . T h i s change was more c l e a r l y demonstrated i f EDTA was i n f u s e d a f t e r calcium gluconate. ( E i g . 22A) The plasma calcium d i d not r e t u r n to normal a f t e r EDTA i n f u s i o n i n the m a j o r i t y of animals. This a l s o may i n d i c a t e t h a t hypercalcemia due to the calcium i n f u s i o n suppressed the pa r a t h y r o i d f u n c t i o n . The r e s u l t was a lowered bone-blood calcium e q u i l i b r i u m o r an impaired m o b i l i z a t i o n mechanism. This lowered e q u i l i b r i u m was n o t i c e d i n some dogs as e a r l y as f o u r hours a f t e r calcium i n f u s i o n . Table IX g i v e s a few examples of changes i n the e q u i l i b r i u m . 1. I n c r e a s i n g the s k e l e t a l a c t i v i t y o f bone by cal c i u m i n f u s i o n i n the parathyroidectomized dog. Rad i o a c t i v e s t u d i e s i n d i c a t e that the r e s e r v o i r o f a v a i l a b l e c a l cium i n the bone i s the calcium i o n s on su r f a c e c r y s t a l s a v a i l a b l e to the blood c i r c u l a t i o n . T h i s r e s e r v o i r o f a v a i l a b l e calcium o r the s k e l e t a l a c t i v i t y of bone can be inc r e a s e d by adding calcium to the r e s e r v o i r . Since an e q u i l i b r i u m i s maintained between the calcium i n the r e s e r v o i r and the calcium i n blood, any r i s e i n the e q u i l i b r i u m l e v e l i n d i c a t e s a r i s e i n s k e l e t a l Ca++ a c t i v i t y ( a Ca++) Calcium i n f u s i o n experiments (continuous and s i n g l e i n j e c t i o n s ) were done on parathyroideetomized dogs. R e s u l t s i n d i c a t e d t h a t the i n c r e a s e i n the bone-blood calcium e q u i l i b r i u m 33. l e v e l was p r o p o r t i o n a l to the amount of calcium i n f u s e d , and t h e r e f o r e to the amount of calcium stored i n the bone. I f m o b i l i z a t i o n from bone occurred, brought about by EDTA i n f u s i o n , the e q u i l i b r i u m l e v e l dropped. The d e f i c i t or drop was p r o p o r t i o n a l to the amount of calcium removed from the blood stream by EDTA, and t h e r e f o r e p r o p o r t i o n a l to the amount o f calcium m o b i l i z e d from the calcium r e s e r v o i r i n bone. Table X shows a r e l a t i o n s h i p between the amount of c a l c i u m added i n t r a v e n o u s l y , and the r i s e i n the bone-blood e q u i l i b r i u m . I f the r i s e i n the e q u i l i b r i u m l e v e l i s measured from the b e g i n n i n g of the continuous i n f u s i o n , there i s a s l i g h t l y h i g h e r r i s e i n the bone-blood e q u i l i b r i u m l e v e l as compared to the same amount i n j e c t e d i n a s i n g l e dose. Therefore the r i s e i n the e q u i l i b r i u m l e v e l was measured from the end o f the i n f u s i o n , f o r the purpose of c a l c u l a t i o n s . ( i n t e r c e p t of e x t r a p o l a t e d l i n e a t " t " , Fig.23) E. The S i z e of the Calcium Storage Pool i n Bone and Rate a t which Calcium i s used f o r Bone M i n e r a l i z a t i o n (see Fig.23) The t o t a l amount of calcium i n f u s e d i n t r a v e n o u s l y i s known. By measuring the r i s e i n the bone-blood e q u i l i b r i u m ("x" mg.%), and knowing the e x t r a c e l l u l a r f l u i d calcium, the s i z e of the calcium storage pool i n bone can be estimated. The slope of the g r a d u a l l i n e i s used to estimate the net r a t e a t which calcium i s l o s t from blood f o r bone m i n e r a l i z a t i o n , ( a c c r e t i o n or i r r e v e r s i b l e uptake i n bone m i n e r a l i z a t i o n , l e s s 34. bone resorption.) The calculations are shown i n d e t a i l i n the Appendix. Table XI shows the estimated calcium storage pool i n bone and the estimated accretion rates of several dogs. Table XII shows results of Bauer, Carlsson and Lindquist i n humans. (4/) Bauer, Carlsson and Lindquist have also studied the k i n e t i c s of turnover of Ca-45 and P-32 i n bone, i n rats. In the young rats, they estimated for t i b i a an accretion rate of 6.2% of the bone Ca/day; a resorption rate of 4.7$ of the bone Ca/day; and exchangeable f r a c t i o n equivalent to 3.0$ of the t o t a l calcium of the bone. ( 3 ) P. E f f e c t of Slow Calcium Infusion. 1. E f f e c t of slow calcium infusion i n a normal dog. When calcium was infused at 4 mg./kg./hr. fo r 9.5 hours, the plasma calcium rose from 8.5 mg.$ to 10 mg.% i n approximately an hour, and maintained this l e v e l u n t i l the infusion stopped. (Fig.8). The calcium excreted i n the urine was 5.25$ of the t o t a l amount injected. That i s , approximately 0.2 mg./kg./hr. of the t o t a l 4 mg./kg./hr. was excreted i n the urine. Since an equilibrium was reached and maintained, a balance of approximately 3.8 mg. Oa/kg/hr. may have been used for net bone mineralization. 2. Ef f e c t of slow calcium infusion i n a parathyroidectomized dog. During long calcium infusions i n two parathyroidectomized 35. dogs, the excretion of calcium was not measured, but the plasma calcium did not reach renal threshold. During one experiment, the infusion rate was 2 mg. Ua/kg/hr., and the plasma calcium rose 3.5 mg.