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Role of vitamin D₃ metabolites in calcium adaptation by rats Miller, David Alexander 1978

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ROLE OF VITAMIN D3 METABOLITES IN CALCIUM ADAPTATION BY RATS DAVID ALEXANDER MILLER B.Sc, University of British Columbia, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHYSIOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1978 <© David Alexander Miller, 1978 In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thesis for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of PHYSIOLOGY The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date JANUARY i 1978 i . ABSTRACT An in vivo assay was used to measure Ca-45 absorption from rat intestine in rats which had been adapted to diets which were either high or deficient in calcium. Results demonstrated that vitamin D was required for adaptation which was found to be strongest in the duodenum. The metabolites of vitamin D3, i .e. 25-OH D3, 24,25(0H)2D3 and 1,24,25(0H)3D3 all showed some adaptation response when supplied as dietary supplements; however, 1,25(0H)2D3 caused an increased Ca-45 absorption which was independent of dietary calcium intake and was not abolished by nephrectomy. Adaptation is defined as an altered efficiency of calcium absorption in response to a change in dietary calcium concentration. These data suggest that the controlled synthesis of 1,25(0H)2D3 is the mechanism which governs the intestinal adaptive response to calcium. Based on these data, an adaptive index was constructed to show each metabolite's effect on adaptation; with 1,25(0H)2D3 causing a 6% calcium uptake within 10 minutes from the duodenum into the blood. A low phosphorus diet was also shown to regulate calcium adaptation, presumably via synthesis of 1,25(0H)2D3, and this synthesis was shown to be independent of parathyroid hormone stimulation. Research Supervisor i i . TABLE OF CONTENTS Historical Introduction to the Vitamin D Metabolites 1. Introduction to Calcium Adaptation 4. Procedure Preparation and Diet of Normal Rats 10. Surgical Methods for Calcium Uptake 10. Determination of Calcium Secretion 12. Method of Analysis 13. Preparation and Surgery for Nephrectomized Rats 13. Preparation and Surgery for Thyroidparathyroidectomized Rats 15. Results Normal Rats 17. Nephrectomized Rats 25. Thyroidparathyroidectomized Rats 28. Discussion 29. Conclusions 42. Bibliography 44. Appendix 53. LIST OF TABLES Table I Composition of Diet Mix 11. Table II Daily Supplement Doses 14. Table III Indices of Adaptation at 10-Minute Ca-45 Uptake (Duodenum) 37. IV. LIST OF FIGURES Figure 1, la. The Effect of Vitamin D Metabolites on Body and Bone Ash Weight. 18, 18a, Figure 2. Comparison of the Effects of Vitamin D Deficiency and Supplementation on Plasma Ca-45 from Li gated Gut Loops. 20. Figure 3. Comparison of the Effects of 25-0H D3, 24,25(0H)2D3 and 1,24,25(0H)3D3 on Duodenal Calcium Uptake. 22. Figure 4. Comparison of the Effects of 1,25(0H)2D3 Administration on Duodenal Ca-45 Transport. 23. Figure 5. Effect of Nephrectomy on Duodenal Calcium Uptake. 26. Figure 6. Effect of Dietary Phosphorus Levels on Duodenal Calcium Uptake in Thyroidparathyroidectomized Rats. 27. V. ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. Cramer, for the original research proposal and his constant help in planning the procedure. His constructive criticism in the data analysis allowed a greater under-standing of the subject, and his encouragement proved quite useful. Secondly, thanks is expressed to Dr. Uskokovic of Hoffmann-La Roche Inc. (Nutley, N.J.) for kindly supplying the vitamin D metabolites used in this thesis and to Mr. K. Henze for preparing and photographing the graphs. Finally, special thanks is given to my father for proof-reading the thesis and to Miss R. Reid whose co-operation in preparing the manuscript was greatly appreciated. 1. Historical Introduction to the Vitamin D Metabolites Several excellent reviews on the development of vitamin D exist in the literature: DeLuca^8, Kodicek 4\ Norman63, therefore only a summary will be given to introduce the metabolites used in this thesis. Vitamin D was discovered by Mellanby57 in 1919 as a fat soluble vitamin which could cure rickets. The role of sunlight in preventing this disease was f irst recognized by Huldshinsky3* and related to the chemical conversion of 7-dehydrocholesterol to vitamin D via ultra violet irradiation of the skin by Windau et a l . 