_——,_.V_‘_.—, .— THE EFFECTS OF PREVIOUS CALCIUM INTAKES ON ADAPTATION TO LOW AND HIGH CALCIUM DIETS IN RATS Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY IOHN D. BENSON 1967 TH it». 7 LI .313; R Y iifiligan sum University Er am‘gma BY “’1 . “IMO & SUN? 3“ BOOK BINDERY INC. LIIIFM RY BINDERS : uue‘-uu 1 ABSTRACT THE EFFECTS OF PREVIOUS CALCIUM INTAKES ON ADAPTATION T0 LOW AND HIGH CALCIUM DIETS IN RATS by John D. Benson Female. weanling rats of the Sprague-Dawley strain were randomly assigned to either a high (0.9%) or low (0.2%) Ca diet for a 60-day preliminary period. At the conclusion of this period. half of each group was switched to the other diet'for a 75-day experimental period. The remaining rats continued on their original diets. According to these pro- cedures rats were fed one of four ways: 1) a low Ca diet throughout both periods (LL). 2) a low Ca diet during the preliminary period followed by a high Ca diet during the experimental period (LH). 3) a high Ca diet during the preliminary period followed by a low Ca diet during the experimental period (HL). or 4) a high Ca diet throughout both periods (HH). Rats adapted to the low Ca diet by increasing the percentage of dietary Ca retained. Likewise. rats adapted to the high Ca diet by decreasing the percentage of the l John D. Benson element retained. The changes in retention were largely due to changes in fecal Ca. indicating that the primary mechanism for altering Ca retention was operating at the intestinal level. The percentage of dietary Ca retained was dependent on the past as well as the present Ca intake. Rats accus- tomed to frugal intakes of Ca (LL and LH) retained a larger percentage of the dietary Ca than rats accustomed to abund— ant amounts of the element (HL and HH) regardless of the present dietary Ca level. The percentage of dietary Ca re- tained was greater in the LL and HL groups than in the LH and HH groups. However. the total amount of Ca retained by these groups (LL and HL) did not equal that of the groups currently ingesting the high Ca diet (LH and HH). Ca reten- tion decreased with advancing age in all groups. Up to 133 days of age. rats accustomed to low Ca in- takes (LL and LH) deposited less Ca and P in their bones than rats which had been used to high Ca intakes (HL and HH). However. at 156 days of age (conclusion of the experiment) the bones of all groups contained equal amounts of Ca and P. The amount of P retained appeared to be directly related to the amount of the element ingested and was not influenced by previous diets. John D. Benson Rats which were fed the low Ca diet throughout the entire experiment (LL) gained significantly less weight than rats (LH and HH) which were given the high Ca diet during the experimental period. The groups did not differ in over- all appearance and vitality. Serum calcium values did not reflect the Ca status of the rats. In fact. the LL and LH groups maintained a higher serum Ca level than the HL and HH groups even though the skeletons of the former groups did not contain as much Ca or P as those of the latter. Serum Ca values increased with advancing age in all groups. The experimental diets (previous or current) did not consistently affect the amount of phosphate found in the serum. Parathyroid activity. as judged by parathyroid gland weight. serum Ca and Pi values. and urinary Pi was greater in the LL and HL groups. Furthermore. the LL group exhib- ited more parathyroid activity than the HL group. This finding supported the belief that increased parathyroid activity was at least partially reSponsible for the in- creased retention efficiency of calcium observed in the LL group. THE EFFECTS OF PREVIOUS CALCIUM INTAKES ON ADAPTATION TO LOW AND HIGH CALCIUM DIETS IN RATS BY John D. Benson A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1967 ACKNOWLEDGEMENTS Sincere appreciation is extended to Dr. Roy Emery and Dr. J. W. Thomas for their guidance during the course of this study and in the preparation of this manuscript. Thanks are due to Dr. H. A. Tucker and Dr. P. Reineke for their many suggestions and for their critical reading of this manuscript. The aid of Max Paape in acquainting this author with the prOper handling of laboratory animals is acknowledged and appreciated. Gratitude is extended to Dr. C. A. Lassiter. chairman of the Dairy department. for providing financial aid in the form of a research assistant- ship. The author is especially indebted to his parents whose many sacrifices and constant interest made graduate study possible. Finally. words seem inadequate to convey the gratitude which is due to my wife. Marie. for her un- yielding assistance in the preparation of this thesis and for her patience. understanding. and moral support through- out the conductance of this study. ii TABLE OF ACKNOWLEDGEMENTS. . . . . . . LIST OF TABLES. . . . . . . . LIST OF FIGURES . . . . . . INTRODUCTION. . . . . . . . . REVIEW OF LITERATURE. . . . . Adaptation . . . . . . . . Low Ca Diet. . . . . . High Ca Diet . . . . . Role of Absorption . . Role of Serum Ca . . . CONTENTS Role of Fecal Endogenous Ca. . Role of Bone . . . . . Role of Parathyroid. . METHOD AND MATERIALS. . . . . Diet . . . . . . . . . . . General Experimental Procedures. . Balance Studies. . . . . . Tissue Samples . . . . . . Chemical Analysis. . . . . Calcium. . . . . . . . Phosphorus . . . . . . Per Cent Ash . . . . . Statistical Analysis . . . iii Page ii vii 29 30 3O 32 33 34 34 35 36 36 Table of Contents/cont. Page RESULTS AND DISCUSSION. . . . . . . . . . . . . . . . 38 Body Weight and Overall Appearance . . . . . . . . 38 Bone . . . . . . . . . . . . . . . . . . . . . . . 42 Calcium Retention. . . . . . . . . . . . . . . . . 46 Phosphorus Retention . . . . . . . . . . . . . . . 56 Serum Calcium and Phosphate. . . . . . . . . . . . 59 Parathyroid Gland. . . . . . . . . . . . . . . . . 64 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . 71 REFERENCES. . . . . . . . . . . . . . . . . . . . . . 74 iv Table 10. 11. LIST OF TABLES Ca Absorption in Rats of Different Ages. . . . . . . . . . . . . . . Experimental Design . . . . . . . . . . . . . Percentage Composition of Experimental Diets. The Effect of Previous and Current Levels of Dietary Calcium on Body Weight Gains. . . . Effect of Previous and Current Levels of. Dietary Calcium on Dry Tibia Weights. . . . Effect of Previous and Current Levels of Dietary Calcium on the Amount of Calcium in the Tibia Expressed as Per Cent of the Dry Bone. . . . . . . . . . . . . . . . . . . . Effect of Previous and Current Levels of Dietary Calcium on the Amount of Phosphorus in the Tibia Expressed as Per Cent of the Dry Bone. . . . . . . . . . . . . . . . . . The Effects of Previous and Current Dietary Calcium Levels and Age on Net Calcium Retention . . . . . . . . . . . . . . . . . The Effects of Previous and Current Levels of Dietary Calcium and Age on Per Cent Calcium Retained. . . . . . . . . . . . . . . . . . Summary of Calcium Balance Data . . . . . . . The Effects of Previous and Current Levels of Dietary Calcium and Age on Calcium Excretion. Page 13 3O 31 4O 43 44 44 47 49 50 52 List of Tables/cont. Table 12. 13. 14. 15. 16. 