$ i n seventeen hours, then promptly f e l l when the infusion stopped. (Fig. 12) The time scale was shortened (Fig. 13), and the net loss of calcium from the blood was measured at various plasma calcium l e v e l s . The rate of disappearance of calcium was found to be proportional to the r i s e i n plasma calcium. Between plasma calcium lev e l s of 8.2 - 9.2 mg.fo, the rate of disappearance of calcium was up to 2.8 mg. Ca/kg/hr. Between plasma calcium l e v e l s of 6.2 -7.2 mg.fo the disappearance rate was as low as 0.7 mg. Ca/kg/hr. Calcium was infused at 3 mg./kg/hr. into the other dog. The plasma calcium l e v e l rose 2 mg.fo and l e v e l l e d o f f . Since the plasma calcium was below renal threshold, the entire 3 mg. Ca/kg/hr. may have been used f o r net bone mineralization. This f a l l s within the range of estimated rate of bone mineralization. (Tables XI and XII) G. Calcium Excretion i n the Urine. Very l i t t l e calcium i s l o s t through the urine normally, for 98-99$ of the calcium f i l t e r e d i n the kidney by the lomerulus i s reabsorbed by the tubules. The calcium excreted i n the urine was measured before and a f t e r calcium infusion, i n both the intact and parathyroidectomized dogs. After calcium infusion i n the normal dog, approximately 36. 5% of the i n j e c t e d calcium was l o s t i n the u r i n e (Table I I ) , so t h a t only a s m a l l f r a c t i o n of the r a t e of disappearance o f calcium from the blood stream can be a t t r i b u t e d to e x c r e t i o n i n the u r i n e . The parathyroidectomized dogs showed a c a l c i u m e x c r e t i o n o f much l e s s than 5% of the i n j e c t e d dose. (Table I I I ) H. E f f e c t of U r e t e r a l L i g a t i o n and Nephrectomy on Removal of  I n j e c t e d Calcium from the Blood Stream. When the u r e t e r s are l i g a t e d the u r i n e p r o d u c t i o n i s terminated, but the kidney may s t i l l c a r r y on i t s metabolism, which i s not the case a f t e r nephrectomy, or l i g a t i o n of the r e n a l a r t e r i e s . The r e l a t i o n between kidney metabolism and c a l c i u m metabolism i s considered important. One theory claims t h a t calcium c i t r a t e i s c a r r i e d from the bone i n t o the blood p o o l . As i t passes through the kidney, the c i t r a t e i s metabolized and the calcium i s l e t f r e e i n t o the plasma. A l s o , the c o n c e n t r a t i o n of c i t r a t e i s thought to have some d i r e c t e f f e c t on the amount of calcium l o s t i n the u r i n e . A f t e r u r e t e r a l l i g a t i o n ( E i g . 15) EDTA and calcium gluconate s o l u t i o n s were i n f u s e d . Both curves were s i m i l a r to those obtained i n the normal animal. A l l of the i n f u s e d calcium must have gone i n t o e x t r a c e l l u l a r f l u i d and the storage p o o l of bone, s i n c e none could escape i n the u r i n e . In the nephrectomized dog, a f t e r c a l c ium i n f u s i o n , the plasma calcium returned to a new and h i g h e r l e v e l , and d i d not show the g r a d u a l f a l l , but maintained the new l e v e l f o r 37. s e v e r a l hours. ( F i g . 14) T h i s seems to i n d i c a t e some change i n the s t a t e of calcium i n the c i r c u l a t i o n , or may be an impairment of the removal mechanism. The change i n the s t a t e of calcium i n the c i r c u l a t i o n i s thought to p a r a l l e l c i t r a t e accumulation a f t e r nephrectomy. However, the method of c i t r a t e a n a l y s i s had not been e s t a b l i s h e d a t the time of these experiments. I . E f f e c t of P a r a t h y r o i d E x t r a c t on Calcium Storage, Calcium  M o b i l i z a t i o n , and Bone-Blood E q u i l i b r i u m L e v e l . A maintenance dose (0.1 u n i t s / k g . / h r . f o r t h i s dog) was administered immediately a f t e r parathyroidectomy. EDTA and calcium i n f u s i o n s were done d u r i n g t h i s maintenance dose. The e q u i l i b r i u m l e v e l was lower a f t e r EDTA i n f u s i o n , showing th a t a maintenance dose o f p a r a t h y r o i d e x t r a c t i s not s u f f i c i e n t to r a i s e the storage p o o l o f calcium i n bone back to the normal l e v e l . The e q u i l i b r i u m l e v e l was returned to normal w i t h the a d d i t i o n of calcium by i n f u s i o n equal to t h a t removed w i t h EDTA, thus r e s t o r i n g the storage pool to i t s previous s i z e . EDTA and calcium i n f u s i o n s were repeated with e x t r a p a r a t h y r o i d e x t r a c t . (1.0 u n i t s / k g . / h r . i n each i n f u s i o n s o l u t i o n ) C a l c u l a t i o n s showed no change i n calcium storage or calcium m o b i l i z a t i o n r a t e s w i t h e x t r a parathormone. However, the e q u i l i b r i u m l e v e l was r e s t o r e d almost to normal a f t e r EDTA i n f u s i o n . Thus a d d i t i o n a l p a r a t h y r o i d hormone was r e q u i r e d to i n c r e a s e the storage p o o l i n bone to i t s previous s i z e a f t e r calcium was removed by acute hypocalcemia. 38. V. iSUMMARY AND CONCLUSIONS 1. When calcium was injected intravenously, i t disappeared rapidly from the blood, moving into bone. Sim i l a r l y when calcium was removed from the blood stream by EDTA infusion, the calcium was restored rapidly to the blood by mobilization from the bone. 2. The rate at which the calcium was stored by the bone agrees closely with the rate at which calcium was mobilized from bone. Calcium storage = 1.20^0.11 mg/min/mg% r i s e i n plasma calcium/M^ Calcium mob. = 1.08^0.10 mg/min/mg% depression i n plasma calcium/M This data indicates the presence of an equilibrium between the ex t r a c e l l u l a r calcium, and the l a b i l e calcium i n bone. The rate at which this equilibrium i s regained i s proportional to the amount of blood c i r c u l a t i n g through the bone and coming i n contact with the l a b i l e calcium f r a c t i o n of bone. 3. Bone blood flow as measured from calcium infusion = 6.46^0.60% cardiac output Bone blood flow as measured from EDTA infusion = 6.37-0.61$ cardiac output. This estimated bone blood flow i s i n general agreement with values derived from clearance studies made with radioactive isotopes. 