1 0 2 . Its role in promoting active calcium absorption from the intestine was introduced by Schacter and Rosen80, Wassermann et a l . 9 8 , and Kimberg et a l . 3 8 . Originally i t was assumed that vitamin D acted without chemical ft! conversion to produce its biological response, Schenck . This concept was reinforced by Kodicek*0, who used radioactive vitamin D3, and found no metabolites or degradation products which were biologically active. Kodicek's results were due to a crude preparation, which had low specific activity and precluded experiments in a physiological range. Purified radioactive vitamin D3 was f irst used by Neville and DeLuca59 and DeLuca et a l . 2 0 which allowed physiological tracing and revealed the presence of other polar metabolites. The f irst metabolite to be discovered was 25-hydroxycholecalciferol (25-0H D3) by Blunt et a l . * . It was found to be forty percent more potent than the parent vitamin D3 in promoting calcium absorption from the intestine and anti-rachitic properties. Its biological action was noted to occur within 6 hours after its administration as compared with the lag time of 10-12 hours for vitamin D3. Radioactive vitamin D3 was shown to accumulate in the liver 60 minutes after intravenous injection, 2. Ponchon and DeLuca"; and here i t is converted to 25-OH D3; a process which has been shown to require nicotinamide adenine dinucleotide; reduced, (NADPH), molecular oxygen and is believed to occur at the microsomal or endoplasmic reticulum site, Bhattacharyya and DeLuca3. The next observation was that there was a loss of H 3 at the C-l position of 25-OH D3 in rachitic animals, Lawson et a l . 4 ^. The resulting product was isolated from Intestine by Holick et a l . 3 3 and the structure was elucidated by Semmler et a l . 8 2 as 1,25-dihydroxycholecalciferol (1 ,25(0H)2D3). This metabolite was found to be 10-15 times as potent as vitamin D3 and 100 times as active as 25-OH in stimulating cultured bone calcium resorption. Doses of 0.5-1.0 ng./gm. tissue were adequate to produce an increased intestinal absorption of calcium, and the response occurred within 4 hours after injection. This metabolite is now believed to represent the active form of vitamin D3 at its biological sites in bone, intestine and kidney. The interest in l,25(0H)2D.j was increased when i t was discovered by Fraser and Kodicek24 that this metabolite was produced solely in the kidney (this was before the structure of 1,25(0H)2D3 was confirmed) and that nephrectomy abolished the conversion of 25-0H D3 to 1,25(0H)2D3, Gray et a l . 3 0 . These nephrectomized animals showed no intestinal calcium transport response to 25-OH D3 or bone calcium mobilization, and i t was concluded that 25-0H D3 must be converted to 1,25(0H)2D3 or a further metabolite before i t is metabolically active. The renal 1-hydroxylase system requires NADPH, ferridoxin, ferridoxin reductase and cytochrome P-450, and i t is believed to react at the renal mitochondrial site, Ghazarian and DeLuca29. These discoveries raised the level of vitamin D from a vitamin to a vitamin-hormone system, because 1,25(0H),D, had a 3. specific tissue for production, and was the metabolically active form which acted at the target tissues of intestine and bone. During the period 1968-1972, several other polar metabolites were isolated by the high resolution chromatographic methods developed by Suda et a l . 8 5 and Holick 3 2. Two of these metabolites which were used 1n the present research are 24,25-dihydroxycholecalciferol (24,25(0H)2D3) and 1,24,25-trihydroxycholecalciferol (1,24,25(0H)3D3). 24,25(0H)2D3 was originally identified as 21,25(0H)2D3 by Suda et a l . 8 5 and subsequently reidentified as 24,25(0H)2D3 by Holick 3 2. This metabolite has been shown to enhance calcium transport in the intestine, but shows no effect on bone calcium mobilization, Boyle et a l . 8 , Henry et a l . 3 ^. The 25-0H-D3-24-hydroxylase system has been found in kidney, Maclntyre et a l . 