17. Summary of Phosphorus Balance Data. . . . . . Phosphorus Excretion Data . . . . . . . Effect of Previous and Current Dietary Levels of Calcium and Phosphorus on Serum Calcium Levels. . . . . . . . . . . . . . . . . . . Effect of Previous and Current Dietary Levels of Calcium and Phosphorus on Serum Phosphate Levels. . . . . . . . . . . . . . . . . . . . Effect of Previous and Current Level of Dietary Calcium on Fresh Parathyroid Gland Weight . . The Effect of Previous and Current Levels of Dietary Calcium on Parathyroid Gland Weight vi Page 58 6O 61 63 65 66 Figure l. 2. LIST OF FIGURES Effect of Dietary Treatments on Body Weights. Effect of Age on Percent Calcium Retained . . vii Page 39 57 INTRODUCTION Parturient paresis. a metabolic disorder of Ca metab— olism in dairy cows. represents a potential economic loss for all dairymen. Although successful means of preventing and treating the disorder have been reported. its etioloqy remains a mystery. The successful prevention of parturient paresis by the prepartum feeding of low Ca diets (Boda and Cole. 1954) stresses the important role that dietary levels of Ca and P play in the disorder. As an explanation for their results. these experimenters prOposed that the low Ca diets conditioned the parathyroid gland to react to the Ca stress imposed by parturition. whereas the feeding of a high Ca diet prior to parturition inhibited the rapid response of the parathyroid gland at parturition. permitting the onset of parturient paresis. The proposed explanation of preconditioning an animal to a low Ca diet in order to combat a later stress (parturi- tion) was almost identical to the concept of adaptation de— scribed by Henry and Kon (1953). wherein rats previously ex- posed to a low Ca diet retained more Ca on that diet than rats which had been previously accustomed to a high dietary Ca level. Unfortunately the role of the parathyroid gland was not investigated in these studies. Since a large number of cows with a history of milk fever were not available and since a low Ca diet in a growing rat should provide a Ca stress similar to parturition. an experiment similar to that of Henry and Kon's but including an evaluation of parathyroid involvement would provide basic information on the possible role of the parathyroids in the prevention of milk fever. Consequently. the questions which this experiment hoped to answer were these: 1) Will an animal adapt itself more efficiently to a low Ca diet. or other Ca stress. if it has first been conditioned to a low Ca diet? 2) What is the role of the parathyroid gland in such an adaptive process? REVIEW OF LITERATURE Le Chatelier's theorem of physical chemistry reads as follows: any alteration in the factors that determine an equilibrium causes the equilibrium to become displaced in such a way as to Oppose as far as possible the effect of the alteration (Hamill. Williams. and MacKay. 1966). Nutritional adaptation obeys this theorem. An animal will react to lower (or higher) dietary levels of a nutrient by conserving (or rejecting) the nutrient in question. Animals must adapt to extreme levels of dietary Ca if they are to survive. Failure to adapt to a lowered Ca intake would produce a Ca deficient skeleton. Eventually the bone stores of Ca would be depleted. causing extremely weak bones. Serum Ca would decrease and tetany would develop. Death would ensue. If an animal failed to adapt to a high Ca in- take. deposition of Ca in bone would continue unchecked. Bone marrow Spaces would be filled with bone. leaving insuf- ficient space for vital hematopoietic functions. Serum Ca levels would rise resulting in a sluggish nervous system. Cardiac or reSpiratory failure would be the likely result. Thus. when subjected to altered intakes of Ca. an animal must (1) maintain its blood Ca level and (2) maintain the normal functions of bone (Malm. 1963). To maintain a normal serum Ca level. an animal can alter its bone accretion or resorption rates. gastrointestinal- absorption rate. and urinary and fecal excretion rates. Adaptation Low Ca Diet.. In a general sense. an animal adapts to a reduced Ca intake by becoming more efficient in the handling of the element. The degree of adaptation is gen- erally measured by Ca retention of per cent utilization of dietary Ca. Growth rate is generally unaffected by dietary Ca levels unless the Ca level is extremely low. Copp and Suiker (1962) fed diets containing 0.037% and 0.40% Ca to rats and did not detect a difference in growth rate. a finding which agreed with the earlier studies of Bell. egpal, (1941). In contrast Sherman and Campbell (1935) reported that rats fed a diet with 0.35% Ca gained significantly more weight than rats fed a diet containing 0.20% Ca. Hansard and Plumlee (1954) fed young rats four levels of Ca (0.013. 0.3. 0.5. 1.0) for 55 days. The rats fed the 0.013% Ca diet weighed significantly less than those on the other three treatments. A more severe restriction of Ca (0.004%) retarded the growth rate of rats after three weeks of the diet (Boelter and Greenberg. 1941). Thus a certain minimal level of dietary Ca exists below which an animal cannot adapt. This lack of adaptation is reflected by reduced weight gains and other inadequately studied physiological responses. The first studies showing adaptation of rats. as measured by increased Ca retention. to low Ca diets were those of Fairbanks and Mitchell (1936). Nicolaysen (1943). and Rottensted (1938). In a more carefully controlled study. Henry and Kon (1953) fed rats a low Ca diet (0.13% Ca) for 21 months. At 12 months their skeletons contained as much Ca as those of control rats on a more generous intake indi- cating a marked degree of adaptation. Lifetime adaptation to a low Ca diet was demonstrated in rats by Nicolaysen (1955). Evidence that dogs can adapt to low Ca diets was presented by Gershoff §£_§l. (1958). Dairy heifers adapted to a low Ca diet by increasing their absorption efficiency of Ca (Lindsey. §£;§1,. 1931). Controlled adaptation studies in human prisoners were performed by Malm (1963). When the Ca in the diet of 22 prisoners was reduced 50%. most prisoners adapted success- fully after an initial period of pronounced negative Ca balance. Two adapted immediately; two did not adapt at all. indicating the individuality of the response. Similar var- iation in individual responses were noted by Nicolaysen (1943). Adaptation to low Ca intakes has been reported in human populations accustomed to low (200 mg/day) intakes of Ca (Nicholls and Nimalasuriya. 1939: Hegsted. g§_§1.. 1952). Advancing age retards the adaptive response (Nicol- aysen gtqgl,. 1953). Nicolaysen §£_§l. (1953) have shown that old rats will adapt only when a severe Ca restriction is imposed. Although some adaptation in old rats was detect- able 10 days after a switch from a Ca rich to a Ca poor diet (Fournier. 1951). a period of four months was needed for adaptation in old rats to equal that of young rats (Henry §£_§l.. 1960). Young rats adapt within a few days (Hansard §;;§1,. 1951). The effects of age on Ca adaptation in the growing animal are confounded by body stores of the element. 0n the basis of studies performed in their laboratory Nicol- aysen gt_§1. (1953) have concluded that old rats adapt only when Ca stores are threatened. Consequently. the effects of age on the adaptive reSponses to Ca can be explained by the body stores of the element. Further discussion of this point will be presented when the role of Ca absorption is considered. Hiqh Ca Diet. If high Ca diets are ingested. hyper- calcemia is prevented either by reduced intestinal absorp- tion of Ca or by rapid skeletal uptake of absorbed Ca. Whereas both mechanisms operate in the young growing animal. only the former functions in older rats (Harrison. 1959). If rapid skeletal uptake of Ca reflects a fixation of Ca on the skeleton rather than in it. precautions must be taken in interpreting short term balance trials (Leitch and Aitken. 