39. 4. The ex t r a c e l l u l a r f l u i d calcium was estimated as 15.73-0.72 mg/kg, and the e x t r a c e l l u l a r f l u i d volume as 203.3-10.4 ml/kg. 5. The quantity of C a + + i n the l a b i l e f r a c t i o n of bone could be increased by calcium infusion. This C a + + a c t i v i t y i n bone was increased i n proportion to the amount of calcium infused, and therefore to the amount of calcium stored i n the bone. The l a b i l e calcium storage pool i n bone was estimated as 2-5 times greater than the e x t r a c e l l u l a r calcium, or approximately 0.2 - ,0.5$ of the t o t a l bone calcium. 6. The net rate of disappearance of calcium from the blood afte r calcium infusion was estimated at 1-2 mg Ca/kg/hr., or 0.15 to 0.35$ of the t o t a l bone calcium per day. These results are similar to values reported f o r calcium accretion by bone i n humans. 40. VI. APPENDIX METHODS USED FOR CALCULATING THE QUANTITATIVE ASPECTS OF CERTAIN  FACTORS INVOLVED IN REGULATION OF PLASMA CALCIUM. A. RATE OF CALCIUM STORAGE IN BONE FOLLOWING INTRAVENOUS INFUSION The calculations below are based on the following assumptions: (a) I t i s assumed that there i s an equilibrium between plasma calcium and a l a b i l e calcium pool i n bone adjacent to the c i r c u l a t i o n . (b) Since no appreciable increase i n soft tissue calcium content of muscle and skin was observed during hypercalcemia (except that due to increase i n e x t r a c e l l u l a r f l u i d calcium), and since less than 5% of the injected calcium appeared i n urine, i t i s assumed that most of the calcium which disappears from blood following intravenous infusion has been taken up by the skeleton. (c) Since 50-80$ of the plasma calcium exchanges across the c a p i l l a r y wall per minute ( i . ), i t i s assumed that the i n t e r s t i t i a l and plasma calcium act as a single pool (ext r a c e l l u l a r calcium pool) with respect to processes occurring over a period of 1-6 hours. (d) Because of the rapid movement of calcium across the c a p i l l a r y wall and the vast excess of the calcium i n the l a b i l e bone pool (as compared to the calcium i n the bone c a p i l l a r y ) i t i s assumed that the a c t i v i t y of calcium ion leaving the c a p i l l a r y i n the bone venules w i l l not d i f f er.'significantly from that i n 41. the l a b i l e bone storage p o o l . T h i s s i t u a t i o n would be analogous to the unloading of CC>2 i n the lungs, and i t would be a n t i c i p a t e d t h at the amount o f calcium taken up by bone would then be p r o p o r t i o n a l to blood flow and the A-V d i f f e r e n c e . i . e . Calcium uptake by bone i n mg/min = k (bone plasma flow i n ml/min) x (A-V) where A and V" are the a r t e r i a l and venous c o n c e n t r a t i o n s o f plasma, calcium i n mg/ml. A i s obtained from the a r t e r i a l or mixed blood sample; V i s assumed to correspond to the plasma i n e q u i l i b r i u m with the l a b i l e bone storage p o o l . In some animals, t h i s bone-blood e q u i l i b r i u m appears to correspond to the p r e - i n j e c t i o n plasma calcium l e v e l ; i n o t h e r s , the l e v e l i s r a i s e d o r lowered d u r i n g the i n f u s i o n . To t e s t t h i s h y p o t h e s i s , the r a t e o f Ca storage or m o b i l i z a t i o n has been determined as d e s c r i b e d below, and p l o t t e d a g a i n s t the A-V d i f f e r e n c e i n ca l c i u m l e v e l as def i n e d above. In almost a l l cases a l i n e a r r e l a t i o n s h i p i s observed, c o n f i r m i n g the h y p o t h e s i s . The slope g i v e s a clearance value which, a c c o r d i n g to F i c k P r i n c i p l e , should be the f u n c t i o n a l bone blood flow. T h i s blood flow may be expressed d i r e c t l y ; i n terms o f body surface area; o r as a per cent o f the average r e s t i n g c a r d i a c output f o r a dog of the same s u r f a c e area. 42. METHOD OF CALCULATION ( F i g . 24) 1. SPECIFIC CALCIUM STORAGE AND MOBILIZATION (a) An e q u i l i b r i u m l i n e r e p r e s e n t i n g the assumed bone-blood e q u i l i b r i u m i s drawn ( u s u a l l y 10 mg$ i n the normal dog). S e v e r a l l i n e s a t each 0.5 mg$ l e v e l o r 0.25 mg$> l e v e l are drawn above the e q u i l i b r i u m l i n e . (b) For each l e v e l the f o l l o w i n g time i n t e r v a l s are measured: t Q a = time o f i n f u s i o n (A M) t ^ = time of recovery to same l e v e l (M C) Storage = t i m e o f s t o r a S e = * c a + *R ( A C ) The storage r a t e may be c a l c u l a t e d u s i n g two assumptions: (1) E x t r a c e l l u l a r calcium a t A = e x t r a c e l l u l a r calcium a t C. (2) Mg calcium s t o r e d i n t g ^ Q r a g e = calcium i n f u s e d i n t ^ a Since the i n f u s i o n r a t e was 10 mg/kg/hr. the Storage r a t e = t ^ x 10 mg x body wt.