5 ^ and probably exists in extra renal sites where ft accounts for 25 percent of 1,24,25(0H)3D3 production, DeLuca^9. The requirement for this enzyme is that the substrate be a vitamin D molecule with a hydroxyl moiety on carbon 25. This allows the conversion of 25-OH D3 to 24,25(0H)2D3 or 1,25(0H)2D3 to 1,24,25(0H)3D3, Kleiner-Bossaller and DeLuca39. Like the 25-0H-D3-l-hydroxylase, the 24-hydroxylase is also found in renal mitochondria but differs in its sensitivity, because i t is not inhibited by carbon monoxide or cytochrome P-450 inhibitors. Both of these metabolites are only 50 percent as potent as vitamin D3 in stimulating intestinal calcium absorption 1n the chick and neither is active in stimulating bone calcium mobilization, DeLuca^8. 4. Introduction to Calcium Absorption Adaptation, as outlined in this thesis, 1s defined as the ability of the body to increase or decrease the absorption, utilization, and secretion of a nutrient as determined by the dietary level and metabolic need of that nutrient. Specifically, this thesis concentrates on the ability of the small intestine to increase or decrease the percent of Ca-45 uptake from a gut site into the blood, and the factors which regulate this adaptation. The ability of the body to adapt to a low calcium diet; i.e. increasing calcium absorption, was f i rst observed by Fairbanks and Mitchell 2 2 and Rottenstein7 7. The f i rst major study of this phenomenon was performed by Nicolaysen 6 0 ' 6 2 . He utilized both in vivo and in vitro studies on rat duodenal-jejunal loops to test various factors on calcium adaptation. It was concluded from these experiments that the adaptation to low calcium diets occurred within 10 days in growing rats, but was not observed to the same extent in rats over two years of age, Kane36. If the rats were maintained on a minimal calcium diet, the increased absorption was continued into adulthood. Vitamin D was required for this adaptation to occur in the amount of 20 I.U./day, and this adaptation could compensate for other dietary inhibitors of calcium absorption, e.g. phytates, Walker and Inrig 9 3 , fatty acids, Nicolaysen6*, and oxalic acid, Lovelace et a l . 4 9 . This adaptation was not abolished by selectively removing the thymus, adrenals or gonads, but could be reduced by parenteral injections of calcium. From these data, Nicolaysen stated that there was an inverse' relationship between the mineralization of bone and the efficiency of calcium absorption. On this basis, he proposed that undermineralized bone secreted a hormone called "the 5. endogenous factor" whose presence or absence determined the adaptation response at the intestinal and renal level. He also noted that vitamin D was the prime regulatory factor, without which the endogenous factor loses practically all of its effect. Kimberg et a l . 3 8 showed that the whole intestine has adaptive properties, but the most pronounced effect was observed in vitro in the duodenum. This adaptation was due to changes in the active transport of calcium which could be blocked by 2-4-dinitrophenol. The kinetics of this study showed that active transport alone, and not simple diffusion into cellular compartments could account for adaptation. The process appeared as a two step sequence with an init ial rapid uptake followed by a slower step, both of which required the presence of 7Q vitamin D to operate at maximum capacity, Schacter et al. . These studies also demonstrated that hypophysectomized animals showed no difference in their ability to adapt to dietary calcium levels, which was confirmed by Krawitt et a l . 4 5 . The cellular components of this adaptation system in the intestine has been the area of intense research during the last decade. The site of adaptation which responds to vitamin D has been given to two cellular structures; the basal lateral membrane of intestinal cells, Urban and Schedl 9 2, Papworth and Patri ck 5 7 and the brush border, Martin and DeLuca54, Walling and Rothman97. The mechanism of calcium uptake at these sites is usually associated with a specific calcium binding protein discovered by Wassermann and Taylor 9 9 and calcium sensitive adenosine triphosphatase carriers (Ca-ATPase), Melancon and DeLuca56. The extent to which these two systems interact to produce the net calcium uptake into the 6. intestinal cell is s t i l l not clear at the present time. The level of calcium binding protein in the intestinal