1959). Trials which indicate an increased Ca retention and presumably increased bone formation may instead only reflect the ability of the skeleton to fix Ca more rapidly than the secretion of digestive juices and urine can remove it. This temporary and superficial fixation might explain in part why summation of retention on short term balance trials yields unrealistically high values. Experiments designed to study adaptation to increased intakes of Ca in animals are few in number. One such study conducted by Gershoff et al. (1958) suggested that dOgs did not retain significantly more Ca when switched from a low to a high Ca diet. The most complete study in humans concerning adaptation to a high Ca intake was that of Malm (1963). who increased the Ca intake of 38 prisoners. and performed bal- ance trials on these individuals. In these trials 15 of the 38 men retained less Ca than on their previously fed low Ca diet: 14 subjects increased their Ca retention; and the Ca retention of 9 men did not change. These reSults indicated the variability of the response to a high Ca diet and that unknown factors were playing a role in the adaptive process. Role of Absorption. Adaptation is strongly dependent on the ability of the intestine to regulate calcium absorp- tion. The intestinal tract has the first Opportunity to pre- sent more Ca to an animal. Young rats on normal intakes of Ca (0.5-l.0%) absorb from 30-60% of the dietary Ca. The amount of Ca retained is determined by the amount fed and the per cent absorbed. An animal can adjust to a low Ca diet by increasing the extent of absorption. Increased absorption in response to a low Ca diet has been shown in rats by Ellis and Mitchell (1933). Fairbanks and Mitchell (1936). Nicolaysen (1943). Rottensted (1938). Henry and Kon (1953). Hansard and Plumlee (1954). and C0pp and Suiker (1962). The same response has been demonstrated in goats by Lengemann (1963). in dogs by Gershoff §£_§1. (1958). and in cows by Lindsay et a1. (1931). Increased Ca absorption was primarily responsible for the successful human adaptation in the studies of Malm (1963) and Nicolay— sen et al. (1953). In vitro methods of assessing Ca absorption have been used to demonstrate the greater absorbing capacity of intes- tines from animals which had been fed diets deficient in Ca. Wasserman (1960) and Dowdle. et al. (1960). established that duodenal gut sacs from rats on low Ca diets tranSported Ca45 to a greater extent than those from rats maintained on high Ca diets. In addition to the greater Ca tran5port through the duodenum. Kimberg. et a1. (1961) presented evidence that rats maintained on a low Ca diet (0.029%) with adequate vita- min D could actively tranSport Ca throughout the entire in- testine. whereas rats on a stock diet could actively trans— port Ca only in the proximal 1/5 of the small intestine. A prOper understanding of the factors regulating Ca absorption is dependent upon a knowledge of the transport mechanism itself. Calcium is absorbed both by active and passive tranSport. The evidence for active transport is as follows. 1) Ca is transferred against concentration and po- tential gradients. 2) Transfer of Ca against a concentration gradient requires oxygen and a metabolizable hexose in the medium. The tranSport is inhibited by nitrogen. low 10 temperature and metabolic poisons. 3) Ca transport is lim- ited by a maximal rate. 4) Potassium ion. 3-methyl-d-glucose. an actively transported hexose. and divalent iron competitively inhibit Ca transport (Schachter. 1963). Further evidence for the active transport of Ca was presented by Schachter and Rosen (1959) and Schachter et a1. (1960) when they established that high energy phosphate was necessary for Ca transport. Adaptation did not occur in the absence of vitamin D (Nicolaysen g£_al.. 1953; Haavaldsen et al.. 1956; Dowdle §t_ .31.. 1960; Kimberg et al.. 1961). Since vitamin D was neces- sary for active transport (Schachter et al.. 1961; Dowdle 2; 31. 1960). any adaptive process may well be Operating by. or at least be dependent upon. a functional active transport system. Disagreement exists regarding the way in which vita- min D affects the Ca active transport system. Schachter g3_ El. (1961) believed that vitamin D action depended upon the existence and maintenance Of the active transport system for Ca. Harrison and Harrison (1960) proposed that the action Of vitamin D was to increase the diffusibility of Ca into the mucosal cell. and that the activation by vitamin D Of active transport might be secondary to an increased availability of Ca to the active transport system. Wasserman (1963) Observed 11 that the vitamin D effect on increased transfer or uptake of Ca by rat and chick intestine was not inhibited by metabolic poisons. incubation under nitrogen. or incubation at depressed temperatures. These observations did not agree with the pro- posal of Schachter et a1. (1961). Wasserman attempted to reconcile the two different proposals with the following considerations. Active transport systems are generally considered as consisting of two components: 1) a "carrier" system which imparts specificity to the system and provides the transported complex with the correct properties for traversing the membrane 2) the "pump“ that supplies energy to drive the substrate carrier up hill If vitamin D was essential for the synthesis and/or operation of the carrier component and not for the energy yielding reaction. the apparent discrepancies in concepts may be resolved.. Cramer and Dueck (1962) Observed a saturation effect on the absorption rate of Ca when they increased the luminal concentration of Ca. These results were suggestive Of a carrier. Further support for the existence of a carrier can be gathered from the data of Schachter et a1. (1961) which demonstrated competitive inhibition of Ca transport by var- ious actively transported sugars. Sugars which were not actively tranSported would not inhibit Ca transport in Schachter's system. 12 Wasserman and Taylor (1963. 1966) administered vita- min D3 to rachitic chicks and found a soluble protein in the mucosal cells Of the duodenum which was capable of binding Ca. Additional evidence that induction of a carrier protein resulted from vitamin D3 treatment was presented by Zull g; al. (1965) and Norman (1965). They demonstrated that simul- taneous administration of actinomycin D and vitamin D3 pre- vented the action Of vitamin D. Furthermore. Norman (1966) has shown that RNA synthesis in the intestinal mucosal cells of rachitic chicks was elevated 50% after vitamin D3 admin- istration. Thus. considerable evidence has accumulated to sug- gest that vitamin D stimulates Ca absorption by inducing a carrier protein. Numerous experiments with rats have substantiated that Ca absorption decreases with advancing age. (Table 1). Comparison between experiments is not justified since so many other factors (level of Ca in diet. vitamin D. etc.) influence Ca absorption. Kimberg et al. (1961) and Schachter et al. (1960) using in vitro techniques have demonstrated that in- testines from young rats transported Ca more rapidly than intestines from Older rats. The percentage of dietary Ca absorbed also decreased with advancing age in cows (Lengemann 13 et al.. 1957: Hansard et al.. 1954). dogs (Liu and McCay. 1953). and humans (Harrison. 