(kg) 60 min = mg/min ^Storage The storage r a t e o f calcium can be c a l c u l a t e d i n mg/min by the above method f o r any plasma ca l c i u m l e v e l above the e q u i l i b r i u m calcium l e v e l . When t h i s storage r a t e i s d i v i d e d by the A - V d i f f e r e n c e i n plasma calcium, a constant 43. i s obtained. I f the storage r a t e i s p l o t t e d a g a i n s t the A - V d i f f e r e n c e , a s t r a i g h t l i n e running through the o r i g i n i s u s u a l l y obtained. ( F i g . 20) The average A - V d i f f e r e n c e i n plasma calcium i s c a l c u l a t e d by the f o l l o w i n g method: (a) The area BAC i s d i v i d e d by t g ^ ^ e to determine the average plasma Ca l e v e l over the p e r i o d A C, (b) Thus the average A - V d i f f e r e n c e (mg%) over the p e r i o d o f A to C = average plasma Ca l e v e l (over A to C) minus the c o n t r o l e q u i l i b r i u m calcium l e v e l When there i s a r i s e i n the bone-blood e q u i l i b r i u m l e v e l , the A - V d i f f e r e n c e i n plasma calcium i s determined as f o l l o w i n g : An assumption i s made th a t the dotted l i n e drawn i n free-hand (Fig.25) corresponds to the Ca++ of the venous blood r e t u r n i n g from the l a b i l e calcium p o o l i n bone. Area PQRS i s d i v i d e d by AC. The answer i n mg.% i s added to the SR mg.% l e v e l . T h i s i s subtrac t e d from the average plasma calcium l e v e l d u r i n g AC. Thus the Calcium Storage Clearance ( i n mg/min/ A - V diff.Ca++ cone. = Storage r a t e Cmg/min) = C 0 N S T A 1 I T Average A - V d i f f . C a + + cone. I f t h i s r a t i o i s expressed g r a p h i c a l l y , a s t r a i g h t l i n e through the o r i g i n i s u s u a l l y obtained. 44. The SPECIFIC CALCIUM STORAGE CLEARANCE i s determined by d i v i d i n g the CONSTANT by the sur f a c e area o f the animal. 2. BONE BLOOD FLOW The bone blood flow may be determined by m u l t i p l y i n g the CONSTANT by 1 0 0 , and expressed as mls/min. The bone blood flow may a l s o be expressed as per cent o f the r e s t i n g c a r d i a c output by the f o l l o w i n g f r a c t i o n : S p e c i f i c Calcium Storage Clearance Cardiac Index ( f o r plasma) The c a r d i a c index f o r plasma = 1 . 6 l i t e r s / M (2.S ) 3. THE EXTRACELLULAR CALCIUM SPACE The e x t r a c e l l u l a r c a l c i u m space i s estimated by the f o l l o w i n g method: (a) The average A - V plasma Ca d i f f e r e n c e (mg.%) i s determined by d i v i d i n g &rea. BDE by the t o t a l time o f i n f u s i o n . (b) The calcium s t o r e d d u r i n g the i n f u s i o n = the A - V plasma Ca d i f f e r e n c e d u r i n g Ca i n f u s i o n x the average storage (mg/min/mg.fo A - V d i f f . ) x i n f u s i o n time (min.) (c) The net a d d i t i o n of calcium to the e x t r a c e l l u l a r p o o l w i l l equal the calcium added d u r i n g t ^ minus the calcium stored d u r i n g t Q . The assumption i s made that there i s a l i n e a r r e l a t i o n s h i p between the i n c r e a s e i n plasma calcium and e x t r a c e l l u l a r f l u i d 45 . calcium d u r i n g the i n f u s i o n . The net a d d i t i o n o f calcium to the e x t r a c e l l u l a r f l u i d p ool i s t h e r e f o r e r e s p o n s i b l e f o r the r i s e i n the plasma calcium a t the end of the i n f u s i o n . Thus the t o t a l e x t r a c e l l u l a r c a l c i u m (mg/kg) = Net a d d i t i o n o f ca l c i u m x c o n t r o l plasma Ca l e v e l d u r i n g the i n f u s i o n r i s e i n plasma c a l c i u m by the end of the i n f u s i o n (d) Assuming the plasma calcium i s 5 mg/kg (5% body wt. at 10 mg.%) the calcium i n the i n t e r s t i t i a l f l u i d may be c a l c u l a t e d . Thus i n t e r s t i t i a l f l u i d Ca = t o t a l e x t r a c e l l u l a r f l u i d c a l c i u m - 5 mg/kg (e) Assuming the i n t e r s t i t i a l f l u i d i s 7 mg.%, the i n t e r s t i t i a l f l u i d volume may be c a l c u l a t e d . Thus the i n t e r s t i t i a l f l u i d volume = i n t e r s t i t i a l f l u i d Ca x 100 ml. 7 mg. (f ) The e x t r a c e l l u l a r f l u i d volume = i n t e r s t i t i a l f l u i d volume + plasma volume 46. 4. EXAMPLE OF THE ABOVE CALCULATIONS(Fig. 26 and Table XIIl) Dog 7-17 i s used as an example. The dog's weight was 2 20.86 kg; the surface area was 0.765 M . The control calcium l e v e l (determined over a one and one-half year period) was 10.3 mg.%. Calcium was infused for one hour at a rate of 212.5 mg. of calcium per hour. (10.17 mg/kg/hr.) (a) Determination of Spe c i f i c Calcium Storage (1) The equilibrium l i n e was drawn at 10.3 mg.% calcium (2) Lines were drawn at each 0.5 mg.% l e v e l ( i . e . 14.0, 13.5, 13.0, 12.5, 12.0, 11.5 mg.fo) (3) For each l e v e l t ^ a , t ^ , and t g ^ o r a g e were determined. e.g. at plasma calcium l e v e l of 14.0 mg.% t C a = 4 min., and t s t o r a g e = 6 min. Infusion rate of calcium was 212.5 mg/hr. = 3.55 mg/min. Therefore storage rate at 14.0 mg.% = 4 min. x 3.55 mg. = 2.365 mg/min. 6 min. (4) Determination of average A - V plasma calcium difference The average plasma calcium l e v e l above 14.0 mg.% 17 x 0.05 = 0.1415 mg.% 6 14.0 + 0.1415 = 14.1415 mg.% 47. The average A - ¥ plasma calcium difference = 14.1415 - 10.3000 = 3.8415 mg.$ (5) The calcium storage clearance = 2.365 mg/min. 3.842 mg.% The S p e c i f i c Calcium Storage clearance = .666 .786 M2 = .870 mg/min/mg.$ A - V d i f f . /M2 (b) Estimation of Bone Plasma Flow Bone plasma flow = 2.365 x 100 = 61.3 ml/min. 3.842 or bone plasma flow = .870 = 5.43$ cardiac output 1.6 (c.) Estimation of E x t r a c e l l u l a r Calcium Space (1) 2383 x 0.05 = 1.96 mg.