mucosa is the f irst macro-molecular constituent of the intestine associated with intestinal calcium transport shown to respond to changes in dietary calcium, Wassermann and Taylor 1 0 0 , Omdahl and Thornton66. This effect has been localized at the brush border and shown to be vitamin D dependent, Taylor and Wassermann88. The Ca-ATPase activity, while related to vitamin D, has not shown the same degree of adaptation to low calcium diets, Taylor and Wassermann89, but this is disputed by Patrick 6 8. The brush border has also been cited as the location for increased calcium uptake following dietary calcium restriction in isolated duodenal cells, and operates independent of these other components of the transport system, Krawitt and Katagiri 4 3, Krawitt 4 2, Spencer et a l . 8 The extent to which the calcium controlling hormones, calcitonin and parathyroid hormone (PTH), influence this adaptation has been the source of much controversy in the literature. Calcitonin has generally been considered to act independent of the PTH system, with its target tissue being bone. No direct action of calcitonin has been demonstrated to increase or decrease calcium absorption by any part of the intestine, Cramer16, Corradino1 4, Lorenc et a l . 4 8 . Its role in adaptation is therefore considered to be minimal or restricted to returning hyper-calcemia states to normal via bone calcium uptake. The action of PTH is less defined with regard to its effect on intestinal absorption. Kimberg et a l . 3 8 stated that thyroidparathyroidectomized animals (TPTX) showed no statistical difference in their ability to absorb calcium in vitro. This was supported by Samrnon et a l . 7 8 , Ahlgren and Larsson1 using in vivo techniques, Kemm37, Shah and Draper8 3, who actually showed 7. a marked inhibition of calcium absorption in TPTX rats. All of these authors agree that the action of PTH is connected with bone calcium mobilization to correct hypocalcemic conditions. During adaptation to a low calcium diet, i t would appear that more pro-PTH is converted to PTH in the parathyroid gland; therefore, the level of control by calcium ions in this system is at the intracellular turnover and degradation of the prohormone, Chu et al J 3 . The discovery of the vitamin D metabolites has given new impetus to the research on calcium adaptation and provides a model which allows the other data to be consolidated. Boyle et a l . 5 demonstrated that animals fed a low calcium diet, increased production of 1 ,25(0H)2D3 which accumulated in the intestine, while a high calcium diet repressed synthesis of 1,25(0H)2D3 and stimulated the synthesis of 24,25(0H)2D3. This implied that the kidney was monitoring dietary and skeletal levels of calcium via serum levels with the control point at 9.6 mg./lOO ml. This discovery led to the idea that the vitamin D metabolites, and their regulation, could represent Nicolaysen's "endogenous factor", Boyle et a l . 6 , Omdahl and DeLuca65, Malm52. The current status of calcium homeostasis and the adaptation phenomenon can be summarized into the following system. Serum calcium levels are maintained at approximately 10 mg./lOO ml. If this level should rise, calcitonin production is stimulated to cause bone mineralization. The rise in serum calcium also stimulates production of 1,25(0H)2D3, and has been shown to exert an inhibitory effect on the secretion of PTH, Care et a l . 1 0 , 1 1 . When calcium levels fal l below this set point value, PTH is secreted. PTH acts directly on bone in a synergistic fashion with 1,25(0H)9D~, Garabedian et a l . 2 8 to cause bone 8. calcium mobilization and elevation of serum calcium. The production of 1,25(0H)2D3 acts directly on intestine to increase calcium absorption and is believed to mediate this response by stimulation of calcium binding protein production at the brush border, Freund and Bronner25, but not at the mitochondrial site, Krawitt et a l . 4 4 A feedback system exists for l,25(0H) 2 n 3, so that when 1,25(0H)2D3 accumulates, the synthesis of 24,25(0H)2D3 is activated, Tanaka et a l . 