1959). Table 1. Ca Absorption in Rats of Different Ages — — _- 1L Age % Ca Absorbed Hansard and Henry and Taylor §£_Ql. Crowder (1952) Kon (1953) (1962) 4 wks 98 93.0 100 6 wks -- -—-- 60 12 wks 57 64 —- 24 wks 46 21 -- 48-72 wks 41 8 20 106 wks 24 -- -- Aging effects on Ca metabolism are confounded with body stores of Ca. In general the body stores of Ca are positively correlated with age. Thus. as noted by Draper (1963). differences in dietary histories may cause animals of the same chronological age to be of a different physio- logical age. Evidence that body stores of Ca were more im- portant in regulating Ca absorption than age was first pre- sented by Rottensted (1938). He fed pairs of rats a high or low Ca diet for three weeks. then switched them all 14 to an intermediate level (0.4%). The rats. all the same age. which had been fed the low Ca diet. stored 90—93% of the in- gested Ca. whereas rats previously fed the higher diet stored 81-86% of the ingested Ca. Nicolaysen (1943) Observed that rats which had been starved such that 5% of their calculated Ca stores had been depleted absorbed more Ca than unstarved rats of the same age. Liu et al. (1941) administered vitamin D to humans with osteomalic (reduced bone Ca) bone. This treatment led to rapid bone salt deposition and absorption values were like those found in infants (well over 70%). Although the increased Ca absorption resulting from this treatment might have been directly related to the increased bone deposition. the direct effect of vitamin D on Ca absorp- tion through the intestine cannot be eliminated. Results such as these led Nicolaysen (1943) to pro- pose the existence of an "endogenous factor." He hypothe-‘ sized that osteoblastic activity in an unsaturated skeleton produces a humoral factor (endogenous factor) which with vitamin D conditions the upper small intestinal mucoSa to increase the transfer of Ca. Gran (1960) repeated the exper- iment of Nicolaysen with parathyroidectomized rats and Ob- tained the same response. Thus the endOgenous factor is not parathyroid hormone. To date. there is no direct experimental 15 evidence to support the existence of an "endogenous factor." The pathways which mediate the intestinal response to lowered bone Ca levels are unknown. The role that endogenous hormones play in this response needs to be established. Earlier work of Bfilbring (1931) and Albright and Reifenstein (1948) and later studies of Gran (1960) and Was- serman (1961) did not demonstrate any changes in Ca absorp- tion when the parathyroids were removed or when parathyroid extract was injected into rats. In contrast to these studies. Talmage andIflliot (1958) demonstrated that parathyroidectomy 2-4 hours prior to experimental use decreased Ca absorption 50% as measured from isolated gut sacs Of rats. Using the same means of assessing absorption. Schachter (1960) Obtained a 30% decrease in Ca transport following parathy parathyroid- ectomy. only if 3 days were allowed to elapse from the time of the Operation to the time of assay. Using in vitro methods. Rasmussen (1959). reported that parathyroidectomy depressed but did not abolish active Ca transport in duodenal segments. Cramer (1963) reported the same result using je- junal fistula techniques. Both Robertson (1942) and Birge §£_§l. (1966) have observed that hyperparathyroid patients absorb Ca to a greater degree than normal euparathyroid patients. 16 As noted by Toverud (1963). interpretation Of changes in Ca absorption of parathyroidectomized animals must be guarded. Parathyroidectomy could have a direct effect on the mechanism of absorption or it could act indirectly through changes in the serum Ca level. A diminished serum Ca level would reduce the endogenous Ca secretion into the digestive juices. Hence parathyroidectomy would inhibit the absorption mechanism but simultaneously. due to the depressed serum level. reduce fecal endogenous calcium and no change in net absorption would be observed. Perhaps this explanation ac- counts for some of the divergent results concerning the ef- fects Of the parathyroids on Ca absorption. Following hypophysectomy there was an initial increase in Ca absorption (4—5 days after hypophysectomy) followed by a marked depression which was alleviated only by the injec- tion of growth hormone (Finkelstein and Schachter. 1962). These results may be useful in understanding the effects Of age on Ca absorption. Glucocorticoid treatment did not de- press Ca absorption in the presence of functioning parathy- roids (Wajchenberg g£_gl.. 1965). Estradiol decreased Ca transport across the intestinal wall when given to either intact or hypophysectomized male rats (Finkelstein and Schachter. 1962). Noble and Matty (1967) and Finkelstein 17 and Schachter (1962) demonstrated reduced Ca absorption in rats receiving injections of thyroxin. Certainly the hor- monal status of the animal plays a role in the regulation of Ca absorption. Parathyroid hormone and growth hormone are apparently necessary for optimal Ca absorption. Additional research may elucidate the effects of these hormones and others upon Ca absorption. Certain dietary constituents affect Ca absorption. Of these. the anion. phosphate is perhaps the most widely studied. The ratio Of Ca to P becomes important to Ca ab- sorption when dietary Ca is limiting. Studies Of Leichser- ring §t_§l. (1951) and Mellanby (1949) on college women demonstrated that Ca retention was depressed when increased P was ingested. When Ca was limiting. a wide P/Ca ratio de- pressed Ca retention in rats (Hansard and Plumlee. 1954). The performance of Hereford calves was poorer when the Ca/P ratio was less than 1:1 (Wise gt_§l.. 1963). Lueker and Lofgreen (1961) varied the Ca/P ratio from 0.8:1 to 6:1 and reported no effect on the amount of Ca absorbed in sheep. Dymsza gt_§l. (1959) and Nicolaysen §t_§l. (1953) could not detect any changes in Ca absorption when increased levels of phosphate were fed to rats. Malm (1953) Obtained the same results in humans. In contrast to these experiments. Young 18 et al. (1966) has shown that Ca absorption was reduced when 4—6 month old lambs were given a phosphorus—deficient diet. Phosphorus supplementation increased Ca absorption. Thus a minimal level of P appears to be necessary for maximal Ca absorption. If the absolute amount of Ca is limiting. fur- ther increases in P will decrease Ca retention. probably due to the formation in the intestinal tract of the relatively unavailable tricalcium—phosphate (Davis. 1963). In the pres- ence of adequate vitamin D and abundant Ca the Ca/P ratio is of minimal importance in Ca absorption. Lengemann and Comar (1961) reported a stimulatory effect on the absorption of Ca45 following oral administra- tion of lactose. The response was the same whether the diet was composed of 0.04% or 1.0% Ca. In an earlier study. Lengemann (1959) demonstrated that intraperitoneal injections of lactose had no effect on Ca absorption. Thus lactose probably acts at the site of absorption. Wasserman et al. (1956) confirmed the stimulatory effect of lactose using the femur content of Ca45 as a measure Of absorption. Chang and Hegsted (1964) observed that the ability Of lactose to stimu- late Ca absorption was lost as rats aged and was not clearly demonstrable after six weeks Of age. 19 Role of Serum Ca. Following absorption. Ca is trans- ported by the plasma tO all parts of the body where it per- forms its vital functions. Plasma Ca exists in three forms: approximately 50% is combined with plasma proteins. approxi- mately 5% is combined with other plasma components. and the remaining 45% is ionized (Guyton. 1963). The three forms are rapidly exchangeable in vitro (Samachson and Lederer. 1958; Visek §£_§l.. 1953; Winget and Smith. 1959). The serum Ca level in young growing rats ranges from 6-12 mg/100 ml. Serum Ca remains constant due to the influ- ence Of parathormone (McLean. 1957) and thyrocalcitonin (Care .§£_§l.. 1967). The latter homone may be of minimal importance in Ca homeostasis. since Bronner §£;§l. (1967) could show no difference in the thyrocalcitonin content of thyroid glands from rats on various Ca intakes. Bronner concluded that thyrocalcitonin may only be of importance when serum Ca is elevated by infusion. The constancy of serum Ca can be i1- lustrated by considering an experiment by Bronner and Aubert (1965) where young male rats were placed on three levels of Ca intakes (0.05%. 0.5%. 1.0%). The plasma Ca level Of these rats remained invariant over a range Of absorption from 4.5 to 83.4 mg/Ca/day. The constancy of serum Ca levels is partially due to its slow rate of absorption from the 20 intestinal tract (Schmidt and Greenberg. 1935). Ca in serum has a biological half-life of 11.4 days (Draper. 