$ 60 (2) Calcium stored during infusion = 1.96 x 0.666 x 60 min. = 78.6 mg. (3) Net addition of calcium to E.C.F. pool = 212.5 mg. - 78.6 mg. = 133.9 mg. = 6.43 mg/kg 48. Total e x t r a c e l l u l a r calcium pool = 6.43 x 10.30 4.00 = 16.55 mg/kg. Plasma calcium = 5 mg/kg. I n t e r s t i t i a l f l u i d calcium = 16.55 - 5.0 = 11.5 mg/kg. I n t e r s t i t i a l f l u i d volume = 11.5 x 100 = 164 ml/kg 7 or I n t e r s t i t i a l f l u i d volume = 164 = 16.4$ body weight 10 Total e x t r a c e l l u l a r f l u i d calcium space = 16.4$ + 5$ = 21.4$ body weight 4 9 . B. ESTIMATION OF LABILE CALCIUM STORAGE POOL IN BOMB When the plasma calcium l e v e l i s r a i s e d by intravenous i n f u s i o n most o f the excess calcium i s taken up by bone, and there i s a r a p i d f a l l to a new e q u i l i b r i u m l e v e l . I t i s assumed that at t h i s t ime, the calcium i n plasma i s once more i n e q u i l i b r i u m w i t h the l a b i l e bone storage p o o l . In most cases (and i n v a r i a b l y i n the parathyroidectomized dog) t h i s new e q u i l i b r i u m l e v e l i s above the o r i g i n a l c o n t r o l v a l u e . At t h i s new l e v e l , some of the i n j e c t e d c a l cium w i l l be accounted f o r by the i n c r e a s e i n e x t r a c e l l u l a r c a l c i u m corresponding to the h i g h e r plasma calcium l e v e l ; however, most w i l l be accounted f o r by storage i n bone. Assuming that the i n c r e a s e i n e x t r a c e l l u l a r calcium and storage p o o l calcium i s d i r e c t l y p r o p o r t i o n a l to the i n c r e a s e i n the plasma calcium l e v e l , i t i s p o s s i b l e to estimate the dimensions of the l a t t e r i n the f o l l o w i n g manner: Assuming an e x t r a c e l l u l a r c a l c ium p o o l of 15 mg/kg at 10 mg.% plasma calcium, an i n c r e a s e o f 1 mg.fo w i l l be a s s o c i a t e d with an i n c r e a s e i n 1.5 mg/kg i n the e x t r a c e l l u l a r c a l c i u m . Since the q u a n t i t y o f calcium s t o r e d i s known, the d i f f e r e n c e w i l l represent calcium taken up by bone. I f i t i s assumed that t h i s i s a l l i n the l a b i l e storage p o o l , and that the i n c r e a s e i s d i r e c t l y p r o p o r t i o n a l to the i n c r e a s e d plasma calcium, the s i z e of t h i s p o o l can be estimated, and equals Ca i n j e c t e d - i n c r e a s e d Ca i n e x t r a c e l l u l a r f l u i d x 10 NEW e q u i l i b r i u m plasma Ca(mg.7o) - c o n t r o l plasma Ca(mg.$) 50. T h i s may be expressed as mg. Ca/kg or as a per cent of the t o t a l bone calcium. METHOD OF CALCULATING THE NET LOSS OF PLASMA CALCIUM AFTER  CALCIUM INFUSION The net l o s s o f calcium from the plasma i s assumed to equal the r a t e a t which calcium i s used f o r bone m i n e r a l i z a t i o n l e s s c a l c ium r e l e a s e d by r e s o r p t i o n . The r a t e of the f a l l i n the e q u i l i b r i u m l e v e l a f t e r c a l c ium i n f u s i o n i s used to estimate t h i s net l o s s . From the above c a l c u l a t i o n s , the slope o f the e q u i l i b r i u m l i n e i s used to measure the net l o s s of calcium i n mg/kg/hr., or as $ of t o t a l bone calcium per day. 51-' EXAMPLE OF CALCULATIONS (Fig.23) 1. Estimation of l a b i l e calcium storage pool i n bone Dog 7-07 i s used as an example. The dog had been parathyroidectomized several days. (a) 9.4 mg. Ca/kg were infused. (b) Rise i n equilibrium l e v e l was 1.2 mg.f> (c) 1 mg.fo r i s e i n equilibrium l e v e l represents 7.75 mg. Ca/kg added intravenously. (d) The ex t r a c e l l u l a r calcium i n Dog 7-07 was 17.5 mg. Ca (10 mg.fo Ca i n blood represents 17.5 mg.Ca/kg i n ext r a c e l l u l a r f l u i d ) (1 mg.f> Ca i n blood represents 1.75 mg.Ca/kg i n ext r a c e l l u l a r f l u i d ) (e) Then 7.75 mg/kg - 1 . 7 5 mg/kg 6.0 mg/kg The l a b i l e calcium storage pool i n bone =6.0 mg/kg x 10 = 60 mg/kg (f) Ratio of bone storage calcium = 60 = 3.4 17.5 E.C.F. calcium 52. That i s , the bone storage pool or the amount of available calcium i n bone i s 3.4 x greater than the E.C.F. calcium. (g) Assume calcium i n bone i s 15 gm/kg (\$) The l a b i l e calcium storage pool = 60 x 100 = 0.40$ of the t o t a l 1 5000 bone calcium 2. Estimation of net loss of calcium from the plasma (a) F a l l i n the equilibrium l e v e l = 5.65 mg.$ - 4.40 mg.% 5.0 hours = 0.25 mg.$/hr. (b) 1 mg.$ r i s e i n equilibrium l e v e l represents 7.75 mg.Ca added to blood. 0.25 mg.% f a l l i n equilibrium l e v e l represents 1.94 mg.Ca/kg/hr. removed from the blood. (c) 1.94 x 24 x 100 = .31$ of the t o t a l bone calcium/day. 15000 Total Calcium 2.5 mM Nondif fusible 0.82 mM J7mM| Globulin Albumin 0.65mM Ionized Calcium 1.33 mM Diffusible 1.63 mM Com-plexes 0.3 mM piexesjv" IqUI ssisa2» Bicarb. POf Cit= Others aCa + + 47XIQ-3 Figure 1A. The state of calcium i n normal serum as calculated from ultrafiltration data and formation constants (22) Figure IB. Photometric t i tration of calcium using Klett Colorimeter and 500 Lambda F i l t er , (values taken from Table i) Figure 2. Continuous delivery infusion machine. Regulation of Plasma Calcium Normal Dog No: 7-41 , 22.2 kg. 5 H 9.86mg.Ca / k g . / h r . 