8 7 and presumably the existing 1,25(0H)2D3 is converted to 1,24,25(0H)3D3. The last major area of regulation is the possible role of PTH in stimulating the production of 1,25(0H)2D3. In contrast to the literature already cited, there are several reports which state that PTH is the prime regulator of the production of 1,25(0H)2D3 in the kidney. Garabedian27 showed that parathyroidectomy decreased the synthesis of 1,25(0H)2D3, and that injection of PTH restored this ability. Garabedian et a l . 2 8 also demonstrated that the adaptation response was equal in the intestine when 1,25(0H)2D3 was supplied in both TPTX and control animals. This hypothesis would supply another feedback loop, in which l,25(0H) 2 n 3 would decrease secretion of PTH as shown by Chertow et a l J 2 . The opposite effect, however, has also been demonstrated, Oldham et a l . 5 * , Dominguez21 and therefore no firm conclusion can be stated. To test the hypothesis that 1,25(0H)2D3 was the prime regulator of the adaptation response, Ribovitch and DeLuca74 proposed that i f an animal was supplied with an exogenous source of 1,25(0H)2D3, then this control point could be bypassed, and no difference in calcium absorption should be observed between high and low dietary calcium intake. Their paper showed that this proposal was valid for calcium adaptation, but 9. had no effect on phosphorus adaptation. The reason for this could be that 1,25(0H)2D3 needs to be further metabolized into a product which acts to control phosphorus absorption, Rlbovitch and DeLuca76. Ribovitch and DeLuca75 have also demonstrated that PTH 1s required in this adaptation process and acts via the stimulation of 1,25(0H)2D3. It was these last set of papers which provided the experimental approach adapted for this thesis. Ribovitch and DeLuca's work is based 80 upon an in vitro assay of Schacter and Rosen as modified by Martin and DeLuca55. This method uses an everted intestinal sac preparation, and measures radioactive calcium fluxes which are expressed as a serosal/mucosal ratio. While this method is convenient, because experimental variables are readily controlled in the bathing solutions, i t could mask interrelationships which exist within the Intact animal. The purpose of this thesis was therefore to util ize an in vivo method which could examine some of the Ribovitch and DeLuca hypothesis of adaptation and provide an independent index of adaptation which would show the effects of any regulatory factors under in vivo conditions. 10. Procedure Preparation and Diet of Normal Rats Male Sprague Dawley weanling rats (3 weeks) were weighed and randomly housed 1n individual free-hanging wire cages. Cages were placed in a windowless room which had yellow incandescent lighting on a twelve hour on-off cycle. All rats init ial ly were placed on a low calcium (0.2%), vitamin D deficient diet as shown 1n Table I. Rats were allowed to feed and drink ad libitum throughout the experimental period. After one week on this diet, rats were divided into groups of 12. Each group received a dally supplement of vitamin D or a given vitamin D metabolite 1n 0.1 ml. propylene glycol via stomach tube, or as intraperitoneal (i.p.) injection in 0.1 ml. ethanol, in doses shown 1n Table,II. Control rats received equal volumes of appropriate vehicle. Supplements were continued until 48 hours prior to surgery. Following two further weeks on these regimens, in vivo Ca-45 uptake was measured in 6 rats of each group. The remaining 6 rats In each group were then placed on a high calcium diet (2.0%) and the vitamin supplements were continued for a further 10 days, when Ca-45 uptake was measured as before. Surgical Methods for Calcium Uptake Rats were weighed and anesthetized with sodium pentobarbital (30 mg./Kg.) by i.p. injection and maintained with the same anesthetic as required. A midline laparotomy was performed and the duodenum or ileum identified. A 4 mm. Incision was made in the antlmesenteric border at the proximal end of the duodenum (or terminal ileum) and a polyethylene cannula (2 mm. I.D., 3 him. O.D.) was inserted Into the lumen and Hgated with 3-0 silk. Flanges at the end of the cannula prevented leakage. 