1963). Due to the slow absorption rate and to the abilities of para— thyroid hormone to regulate serum Ca. few studies have demon- strated effects of low or high dietary Ca intakes on serum Ca. Only when rats were fed a diet containing less than 0.004% Ca was a marked depression of serum Ca noted (Boelter and Greenberg. 1941). Role of Fecal Endogenous Ca. The principle excretory routes of Ca are the feces and the urine. Ca which has been absorbed and reexcreted in the feces is called "fecal endogenous calcium." Regulation of endogenous excretion of Ca could play a significant role in the adaptation process. Fecal endogenous Ca may arise from gut wall secre— tions (exsorption) or digestive juices or both. Exsorption may represent secretion from gut wall glands or leakage of Ca from gut epithelial cells. but apparently it does not represent secretion directly through the mucosa from the plasma (Cramer. 1963). Since only a small. invariant amount Of Ca passes through the mucosal cells into the gut. exsorp- tion is probably of minimal value in Ca homeostasis and hence in the adaptive process. 21 Ca secreted with the digestive juices comprises the bulk of fecal endogenous Ca. Gran (1960) reported that the amount of Ca secreted with the digestive juices was linearly correlated with the calculated ionized Ca in the plasma. In contrast. no increase in fecal endogenous Ca was found when Ca was injected directly into the veins of humans (McCance and Widdowson. 1939; Baylor §E_§l.. 1950). Blau §£_§l. (1957) Observed that the fecal endogenous Ca of two humans was independent Of the level of Ca intake. Hansard and Plumlee (1954) recorded fecal endogenous Ca values of 8. 23. 41. 50 mg/kg body weight/day for rats fed diets containing 0.013. 0.3. 0.5. and 1.0% dietary Ca respec- tively. Studies with rats (Hironka §t_§l.. 1960) demonstrated an increase in fecal endogenous Ca with age. Since increased dietary Ca and advancing age both reduce the amount of Ca absorbed. the possibility exists that these factors affect fecal endogenous Ca values not by directly altering the se- cretion rate of endogenous Ca buthy inhibiting reabsorption of the element. In this regard Bronner (1964) stated that there are no reports on agents that might cause an augmented loss of fecal endogenous Ca without acting on Ca absorption. Urinary Ca Excretion. Since Ca is secreted into the urine its regulation could play an essential role in 22 homeostasis and in an animal's response to diets with altered Ca levels. There is evidence to show that urinary Ca is related to Ca intake. Knapp (1947) in a statistical study involving (M36 humans. aged 1-80 years. derived a correlation between Ca intake and urinary Ca (r = +.634). From the results of tfliis survey he concluded that the quantity of urinary Ca was deqpendent upon an endogenous factor or factors and on Ca in- tjflce per unit body weight. Nicolaysen §£_§l. (1953) conducted a Ilong term study on 30 men consuming two levels of Ca. On iJitakes Of 900 mg Ca the urinary Ca value was 230 mg. When Iflie Ca intakes were halved. the urinary Ca dropped to 207 mg C61. When the individual variations were considered the author CCuicluded that the urinary Ca level was an individual constant. L’Ialm (1963). in a study with prisoners on rather constant Ca iJitakes. confirmed that a highly significant correlation e>OQ.EDHOHmO humumHQ DQOHHOO paw msoH>mHm mo muommmm 039 .m magma 48 The rats Offered the low Ca diet. however. were utilizing the available Ca much more efficiently than the rats fed the high Ca diet (Tables 9 and 10). Whereas the rats given the low Ca diet were ingesting only 25% as much Ca as the rats on the high Ca diet. they were retaining 40-60% as much Ca as the rats fed the high Ca diet. The greater utilization of Ca exhibited by the rats on the low Ca diet represents adaptation. Analyses of balance data during the experimental period revealed that Ca retention was governed primarily by the level of Ca in the current diet and secondarily by the Ca level in the previous diets. As shown in Table 10. groups receiving the high Ca diet during the experimental period (LH and HH) retained approximately twice as much Ca as groups receiving the low Ca diet during that period (LL and HL). thus illustrating the effect of the current intake of Ca on its retention. As was the case in the preliminary period. the groups currently receiving the high Ca diet (LH and HH) retained Ca with much less efficiency (Tables 9 and 10). The effect of the previous dietary Ca levels on cur- rent Ca retention can be demonstrated by comparing those groups which were fed the same Ca level during the experimental 49 .m OHQMB CH 66 0866 mCOHumcmemp DOOEHOOHBQ .mxmm3 mHImH mean How m. z .meOB HHIB 6006 How OH H z .HOHHH OOCOHOQ hmpIm m :0 ©0669 OHM mosam>m 6.6 + 6.6H 6.6 H 6.66 6.6H H 6.66 6.6 H 6.66 66H 6.6 H H.6H 6.6 H 6.66 6.6 H 6.66 6.6 H 6.66 6HH 6.6 H 6.66 6.6 H 6.66 6.6 H 6.66 6.6 H 6.HH 66H 6.6 H 6.Hm 6.6 H 6.66 6.6 H 6.66 6.6 H 6.66 66 66 gm 6A an 6.6 H 6.66 6.6 H 6.66 6H 6.6 H 6.Hm 6.H H 6.66 66 6.6 H 6.66 6.6 H H.66 66 I I I I I I I I I I I I IpmsHmumm mo *1 I I I I I I I I I I I I I I I 6%66 OOHHOm anm 30A OOCOHmm HO mchsHmOm £6 HOOEHOOHB um 0mm Amm + CMOEV OwsHmumm EDHOHOO HCOO men so mmé ps6 EOHOHOO SHODOHQ m0 6H0>0q HOOHHDO UGO msOH>OHm mo muommmm 038 .m OHQOB 50 Table 10. Summary of Calcium Balance Data Treatmentb Calcium Net CaC % Cad Intake Retention Retained ——————— “'mg Ca/rat/5 days-'—'—“—'—'—'— Preliminary Perioda Low 153.3 123.2 f 4.2 76.7 i 1.5 High 680.5 263.0 i 13.0 34.4 i 1.7 Experimental Periode LL 170.7 114.4 i 4.8 66.8 i 2.6 HL 166.5 89.6 i 5.2 53.7 i 2.8 LH 751.4 222.8 1 22.9 33.9 i 3.7 HH 801.3 197.6 1 20.6 24.1 i 1.8 aValues for this period are the means 1 SE Of 3 balance trials conducted at 48. 62. and 76 days of age (10 rats at each age). bTreatment designations are same as in Table 5. c High > Low at P < .01. LH and HH > LL and HL at P < .01. dLow > High at P < .01. LL and HL > LH and HH P < .01 by orthogonal contrasts. LL > HL P < .05 by orthogonal contrasts. LH > HH P < .05 by orthogonal contrasts. eValues for this period are the means 1 SE of 4 balance trials conducted at 90. 104. 118. and 132 days of age. 51 period but had been fed different levels of Ca during the preliminary period. In other words compare HL with LL and LH with HH in Tables 8 and 10. The LL group retained more Ca than the HL group between 90 and 132 days of age. and likewise the LH group retained more Ca than the HE group be- tween 90 and 118 days Of age. The differences in Ca reten- tion between groups currently being fed the same level Of Ca (LLvs HL and LH vs HH) was not as large as the difference between groups currently being fed different levels of Ca (HL and LL vs LH and HH). This finding demonstrated that Ca retention was affected more by the current dietary Ca level than by the previous dietary level of Ca. The greater Ca retention of the LL group when com- pared tO the HL group. and the LH group when compared to the HH group. was not due to differences in intake. as in the preliminary period. but was a factor of increased percent retention (Table 10). An increased per cent retention could be interpreted as an adaptive mechanism to an altered Ca in- take and reflects the ability of an animal to retain Ca in proportion to its need. The excretion data presented in Table 11 show clearly that most of the Ca was excreted via the feces. Therefore regulation of Ca retention occurred at the intestinal level. most likely by changes in absorption. 52 Table 11. The Effects of Previous and Current Levels of Dietary Calcium and Age on Calcium Excretion (Mean 1 SE) Age . Treatmenta 'Urine Cab Fecal Cab days mg mg 48 Low 59.1 i 5.2 7.9 i 1.6 62 Low 27.6 t 1.3 8.1 i 2.4 76 Low 9.7 i 0.7 7.1 i 1.7 48 High 46.4 i 4.9 380.2 i 29.5 62 High 22.5 i 1.7 411.7 t 10.6 76 High 20.3 i l 2 452.1 i 17.3 90 Low-Low 8.4 t 0.5 14.5 i 7.4 104 Low-Low 12.0 i 1.7 37.8 i 11.1 118 Low-Low 13.3 i 3.2 53.1 i 12.2 132 Low-Low 12.4 i 2.0 66.9 i 4.3 90 High-Low 5.3 i 2.9 53.2 i 5.2 104 High-Low 7.4 i 1.4 60.6 i 8.8 118 High-Low 10.1 i 0.7 68.0 i 10. 