2 3 4 5 Time in Hours. Figure 4. Calcium gluconate infusion i n a normal dog, showing a constant bone-blood equilibrium level. Regulation of Plasma Calcium Normal Dog No: 7-15, 18.13 kg. mg°/o Ca I 10.25 mg. Ca /kg./nr. ' c a ' / 7 // T I 1 1 1 1 1 1 0 1 2 3 4 5 6 7 Time in Hours. Figure 5. Calcium gluconate infusion i n a normal dog, showing a rise i n the bone-blood equilibrium level Figure 6. Calcium Regulation. Effects of EDTA and Ca infusion when EDTA i s infused prior to Ca. 2i Figure 7. Calcium Regulation. Effects of Ca and EDTA infusion when Ca i s infused prior to EDTA. Regulation of Plasma Calcium Normal Dog No: 7 - 7 0 , 7.5 kg. mg.% Ca 1 0 1 • t - * — > • • — r 5 . 2 6 % of in jec ted Calcium was excreted in the U r i n e - . //1// /// / / / / 12 Time in Hours. Figure"B. Calcium Regulation. Effects of slow calcium infusion i n the normal dog. Ca Storage in Bone Parathyroidectomized Dog 10.7 kg. E M — I Plasma _ = ^ = = = Co mg % 15 "l Figure 9. Effect of calcium infusions in parathyroidectomized dog. The bone-blood equilibrium rose in proportion to the total amount of calcium infused. Calcium Storage in Bone Pcirathyroidectomized Dog No: 7 — 5 6 , 13.5kg. Figure 10. Effect of calcium infusion i n parathyroidectomized dog. There i s no rise in the bone-blood equilibrium level . Calcium Storage in Bone Parathyroidectomized Dog No: 7 — 0 7 , 13kg. Plosmo Ca mg.% 10 n 5H Single Dose of Ca 9.4 mg./kg. Single Dose of Ca 7.0 m g . / k g . 0 0 2 3 4 5 6 7 Time in Hours. Figure 11. Effect of single injections of calcium i n parathyroidectomized dog. Figure 12. Effect of slow calcium infusion in a parathyroidectomized dog. Slow Calcium Infusion in Parathyroidectomized Dog No: 7 - 5 4 , 20.8 kg. Plasma Ca mg. % 10 2 mg. / k g . / nr. Ca T 1 5 10 15 2 0 25 Time in Hours. —r— 3 0 35 Figure 13. Effect of slow calcium infusion i n a parathyroidectomized dog (Figure 12 - same experiment) Dog No: 7 - 4 9 , 22.7 kg. Parathyroids Intact. mg.% Ca = = = = = = Bilateral Nephrectomy —•—1 1 \ V 1 1 i * — 1 — 1 — i , , Time in Hours. Figure 14. The effect of nephrectomy on calcium mobilization, calcium storage and calcium level . Dog No: 7 — 4 8 , 25.4 kg Para thyro ids In tac t . Figure 15. The effect of ureteral ligation on calcium mobilization, calcium storage and calcium level. I Effect of Parathyroid Extract on Calcium Level, Mobilization and Storage Dog No. 7 - 5 0 ( EZ.7 kg.) 0 5 10 15 20 25 30 33 Time in Hours. Figure 16. The effect of parathyroid extract on calcium level, mobilization and storage. Calcium Storage in Bone. Parathyroidectomized Dog No: 7 - 6 8 , 13.6 kg. Plasma Ca mg.% 10 Point at which an Equilibrium appears to be established between Ca in Bone Storage Pool and Plasmo Calc ium. • • • 9 X — Carot id Artery Plasma Ca • — Femoral Vein Plasmo Ca 0 — Marrow Plasmo Ca 15 mg. / kg . / hr . ''A.Ca,///, 4 T ime 6 Hours. 10 Figure 17. Showing the difference between arterial , venous, and marrow samples during calcium infusion. Plasma Ca mg. % Regulation of Plasma Calcium in normal Dog N o : 7 - 4 4 , 12.7 kg. 10 x-t-0 Femoral Artery Plasma Ca Jugular Vein Plasma Ca Femoral Vein Plasma Ca E D T A —i— 10 1 12 Time in Hours. Figure 18. Showing difference between femoral artery, femoral vein, and jugular vein samples during EDTA and calcium infusions. Crystal of Bone Sal t . Figure 1 9 . Proposed labile calcium store on surface of bone crystals in equilibrium with the blood calcium. Calcium Mobilization Prior To Calcium Storage. Normal Dogs. x x xSpecific Storage. 0 o — o Specific Mobilization. 7 - 4 4 7 , — 46 7 — 45 Plasma Calcium Rise or Depression mg. % ( A - V Difference) Figure 20. Showing calculated calcium mobilization and calcium storage rates when EDTA infusion was carried out prior to calcium infusion. The slopes of the line represent the effective bone-blood flow. Calcium Storage Prior To Calcium Mobilization. x x — x Specific Storage. o—0---0 Specific Mobilization 7—13 7—14 Plasma Calcium Rise and Depression mg. % ( A - V Difference ) L Figure 21. Showing calculated calcium mobilization and calcium storage rates when calcium infusion was carried out prior to EDTA infusion. The slopes of the lines represent the effective bone-blood flow. Calcium Mobilization Prior To Calcium Storage: 6 -i c 4 -1 6 J.V. = Jugular Vein Samples F.V. = Femoral Vein Samples Dog 7 - 4 4 ( J . V . ) Dog 7 - 4 4 ( F. V ) Dog 7 - 4 5 ( J . V ) Dog 7 - 4 8 , ( J .V . ) §> ' 1 1 i!2 i!5 2!o I I I I 2l2 2l5 2<a 3>a I "1 I r O 1.2 14 1.5 2.0 ' l:> 2.0 25 3.0 3.5 Z Z 2 5 Z'8 3 0 2.0 2.4 2.8 3.0 O 55 Calcium Storage Prior To Calcium Mobilization Storage Mobilization Depression of Rise in Plasma Calcium in m g . % . Figure 22. When EDTA was infused prior to calcium infusion, the calculated mobilization and storage rate was almost identical . I f calcium was infused prior to EDTA infusion, the mobilization rate and storage rate were similar i f expressed as mg/min/mg.$ rise or depression in the plasma calcium, (that i s the slopes were identical) . However, the mobilization rate i s less i f expressed as mg/min. at a definite depression in the plasma calcium level . This corresponds to the different intercepts of the storage and mobilization rates in Figure 21. I i 0 Storage Prior To Calcium Mobilization E Dog 7-13 Mg. Ca added 100 mg. and removed 7 - 1 5 196 mg. 7 - 1 7 230.8 mg. Calcium Mobilization Prior To Calcium Storage. Rise in Equilibrium level after Calcium infusion t 1 7 - 4 4 7 - 4 5 7 - 4 6 131 mg. 163 mg. 272 mg. "1 Fall in Equilibrium level after EDTA infusion 7 - 4 8 254 mg. Figure 22A. Showing changes i n bone-blood equilibrium level after EDTA arid calcium infusions. Calcium Storage. Figure 23 . Calcium storage i n bone Calcium Storage Constant Bone-Blood mg. % Ca Equilibrium. 