11. Table I  Composition of Diet Mix (per 1 Kg. of diet) Starch 620.5 g Casein 200 g Alpha-Cell (Cellulose) 37 g Vegetable oil + 2000 I.U. Vitamin A 80 g Trace Mix 10 g Vitamin Mix 10 g NaCI 10 g KC1 15 g MgS04„ • 7H20 3 g Na2HP04 13.5 g Choline,Chloride 1 g Trace Mix (per 1 Kg. mix) Fe Citrate 100 g CuS04 • 5H20 13 g MnS04 • 4H20 4 g ZnS04 • 7H20 2 g CoCl2 • 6H20 1 g Kl 1 g Starch 879 g Vitamin Mix (per 1 Kg. mix) Thiamine HC1 1.0 g Riboflavin 1.0 g Alpha Tocopherol 1.0 g Folic Acid 1.0 g Pyridoxine 1.4 g Niacin 3.0 g Calcium Pantothenate 3.0 g I-Inositol 10.0 g Ascorbic Acid 20.0 g Vitamin B-j2 0.01 g Starch 958.59 g For Low Calcium Diet + 0.2% CaCl For Normal Calcium Diet + 1.2% CaCl For High Calcium Diet + 2.0% CaCl 12. At a distance of 5 cm. distal to this cannula, a second cannula was inserted to produce an isolated gut loop with an intact blood supply, and inflow and outflow cannulae. The body wall was closed around the gut loop. Lamps maintained body temperature at 38° C. Original gut contents were flushed out, using 10 ml. warmed isotonic saline followed by air. Each cannula was sealed with an inner plastic plug so that dead space was eliminated in the system, and the rat was allowed to equilibrate for 20 minutes. Plugs were then removed and the loop flushed out again with saline and air. Warmed isotonic Ca-45 solution (0.25 ml. with 20 micro-Ci./ml.) was injected into the loop and plugs replaced. Blood was sampled via the tail vein at 5, 10, 20, 30 and 40 minute intervals following Ca-45 injection, and collected into heparinized capillary tubes (4 per sample). At the conclusion of the experiment, the rat was sacrificed with an overdose of sodium pentobarbital and the tibia bone of the left hind leg removed as an index of bone retention. The remaining Ca-45 solution in the gut loop was then collected and prepared for liquid scintillation counting. Determination of Calcium Secretion Rats were prepared for calcium studies as previously described; however, only isotonic saline was placed in the lumen of the ileal or duodenal loop. The rats were then given an i.p. injection of isotonic Ca-45 (q.25 ml. with 20 micro-Ci./ml.) and after either 10 or 40 minutes (group 1 and 2), the gut contents were flushed out with air. The radioactivity in this solution represented blood to gut net secretion. The gut loop was dissected free and prepared for ashing. 13; Method of Analysis Capillary tube blood samples were capped (Critocaps: Sherwood Medical Industries Inc., St. Louis, Mo.) and centrifuged for 10 minutes (Micro-Capillary Centrifuge: International Equipment Co., Needham Heights, Mass.). Plasma was then collected from the four samples into a 50 microliter pipette (Becton Dickinson Co., Parsippany, N.J.) and pipetted onto number one Whatman chromatography paper, cut in 8 x 4 cm. strips and folded into a corrugated pattern. This was dried under a heat lamp and placed into a scintillation vial containing 10 ml. fluor-toluene solution. The radioactivity was measured in a liquid scintillation counter (Beckman LS-233) with background automatically subtracted. Efficiency error of each sample was corrected by use of a suitable quench series and B/A channels-ratio method. The tibia bones and gut loops were cleansed of adhering tissue, defatted in a 2:1 chloroform, methanol solvent and dried for 24 hours at 104° C. Samples were then ashed in a muffle furnace at 550° C. for 24 hours. Samples were weighed and dissolved in 4 ml. 3 M. HC1, and 100 microliters prepared for liquid scintillation counting. Plasma radioactivity is expressed as percent of dose of Ca-45 init ia l ly placed in the gut loop per ml. plasma (x ± SEM). Secretion data are expressed as percent injected dose appearing in the lumen per ml. gut contents (isotonic saline). Bone ash data and net weight gain of the rats are expressed as g. per ng. metabolite used in the supplement. Preparation and Surgery for Nephrectomized Rats Rats chosen to undergo bilateral nephrectomy (NX), were housed and 14. Table II  Daily Supplement Doses Dose of Vi tami n Group Supplement and Administration or Metabolite/rat control 1 P.G. ; oral -control 2 Ethanol ; i.p. -1 P.G. + Vitamin D3 ; oral 625.0 ng. 2 P.G. + 25-OH D3 ; oral 25.0 ng. 3 P.G. + 24,25(0H)2D3 ; oral 25.0 ng. 4 P.G. + 1,24,25(0H)3D3 ; oral 25.0 ng. 5 P.G. + 1,25(0H)2D3 ; oral 12.5 ng. 6 Ethanol + 1,25(0H)2D3 ; i.p. 12.5 ng. NX P.G. + Vitamin D3 ; oral 250.0 ng. TPTX P.G. + Vitamin D3 ; oral 250.0 ng. P.G. = Propylene Glycol NX = Nephrectomized TPTX = Thyroidparathyroidectomized prepared on the similar diet as shown in Table I, and supplemented as shown in Table II, on the same schedule as normal rats until 