132 High—Low 9.6 i 2.3 93.7 i 8.6 90 Low—High 16.2 i 3.5 372.7 1 12.0 104 Low-High 19.6 i 3.8 474.9 1 33.8 118 Low-High 26.0 i 2.2 531.4 1 32.0 132 Low—High 14.5 i 3 8 631.7 1 45.3 90 High-High 19.7 i 4.5 517.8 1 11.0 104 High-High 16.7 i 4.3 535.6 i 17.8 118 High-High 24.1 i 5.7 630.4 1 25.6 132 High-High 20.8 i 4.6 649.7 1 36.7 a . . Treatment de51gnations same as for Table 5. bN N 10 for ages 48-76 days. 5 for ages 90-132 days. 53 Although rats on the high Ca diet excreted 2 times as much Ca via the urine as those on the low Ca diet. they excreted 10 times more Ca in the feces than did the rats on the low Ca diet. Even though urinary Ca values were affected by Ca intake. a finding which agreed with the results Of Rot- tensted (1938). Knapp (1947). Vermeulen (1959). and Malm (1963). they were not significantly important in the adap- tive process. As mentioned earlier. the amount of Ca in the tibias Of the rats fed the high Ca diet had attained a maximal value at the end of the preliminary period. whereas the tibias from rats on the low Ca diet did not reach a maximal value until between 133—156 days of age (Table 6). Conse- quently. the groups fed the low Ca diet during the prelimin— ary period did not have maximal Ca stores at the conclusion of the period. Nicolaysen (1943) suggested that Ca absorp- tion is regulated by a humoral factor from the osteoblasts which he has termed "endOgenous factor.“ This factor would be secreted during periods when Ca is being rapidly deposited in the bone. such as must have occurred for the LH and LL groups during the experimental period. since the tibias from these groups contained as much Ca as the other groups at the end of the experiment. According to Nicolaysen's hypothesis 54 the endogenous factor on the intestine to increase Ca absorp— tion. The greater extent of Ca absorption exhibited by the LL group when compared with the HL and by the LH group when compared with the HH group could be explained by Nicolaysen's hypothesis. These results. however. present no direct evi— dence for such a factor. Additional evidence that body stores of Ca control Ca absorption has been reported by Rot- tensted (1938) and Henry and Kon (1953). The manner in which the absorption of Ca could be increased at the intestinal level is Open to question. One possibility is that some stimulation resulting from the low Ca stores in the bone could induce a carrier protein which would serve to transfer more Ca across the intestine. Wasserman and Taylor (1963. 1966) have shown that vitamin D induces such a protein when 3 given orally to chicks. It has also been shown that Ca ab- sorption will not increase in response to a previous low Ca diet if the animal is deficient in vitamin D (Nicolaysen. 1953). The success of the adaptive response can be seen by again consulting Table 6 (page 44). where at the end Of 156 days of age the bones from all treatments contained equiva- lent amounts Of Ca. This same type Of reSponse to a low Ca diet. whereby animals retained Ca more efficiently Over a 55 longer period of time such that ultimately their bones con- tained as much Ca as rats fed a control diet. has been de- scribed by Sherman and Boohrer (1931). Lanford gt_§l. (1941). and Henry and Kon (1953). The rapidity of the adaptive reSponse can be seen by studying Table 8 (page 47). The largest differences in net retention among groups currently being fed the same diet occurred at 90 days of age or immediately after the change in diet. The differences became progressively smaller as age advanced. Such data may imply that the rats given the low Ca diet have Optimally conditioned their bodies to re— tain as much Ca as possible. whereas the group on the high Ca diet have not undergone such a conditioning process since their diet has always provided abundant amounts of Ca. Thus. when the group on the high Ca diet was suddenly switched to the low Ca diet. they simply could not make the necessary metabolic changes quickly enough to absorb Ca with the same efficiency of the preconditioned LL group. On the other hand. the reduced Ca absorption observed in the HL group should not be interpreted strictly as inefficient absorption. but rather a protective device against excessive Ca absorp- tion since this group has stored sufficient amounts Of Ca. 56 The retention of Ca decreased linearly with age in all treatments (Figure 2 and Table 8). Diminished Ca reten- tion as a result of advancing age has been previously demon- strated by other investigators as outlined in Table 1. Since age was positively correlated with body stores of Ca. this factor and not age itself was probably responsible for the reduced Ca retention at the later ages. On the basis of these data as well as that of the studies mentioned. the conclusion that submaximal body stores Of Ca were responsible for the increased retention and per cent utilization exhib- ited by the LL group seems unavoidable. Phosphorus Retention As shown in Table 12. no adaptation to the lower dietary P level was evident. During both the preliminary and the experimental periods. the amount of P retained was directly related to the amount ingested. Unlike Ca. the previous diet did not influence P retention or per cent utilization. Since both diets were adequate in P content. according to N.R.C. bulletin 990. there was little reason to expect adaptation to these levels of P. Although between 104-132 days of age significantly less P was found in the 96 Ca RETAINED 45 35 30 57 [IIFT] IIUUII {‘1 l’ l I | I’ I I II ] l l l I 90 I04 I I8 I32 AGE - DAYS Fig. 2. Effect of age 0n 1 Ca retained. Each point represents seen of 20 rats (5 from each of the 4 treatments). Response was significantly linear at the .05 level. 58 Table 12. Summary of Phosphorus Balance Data Period Treat- P In- P Re- ., %.P ment take tained Retained Preliminarya Low 298.5 97.3 i 6.2 32.0 i 1.8 Preliminary High 689.5 201.8 f 12.6 28.7 i 1.4 Experimentalb LL 269.1 70.8 i 6.7 26.2 i 2.6 Experimental HL 262.6 70.0 i 9.2 26.5 i 3.3 Experimental LH 702.2 163.8 f 22.0 22.6 i 2.6 Experimental HH 754.1 180.4 1 25.2 23.1.: 2.6 aValues for this period are the means t SE of 3 balance trials conducted at 48. 62. and 76 days of age (10 rats at each age). bValues for this period are the means 1 SE of 4 balance trials conducted at 90. 104. 118. and 132 days of age (5 rats at each age). bones of the rats which had received the low Ca and P diet (LL and LH) rather than the high Ca and P diet (HL and HH) during the preliminary period (Table 7). the LL and LH groups did not retain more P nor utilize the element any more efficiently than the HL and HH groups (Table 12). These results supported the hypothesis that P absorption was not controlled by P stores and was solely dependent on the level of P ingested. 