7 6 5 Ca Rising Bone — Blood Ca Fi&ure 24 and. 25. Method of calculating calcium storage or mobilization. Example: Calcium Storage Dog No :7- i7 , 20.86 kg. SA= 0 .765 m z Plasma Ca mg. % Sum of A r e a s : I 2 5 ( T T ) 8 I 3 8 ( 1 3 6 ) I 55 7 5 ( 3 7 6 ) 2 3 I I 4 5 ( 7 5 2 ) 3 0 5 3 4 I ( 1 3 9 8 ) 3 7 9 3 8 6 ( 2 1 6 3 ) 12 AM T i m e in Hours. ! Figure 26. Method of calculating calcium storage. TABLE I. Mis. .01 $ versene light absorption titrated 0,0 258 1.0 166 1.2 152 1.4 140 1.6 126 1.8 112 2.0 97 2.2 91 2.4 89 2.6 87 end point = 2.08 ml Values for t i trat ion of calcium standard with 0.01 fo Na^DTA TABLE I I . Urine calcium excretion i n the normal animal a f t e r calcium infusion. Dog 7-12 7-13 7-14 7-15 7-17 7-19 7-70 Rate of calcium Duration of infusion mg./kg./hr. calcium infusion 10 mg./kg./hr. one hour 4.0 mg./kg./hr. 9.5 hours Urine calcium expressed as $ of calcium injected 3.4$ (approximately) *5.0$ *5.0$ " *5.0$ " *5.0$ *5.0$ " +5.26$ (accurate) urine c o l l e c t i o n by catheterization + u r i n e c o l l e c t i o n by canulation of one ureter TABLE III Urine calcium excretion i n the parathyroidectomized animal, (accurate Ca analysis urine c o l l e c t i o n by canulation of one ureter) Dog Rate of calcium Duration of infusion mg./kg./hr. Ca infusion EM-1 10 mg./kg./hr. one hour 7-67 12.95 mg./kg./hr. 1.5 hr. 7-68 15.0 mg./kg./hr. 1.5 hr. Urine Ca expressed as $ of Ca injected. 0.144$ 3.19$ TABLE IV Specific calcium storage i n normal dogs, with estimated bone blood flow and extracellular calcium. Dog Body Wt. Kgms. Area M2 Specific Ca Storage ag/Mn/mgm* rise/M Estim. bone blood flow mls/min. Bone blood . flow as $> card, output I . S . F . E . C . F . calcium calcium mg/kgm mg/kgm E . C . F . volume $ of body wt E . C . F . volume ml/kg • 7-11 27.0 0.94 2.11 198 13.20 14.40 19.4 25.4 256 7-12 32.5 1.09 0.81 89 5.06 11.30 16.3 21.2 211 7-13 10.0 0.46 1.26 58 7.42 9.30 14.3 18.3 183 7-14 23.6 0.85 0.89 76 5.23 6.75 11.7 14.7 147 7-15 18.1 0.71 1.13 80 7.06 12.30 17.3 22.6 226 7-16 15.0 0.61 1.02 62 6.35 8.80 13.8 17.5 175 7-17 20.9 0.77 0.87 67 5.43 11.55 16.5 21.4 214 7-21 14.5 0.51 0.91 46 5.70 10.30 15.3 19.7 197 7-39 23.6 0.85 1.32 112 7.56 11.05 16.2 20.9 208 7-41 27.2 0.90 1.13 102 6.62 8.60 13.5 17.2 172 7-44 12.7 0.54 0.77 42 4.80 5.70 10.7 13.2 132 7-45 16.3 0.67 0.61 41 3.82 14.70 19.5 25.8 257 7-46 27.2 0.95 0.86 82 5.37 11.70 16.6 21.6 216 7-43 25.4 0.90 1.16 104 6.80 14.25 19.1 25.3 252 Averages ± S.E. 1.20±0.11* 6.46±0.60* 15.73±0.72* 203.3±10.4 Based on the value for cardiac output i n the dogs ( £ - 5 ) Specific Calcium Mobilization i n Normal Dogs Dog 1 'Body 1 Surface Spec. Ca mobilization^ # wt. area mg./min./mg. f l / /" kg. sq.m. EM-1 12.2 0.530 1.060 7-07 17.2 0.675 1.210 7*39 27.2 0.945 1.28 7-44 12.7 0.540 1.07 7-45 16.3 0.650 0.62 7-46 27.2 0.945 0.83 7-48 25.4 0.895 1.02 7-50 22.7 0.825 1.57 1.08±0.103 TABLE V l with Estimated Bone Blood Flow and Extracellular Calcium. Estim. bone Bone blood Extracellular Extracellular blood flow flow as $ Ca mg./kg. volume ml./min. cardiac output ml./kg. 56.18 6.23 14.05 177 82 7.1 20.80 261 123 7.6 16.10 209 58 6.3 -40 3.6 -78 4.8 19.10 253 92 6.0 23.7 328 130 9.2 22.7 305 6 .37±0.61 19.4±1.7 255±8 TABLE VI EDTA Infusion Prior to Calcium Infusion. Estimation of Calcium Storage and Calcium Mobilization Rates. Dog Calcium storage mg/min/mg# Calcium mobilization ' mg/min/mg$ Diff . mg/min/mg$ Specific storage Specific mobilization Diff . mg/min/ 7-44 0.3995 0.515 0.1145 0.742 0.954 0.212 7-45 0.4065 0.4096 0.0031 0.612 0.616 0.004 7-46 0.8120 0.778 0.0340 0.812 0.778 0.034 7-48 1.034 0.914 0.1200 1.156 1.022 0.134 TABLE VII EDTA Infusion Prior to Calcium Infusion. Estimations of Bone Blood Plow, E .C.P . Ca, etc. from both EDTA Curves and Calcium Curves. Dog. Io.7-45 Spec. Spec. Bone f Ex-ce l l . I .S .P.- I .S.P. E .C.P . j mob.' stor. blood Ca Ca volume volume flow mg./kg. mg./kg. $ body % body $ C O . weight weigit Calculations made from EDTA curve 0.616 3.62 19.10 14.17 20.2 25.13 Calculations made from calcium curve 0.612 3.82 19.45 14.70 21.0 25.75 TABLE VIII Calcium infusion prior to EDTA infusion. Estimation of calcium storage and calcium mobilization rates. Dog Ca storage: mg/min/mg# rise i n plasma Ca. Ca mobilization: mg/min/mg# depression i n plasma Ca. Differ. mg/min/ mg% Specific storage: mg/min/ mg^/cr Specific* mobilization^ mg/min/mg$/fa Difference: mg/min/mg /^M 7-13 0.5815 0.4625 0.1190 1.260 1.005 0.255 7-14 0.7550 0.6020 0.1530 0.890 0.710 0.180 7-15 0.7970 0.6950 0.1020 1.130 0.986 0.144 7-17 0.6660 0.6204 0.0458 0.870 0.812 0.068 0.1048 0.162 Dog 7-13 7-15 7-17 TABLE IX Calcium infusion prior to EDTA infusion, showing changes i n the bone-blood calcium equilibrium. Mg.Ca added during Ca infusion. 100 196 230.8 Mg. Ca removed during EDTA infusion. 100 196 230.8 Mg.$ rise in plasma Ca during Ca infusion. 3.24 4.63 4.47 Mg.$ f a l l in plasma Ca during EDTA infus. 4.10 5.55 4.40 rise i n equilibrium level after f a l l i n equilibrium level after Ca infusion. EDTA infusion. 