48 hours prior to surgery. Rats were anesthetized as previously described and a midline laparotomy performed. Each kidney was identified and the renal vessels and ureter were ligated with 4-0 silk soaked in chlorhexidine diacetate (Hibitane: Ayerst, Montreal) as a gut antiseptic. Both kidneys were then dissected free and removed. The abdominal muscle layers and skin were sutured together separately using 3-0 silk with both continuous 15. and reinforcing interrupted sutures. An i.p. injection of either 12.5 ng. 1,25(0H)2D3 or 25.0 ng. 25-0H D3 in 0.1 ml. propylene glycol was administered as a pulse dose, with control rats receiving only the vehicle (propylene glycol). The rat was allowed to recover for 10 hours, and supplied with isotonic saline to drink. At the end of this period, the rat was reanesthetized with sodium pentobarbital, sutures removed, and Ca-45 uptake from duodenum was performed as previously outlined; however, the tibia was not removed. Preparation and Surgery on Thyroidparathyroidectomized Rats Rats of the same strain and age as normal rats were randomly divided into two groups, and housed in individual cages as previously described. Both groups were placed on a normal calcium (1.2%) diet; however, one group's diet was phosphorus deficient (0.01%), while the other group's phosphorus content was sufficient, as outlined in Table I (0.3%). Both groups received a daily supplement of vitamin D3 as shown in Table II. After 3 weeks on this schedule, a blood sample from each rat was collected via tail vein and serum phosphorus was analysed in an atomic absorption flame emission spectrophotometer (Jarre!-Ash J.A. 82-270 #280). All rats then underwent TPTX. Surgery was performed using sodium pentobarbital as anesthetic, blunt dissection removal of the thyroid and parathyroid glands, and the wound was closed with interrupted 3-0 silk sutures. All rats received 5 micro g. L-thyroxine/100 g. body weight/day i.p. (Nutritional Biochemical Co., Cleveland, Ohio) for the remainder of the experiment. The effectiveness of the TPTX was tested 48 hours after surgery by measuring serum calcium, by atomic absorption, in the presence of 0.1% LaCl 3. A value of serum 16. calcium<7.0 mg./lOO ml. was considered a successful TPTX rat, and all rats with higher serum calcium were not included 1n the data. All rats then received 1.0% calcium gluconate in the drinking water for the following 2 days, after which they were given normal water. Half the rats of each group were then placed on a low calcium (0.2%) diet, while the other half received a high calcium (2.0%) diet. Phosphorus content of these diets, and vitamin D3 supplements for both groups were identical to the schedule given prior to surgery. After 10 days on these diets, each rat was given an i.p. pulse dose of 25 ng. 25-OH D in 0.1 ml. propylene glycol, 24 hours prior to surgery. Plasma Ca-45 uptake from Iigated duodenal loops was then measured in all rats. All statistical analysis was calculated using the Student T test with the mean and standard error of the mean expressed (x + SEM). In a l l graphs which show percent Ca-45 in plasma as a function of time, i t i s assumed that these values r e f l e c t a method of measuring i n te s t ina l absorption. Although the plasma calcium level i s a measurement of a l l factors which e f fec t calcium metabolism including release of calcium by bone and excretion by kidney, i t i s s t i l l assumed to be a va l id ind icat ion of absorption. This i s because the maximum amount of plasma Ca-45 occurs at 10 minutes which i s too rapid for these other factors to influence th i s level and the analysis of the Ca-45 leaving the gut lumen (appendix) shows corre lat ion with the Ca-45 appearing on the serosal side for both the low and high calcium d ie t s . 