59 Urine serves as an important excretory route for P as shown in Table 13. Urinary phosphate excretion equalled or exceeded fecal phOSphate excretion in those groups receiv- ing the low Ca diet. Current levels of dietary P were re- flected by the amount Of P excreted in the urine. The effect of the previous treatment on urinary P excretion during the experimental period was not as great as that of the current diet but may be of importance when discussed in relation to parathyroid activity. This discussion will be delayed until the parathyroid gland is considered. Serum Calcium and PhOSphate The analyses of serum Ca values revealed three note- worthy findings.. They appear in Table 14 and are: 1) serum Ca increased significantly with age. 2) rats fed the low Ca diet during the preliminary period maintained higher serum Ca values during the experimental period than rats fed the high Ca diet during the preliminary period (LL vs LH and HL vs HH). and 3) the current Ca intake had no effect on the serum Ca level (LL vs LH and HL vs HH).» The level of serum Ca is generally considered to be independent of age though few controlled studies have been reported to test this 60 Table 13. Phosphorus Excretion Data Age Treatmenta Urinary Pb Fecal Pb days - - - -mg/rat/5 days- - — - 48 Low 187.8 48.0 62 Low 139.0 42.2 76 Low 135.8 49.1 48 High 196.2 294.9 62 High 174.6 304.8 76 High 176.9 315.6 90 Low-Low 128.0 55.2 104 Low-Low 127.6 .75.3 118 Low-Low 124.0 76.2 132 Low-Low 118.0 89.2 90 High-Low 76.4 76.8 104 High-Low 106.8 83.4 118 High-Low 128.8 92.7 132 High-Low 103.6 100.5 90 Low-High 169.6 288.0 104 Low—High 176.2 334.7 118 Low-High 204.4 373.8 132 Low-High 185.6 421.4 90 High-High 161.2 346.9 104 High-High 138.4 374.7 118 High-High 189.2 436.1 132 High-High 190.8 457.5 aTreatment designations are same as Table 5. bN = N 10 for ages 48-76 days. 5 for ages 90—132 days. 61 6n HO>OH mo. um HCOHOMMHO hausmOHchmHm mum .m manna mm 0866 msoHumstmOp uswEummueo .mocmHHm> mo mHmNHmCm 6:608 OODQOHOBCO >9 HO>0H mo. um HOOHOHHHU hausmOHMHsmHm 0H6 musOEummnu HCOHOHHHO How mwsam> EOHOHOO EOHOm Q .OOOOHH6> mo mHmhamcm 62608 UODSOHOBCS 60mm HOOHOHMHU How mmsHm> ESHOHOO EOHOmm A66 66.6 H 66.6 A66 66.6 H 66.6 A66 6H.o H 66.6 166 6H.o H 6.6H 666 A66 6o.H H 66.6 A66 66.H H 66.6 AOHV 66.6 H 6H.6 A6HV 66.6 H 66.6 66H A66 66.6 H 66.6 A66 66.6 H 66.6 A66 66.6 H 66.6 A66 66.6 H 6H.6 66H A66 HH.6 H 66.6 A66 66.6 H 66.6 A66 66.6 H 66.6 A66 6H.o H 66.6 6HH A66 H6.o H 66.6 A66 6H.o H 66.6 A66 66.6 H 66.6 A66 6H.o H 6.6 66H A66 66.6 H 66.6 A66 HH.o H 66.6 A66 66.6 H 66.6 166 H6.o H 6.6 66 I I I I I I I I I I I I IHE ooa\mu mEI I I I I I I I I I I I 6668 66 am 66 an Omfl on DCOEHOOHB Amm H smwev 6HO>OA SOHOHOO Esuwm so msuonmmozm paw ESHOHMO HO 6H0>OA SHMHOHQ HGOHHOO ps6 msoH>0Hm m0 uommmm .66 06968 \ 62 hypothesis. Widdowson and Dickerson (1964) reported that serum Ca increased with age in humans. Reasons that a pre- viously high Ca intake should depress the serum Ca level up to 156 days of age. or 75 days after the high Ca diet was last fed. remain unknown. Rats which were fed the same diet during the preliminary period but different diets during the experimental period (LL vs LH and HL vs HH) did not have dif- ferent serum Ca values (Table 14). Serum Pi values were generally unaffected by treat- ment. However. at 118 days of age the HL and HH groups had a higher serum Pi value than the other groups (Table 15). This difference was not evident at-104 days nor at 132 days and hence was probably not physiologically significant. serum Pi increased to a maximal value at 118-132 days and then decreased sharply in all treatments. Harrison (1941) reported that serum Pi decreased with age. Perhaps this de- crease was just becoming evident at 156 days of age. Unlike Ca. the concentration of P in the red blood cell is high. Consequently. varying degrees of hemolysis would distort the true serum Pi value. Although precautions were taken to avoid hemolysis. it did occur in practically every sample. Thus. the true significance of the serum Pi values reported herein cannot be ascertained. 63 .m magma mm 0866 6OOHumsmH60O OOOEHMOHBM 66.6 H 6H.6 66.6 H 6.6 66.6 + 66.6 H6.o H 66.6 66H 6o.H H 66.HH 66.H H 6.HH 6H.H H 66.6H 66.6 H 66.6 66H 6H.H H 66.6H 66.6 H 6.6H 66.6 H 66.6 6o.H H 6H.6 6HH 6H.H H 66.6 66.6 H 66.6 66.6 H 66.6 66.6 H H6.6 66H I I I I I I I I I I I I I I I HE ooa\mE I I I I I I I I I I I I I I I I 66 66 66 Ha mmd OHCOEHMOHB Amm H QMOEV 6H0>OA Oumnmmonm Esuwm so msuosmmonm pcm EDHOHMO mo 6HO>OA mHmHOHO HOOHHOO pcm msOH>OHm mo Hummwm .ma manms 64 Parathyroid Gland The role of parathormone. a hormone secreted by the parathyroid glands. in maintaining serum Ca levels is well established. Whether this hormone plays a dominant role in increasing Ca retention during long periods of Ca depriva- ‘tion is not firmly established. The results of Luce (1923). Ham §;_§l, (1940). Harrison and Fraser (1960). and Au and Raisz (1965) indicated that low intakes of Ca stimulated. parathyroid activity. Nordin (1960). however. concluded that low Ca diets did not affect parathyroid activity and that a bone-tissue fluid equilibrium was responsible for correcting small changes in serum Ca. The size and weight of the parathyroid gland pro- vided an estimate Of the activity of that gland (Harrison and Fraser. 1960). In this experiment parathyroid weights were generally heavier in those rats (LL and HL) which were fed the low Ca diet during the experimental period (Table 16). The variance among samples within a group was rather large (Table 16). The large variance was probably due to the lack of an extremely precise weighing technique and to individual differences arising in part from differences in body weight. When some of this variation was removed by 65 Table 16. Effect of Previous and Current Level of Dietary Calcium on Fresh Parathyroid Gland Weight (mean t SE) Treat- Age ment 118 days - -156 days ‘ "238 days _____________ Hg - _ _ _ _ _ _ _ _ _ _ _ LL 123.3 6 5.6(3) 154.7 ¢ 22.1(10) 169.9 i 15.1(5) HL 111.0 1 15.1(3) 157.0 : 13.3(7) 107.5 f 16.1(5) LH 81.5 i 5.4(3) 137.1 1 22.6(8) 130.8 f 17.9(5) HH 104.5 1 10Y8(3) 138.1 1 22.7(3) 141.7 6 21.9(6) aTreatment designations same as for Table 5. b . Values in parentheses represent number of rats. expressing the parathyroid gland weights as ug of parathy- roid tissue (air—dried) per gram of body weight. a procedure which is necessary according to Stott and Smith (1964). and weights were pooled over all ages. significant differences due to treatment did occur (Table 17). As expected. the LL group possessed the highest parathyroid gland weight: body weight ratio (Ptg/Bw) which was significantly larger than that Of all other groups. The Ptg/Bw of the HL group was significantly greater than that of the LH group indicating that the present diet of the HL group was sufficiently low in Ca to stimulate parathyroid gland size. Since the 66 Ptg/Bw ratio of the LL group was significantly larger than that of the HL group. the previous diet was affecting the weight and presumably the activity Of the parathyroid glands of these rats. The greater parathyroid weight of the LL group contrasted to the HL group can most likely be attributed to a longer exposure to the low Ca diet. Whereas the LL group had been fed a diet poor in Ca for a 60-day pre- liminary period plus a 75—day experimental period. the HL group was fed the low Ca diet only during the last 75 days of the experiment. Table 17. The Effect Of Previous and Current Levels of Dietary Calcium on Parathyroid Gland Weight (Hg/9 bOdY wt.) E Treatmenta N Gland Weight LL 21 68.7b HL 15 62.7C HH 12 57.0Cd LH 16 53.0d a . . . . . Treatment deSignations same as in Table 5. d . . . . . bc Values With the same superscript are not Significantly different by Duncan's multiple range test. 67 The failure for the Ptg/Bw ratios Of the HE and LH groups to differ probably was because the high Ca diet fed during the experimental period repressed the parathyroids of both groups regardless of previous intake. whereas the low Ca diet being given the other 2 groups (LL and HL) was stim- ulating parathyroid activity. Although previous workers have demonstrated a correlation between parathyroid activity and weight of the gland. one must realize that the size of tale-3;. '\ an endocrine gland can provide only a crude estimate of its activity. The classical effects of parathormone are hyperphos- phaturia. hypophosphatemia and hypercalcitemia. Examination of these parameters provided further indirect evidence for parathyroid activity. These parameters were affected by the current level of Ca