0.973 2.37 0 1.3 3.25 1.60 TABLE X Showing rise i n Bone-Blood Calcium Equilibrium after continuous or single injection of Calcium in the Parathyroidectomized Dog No.EM-1 Rise 1 in bone-blood equilibrium (mg#) Continuous infusion (5 mg.Ca/kg) - measured from beginning of infusion 1.55 M " (10 mg.Ca/kg) " " • " " 2.80 " " ( 5 mg.Ca/kg) ». " e n d " " 1.45 " " (10 mg.Ca/kg) " » n H « 2 .00 Single dose ( 5 mg.Ca/kg) - - 1.40 " " (10 mg.Ca/kg) - - 2.05 TABLE XI Dog Wt. Area Estim. E . C . F . Bone storage Ratio of Storage Rate at which Rate at which Ca i s kg. sq.m. bone blood Ca. mg/kg pool mg.Ca/kg storage pool as Ca i s used for used for bone mineral-flow # card. pool/E.C.F. f> total bone mineral- ization as $> of total output calcium bone Ca ization, mg Ca/ bone Ca/day. kg/hr. EM-1 12.2 0.53 6.23 14.1 36 2.57 0.1295 1.0 0.087 7-07 17.2 0.675 7.12 20.8 60 3.40 0.4000 1.94 0.310 7-39 23.6 0.85 7.56 16.1 45 3.00 0.3000 0.915 0.100 7-67 14.1 0.58 80.3 4.59 0.5360 1.955 0.314 7-68 15.4 0.62 65.8 3.76 0.4380 2.14 0.343 TABLE XII Bauer, Carlsson and Lindquist's results i n human studies by means of radiocalcium (4-) Age of human Weight Exchange Ca mg/kg. Accretion rate mg/kg years 0.5 4.3 199 2.72 5.12 6.9 155 2.20 1.5 12.9 233 1.93 11 32.0 161 1.02 22 61.0 125 0.54 25 60.0 77 0.32 26 70.0 73 0.28 43 58.0 79 0.26 51 65.0 78 0.23 56 56.0 70 0.35 57 90.0 68 0.20 58 70.0 62 0.29 60 65.0 67 0.36 63 86.0 88 0.21 TABLE XIII Summary for calculation of a l l 0.5 mg.$ levels Mg.# Ca storage Mg/min. Mg/min. Mg.# Mg.# Storage plasma (min) (min) Ca Ca average average mg./min./mg.$ level added stored Ca level Ca rise rise i n plasma Ca. (A-V d i f f . ) (A-V di f f . ) 14.0 4 6~ 14.2 2.365 14.1415 3.8415 0.616 13.5 12 18 42.6 2.365 13.8780 3.5780 0.661 13.0 19 30 67.5 2.250 13-6275 3.3275 0.675 12.5 27 46 96.0 2.085 13.3185 3.0185 0.681 12.0 34 64 120.8 1.890 13.0920 2.7920 0.677 11.5 42 90 149.0 1.660 12.7050 2.4050 0.688 rise i n serum calcium origin i s obtained: (Fig.20) BIBLIOGRAPHY 1. Armstrong, W. D., Johnson, J. A., Singer, L., Lienke, R.I. and Premer, M.L. Rates of transcapillary movement of calcium and sodium and of calcium exchange by the skeleton. Am. J. Physiol., 171: 641, 1952. 2. Bauer, G. C , Carlsson, A. and Lindquist, B. Some properties of the exchangeable bone calcium. Acta  physiol. Scandinav., 35: 67, 1955. 3. Bauer, G. C , Carlsson, A. and Lindquist, B. Evaluation of accretion, resorption and exchange reactions i n the skeleton. Kgl. Fysiograf. Sallskap. Lund Forh., 25: 1, 1955. 4. Bauer, G. C , Carlsson, A.and Lindquist, B. Bone s a l t metabolism i n humans studied by means of radiocalcium. Acta. Med. Scand., 15.8: 143, 1957. 5. Clark, E.P., and C o l l i p , J.B. A study of T i d s a l l method fo r determination of blood serum calcium with a suggested modification. J. B i o l . Chem., 63: 461, 1925. 6. Copp, D. H. Report, on N.R.C. Project M.P. 320, Jan.,. 1954. 7. Copp, D. H. Calcium and phosphorus metabolism. Am. J. Med., 21: 275, 1957. 8. Engfeldt, B. Studies on parathyroidal function i n r e l a t i o n to hormonal influences and d i e t e t i c conditions. Acta. Endocr. Kbn. Suppl., 6: 7, 1950. 9. F a l e s , F. H. A micromethod f o r the determination of serum calcium. J . B i o l . Chem., 204: 577, 1953-10. Finean, J . B., and Engstrom, A. The low-angle s c a t t e r of x-rays from bone t i s s u e . Biochim. e t . biophys. a c t a , 11: 178, 1953. 11. F r e d e r i c k s o n , J . M., Honour, A. J . , and Copp, D. H. Measurement of i n i t i a l bone clearance of Ca-45 from blood i n the r a t . F e d e r a t i o n Proc., 14: 49, 1955. 12. H a r r i s o n , H. E. The i n t e r p r e t a t i o n of c i t r a t e and calcium metabolism. Am. J . Med., 20: 1, 1956. 13. Hines, J . R. and Freeman, S. E f f e c t s of r e n a l and p a r a t h y r o i d f u n c t i o n on disappearance of i n t r a v e n o u s l y i n j e c t e d c a l c ium c h l o r i d e . Am. J . P h y s i o l . , 171: 114, 1952. 14. Howard, J . E. The metabolism of calcium and phosphorus i n bone. B u l l . New York Acad. Med., 27: 24, 1951. 15. Howard, J . E. Calcium metabolism, bones and calcium homeostasis. A review of c e r t a i n c u r r e n t concepts. J . C l i n . E n d o c r i n o l . Metabolism, 17: 1105, 1957. 16. K o e l l i k e r , A. Die normale Resorption des Knochengewebes und i h r e Bedeutung f u r d i e Entstehung der typrschen Knochenformen. L i e p z i g : F. C. ¥. Vogel. 1873. 17. lawson, 0. H., Overbey, D. T., Moore, C. J . and Shadle, 0. ¥. Mixing of c e l l s , plasma and Dye T-1824 i n the c a r d i o v a s c u l a r system of b a r b i t a l i z e d dogs. Am. J . P h y s i o l . , 151: 282, 1957. 18. Lehmann, J. A photo-electric micro method for the di r e c t t i t r a t i o n of calcium i n serum with ethylenediamine tetraacetate. Scand. J. C l i n , and Lab. Investigation, 5_: 203, 1953. 19. M i t c h e l l , H. H., Hamilton, T. S., Steggerda, F. R. and Bean, H. W. The chemical composition of the adult human body and i t s bearing on the biochemistry of growth. J. B i o l . Chem., 158: 625, 1945-20. McLean, F. C. The parathyroid hormone and bone. C l i n i c a l Ortheopaedics, 9_: 46, 1957. 21. Jtfeuman, W. F. and Neuman, M. W. Postulates regarding structure and metabolic function of bone. Am. J. Med., 22: 123, 1957. 22. Neuman, W. F. and Neuman, M. W. The chemical dynamics of bone mineral. The University of Chicago Press, 1958. 23. Pharmacology, Physiology, Biochemistry and Toxicity of Versenes, Bersworth Chemical Co., Framingham, Mass., 1952. 24. Poulos, P. P. The renal tubular reabsorption and urinary excretion of calcium by the dog. J. Laborat. C l i n . M., 49_: 253, 1957. 25. Wiggers, H. C. Cardiac output and t o t a l peripheral resistance measurements i n experimental dogs. Am. J_. Physiol., 140: 519, 1944. 26. Wolf, A. V. and Stanley, M. Effe c t of intravenous calcium s a l t s on renal excretion i n the dog. Am. J. Physiol., 158: 205, 1949. 

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