and P intake. so only comparisons between groups which were fed the same dietary levels of these ele— ments had any meaning with regard to parathyroid activity. Reexamination of Table 13 (page 59) indicated that the LL group excreted more P in the urine than did the HL group. The serum Ca level of the LL group were also significantly higher than in the HL group (Table 14). Also the LH group maintained a higher serum Ca value than the HH group. al- though parathyroid gland weights did not indicate any 68 differences between these two treatments. Rats fed a low Ca diet during both periods (LL) had a lower serum Pi value than rats fed a high Ca diet followed by a low Ca diet (HL). Likewise. rats fed a low Ca diet during the preliminary period but a high Ca diet during the experimental period (LH) possessed a lower serum Pi value than rats fed a high Ca diet (HH) during both periods (Table 15). The parathy- roids of the LL group appeared more active than those of the HL group since the LL group had a larger Ptg/Bw ratio. ex- creted more Pi via the urine. and maintained higher serum Ca and lower serum Pi values than the HL group. The parathy- roids Of the LH group were not heavier than those of the HH group. Thus. on the basis Of the parameters studied. one can only conclude that these groups (HL and HH) did not dif- fer in parathyroid activity. The levels of Ca and Pi in the serum and urine were probably affected in these groups by factors other than parathormone. The finding reported herein that a previously fed low Ca diet could stimulate parathyroid activity when ani- mals were exposed to a Ca stress (continued low Ca diet). may aid in explaining the mechanism whereby parturient pare— sis is prevented by the prepartum feeding of a low Ca diet. In light Of these results a prepartum low Ca diet could 69 indeed condition the parathyroid gland such that it would be more active at a later date. As reported in this study. animals preconditioned to a low Ca intake retained more Ca than animals previously accustomed to an abundant intake of the element. If the prepartum low Ca diets fed to cows' evoked the same response. the increased retention of Ca could present enough Ca to the plasma to prevent the onset of parturient paresis. One of the more important actions Of parathormone in the bovine is to stimulate Ca absorption (Payne et al.. 1965). Consequently. a more active parathy- roid at the time of parturition could prevent Ca levels from decreasing to the extent that brings about parturient pare- sis. However. in this study the greater Ca retention of the LL group was better explained not by an increase in parathy- roid action per se. but by a controlling factor from the bone which stimulated Ca absorption when bone stores Of Ca were not completely saturated. Although the parathyroid gland may participate in this response. Gran (1960) has dem- onstrated that parathyroidectomized rats were as capable of responding to a previously fed low Ca diet as intact rats. Consequently. low Ca diets fed prepartum could prevent par- turient paresis by the following sequence of events: 1) pre- partum low Ca intake decreases total retention of the element. 70 2) serum Ca level is maintained by bone resorption under the influence of parathormone (or by tissue—bone-fluid equilib- rium). 3) continued resporption of Ca from bone leaves a skeleton depleted in Ca. 4) reduced bone Ca triggers an in- testinal mechanism to absorb more Ca. 5) increased Ca ab- sorption allows the animal to meet the severe Ca stress at parturition. preventing the onset of parturient paresis. The validity of the preceeding hypothesis is dependent upon the degree to which the body stores of Ca are affected by a prepartum low Ca diet. and whether bone stores of Ca control its absorption in the bovine to the same extent as they do it in the rat. Furthermore the studies reported herein con- cerned a growing animal and not a pregnant one. The hypothe- sis needs to be tested further by feeding animals with known dietary histories a low Ca diet prepartum and evaluating the effects of such a diet on Ca absorption. bone Ca. and para- thyroid activity at the time Of parturition. SUMMARY AND CONCLUSIONS Rats adapted to a low Ca diet by increasing the per- centage of dietary Ca retained. Likewise. rats adapted to a high Ca diet by decreasing the percentage of the element re- tained. The changes in retention were largely due to changes in fecal Ca. indicating that the primary mechanism for con- serving or rejecting dietary Ca was Operating at the intes- tinal level. The percentage of dietary Ca retained was de- pendent on the past~as well as the present Ca intake. Rats accustomed to frugal intakes of Ca (LL) retained a larger percentage of the dietary Ca than rats accustomed to abund- ant amounts of the element (HL) even though both groups were ingesting the same low Ca diet. Similarly. rats previously acclimated to a low Ca diet (LH) retained more Ca than rats previously exposed to a high Ca diet (HH) when both groups were being fed the same high Ca diet. Although «w the percentage Of dietary Ca retained was increased in the groups being fed the low Ca diet (LL and HL). the total amount of Ca retained by these groups did not equal that of the groups currently ingesting the high Ca diet (LH and HH). 71 72 Groups accustomed to low Ca intakes (LL and LH) de- posited less Ca in their bones than rats which had been used to high Ca intakes (HL and HH) up to 133 days of age. The greater retention efficiency of the LL group when compared to the HL group and the LH group when compared to the HH group can be explained if diminished bone Ca stimulated Ca absorption through the intestine as proposed by Nicolaysen (1943). Furthermore. Ca absorption decreased linearly with advancing age in all groups. Since age is positively corre— lated with bones stores of Ca. Nicolaysen's theory of bone Ca regulating absorption would also explain this result. The adaptive reSponses to both the low and high Ca diets were successful. because after 156 days (conclusion of the experiment) the bones of all groups contained equal amounts of Ca and P. Thus. rats accustomed to a Ca poor diet deposited Ca in their skeletons at a slower rate but for a longer period of time than rats which had been accli- mated to a Ca rich diet. The amount of P retained appeared to be directly re— lated to the amount of the element ingested and was not in- fluenced by previous diets. Rats which were fed the low Ca diet throughout the entire experiment (LL) gained significantly less weight than 73 rats (LH and HH) which were given the high Ca diet during the experimental period. The groups did not differ in over- all appearance and vitality. Serum Ca values did not reflect the Ca status of the rats. In fact. the LL and LH groups maintained a higher serum Ca level than the HL and HH groups even though the skeletons of the former groups did not contain as much Ca or P as those Of the latter. Serum Ca values increased with advancing age in all groups. The experimental diets (pre- vious or current) did not consistently affect the amount Of phOSphate found in the serum. Parathyoid activity. as judged by parathyroid gland weight. serum calcium and phosphate values. and urinary phosphate. was greater in those groups fed the low Ca diet (LL and HL). than in those fed the high Ca diet (LH and HH). Furthermore. the group which was fed the low Ca diet through- out the entire experiment (LL) exhibited more parathyroid activity than the group (HL) which had received the high Ca diet prior to the experimental period. This finding supported the belief that increased parathyroid activity was at least partially responsible for the increased retention efficiency of Ca Observed in the LL group. 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