17. Results Normal Rats Fig. 1 shows the effect of vitamin D3 metabolites on both bone ash and body weight. In each case the values for rats fed high calcium diet were greater, though the differences are not statistically significant. Each metabolite produced a significant body weight increase in groups fed diets either low or high in calcium (p<0.05 for 24,25(0H)2D3 -p<0.001 for 1,25(0H)2D3 as an i.p. dose). There was no statistically significant difference (p>0.05) between the body weight increases of 24,25(0H)2D3 and 1,24,25(0H)3D3. Body weight in those receiving 25-OH D3 was significantly greater (p<0.02) than those receiving 24,25(0H)2D3, but showed no significant difference over those receiving 1,24,25(0H)3D3 (p>0.05). Body weight of rats administered 1,25(0H)2D3 was significantly greater (p<0.01 - p<0.05) than that of rats receiving the other metabolites, and 1,25(0H)2D3 as in i.p. dose was significantly greater (p<0.05) than the oral dose of the same metabolite. Fig. 1 shows the effect of these metabolites on bone ash weight. There was no statistical difference (p>0.05) between the bone ash of control rats and that of rats treated with 24,25(0H)2D3 or 1,24,25(0H)3D3. Tibia ash weight of rats receiving 25-OH D3 was significantly increased (p<0.05) over ash weights of either the previous two metabolites or the control. Ash of 1,25(0H)2D3, whether administered i.p. or orally, showed a significant increase (p<0.001 - p<0.02) compared to bone ash of rats treated with all other metabolites, and the i.p. dose also had greater effect than the equivalent oral dose (p<0.05) on bone ash. Fig. 2 shows the effect of vitamin D3 on intestinal Ca-45 uptake. Part A of the graph shows the control rat results which received no .18. Effect of Vitamin D Metabolites on Bone and Body Weight CO < c o CD E 6.0 -t 4.0 H 2.0 H 0.0 I I Low Ca Diet H High " o o * ' v — , — ' * — „ — ' >. J 24,25 1,24,25 25 1.25 1.25 (0H) 2 (0H) 3 OH (0H) 2 (0H) 2 D 3 D 3 D 3 Oral I.P 03 a a> \— o c 6.0 n 4.0 H f 2.0 0.0 Fig. 1 The Effect of Vitamin D Metabolites on Body and Bone Ash Weight The low calcium diet values are calculated for the 3 week assay period on 0.2% calcium. High calcium diet (2.0%) values are calculated for the following 10 days. Values are x + SEM with N=6. Control rats received no supplement and other rats received the metabolite and dose shown in Table II. The upper half shows bone ash in mg. per ng. metabolite. The lower half shows % body weight increase per ng. metabolite. 18a. Effect of Vitamin D Metabolites on Bone and Body Weight. n Low Ca Diet • High Fig. la The Effect of Vitamin D Metabolites on Body and Bone Ash Weight This figure shows the original data of Figure 1 before bone or body weight increases were expressed per ng metabolite. All parameters are therefore identical with Figure 1 with x + SEM. 19. vitamin D supplement. Neither duodenal nor ileal values show any significant difference (p>0.05) between low and high calcium diets. Duodenum transferred Ca-45 at significantly greater rates (p<0.01) than ileum. The time course of the duodenal uptake of calcium is apparently maximum at 10-15 minutes; conversely, the uptake of calcium from the ileum is slower, with maximum values at 25-35 minutes. Part B of the figure illustrates the effect of vitamin D3 supplementation on plasma Ca-45. Those values after instillation of Ca-45 in ileum showed no significant difference (p>0.05) whether rats were fed low or high calcium diets. The duodenal Ca-45 transfer showed the adaptation response, because there was a significant difference (p<0.01) between the low and high calcium diet effects at all time intervals. The duodenal effects are all significantly greater than the corresponding ileal effects, with the duodenal low calcium diet causing the greatest difference (p-^0.01 for high diet and p<0.001 for low diet). The time course for both duodenum and ileum Ca-45 transfer into plasma is similar to the description given in Part A, with the maximum uptake from duodenum at 10-15 minutes, and 25-35 minutes for ileum. When Parts A and B of the figure are compared, i t is noted that there is no difference (p>0.05) between ileal rates in either vitamin D deficient (control) or vitamin D metabolite supplemented rats. There is also no difference (p>0.05) between duodenal rates for rats fed the high calcium diet compared to rats fed a low diet. Between the duodenal values for the low calcium diet, there is a significant difference (p<0.05) for all time intervals. Fig. 3 shows the effect of 25-0H D 3 > 24,25(0H)2D3 and 1,24,25(0H)3D3 on duodenal Ca-45 transfer. All three metabolites caused a significantly I 20. j Effects of Vitamin D x H i gh " o a. = B. Supplementation ifi 4.0-\ Fig. 2 Comparison of the Effects of Vitamin D Deficiency and Supple- mentation on Plasma Ca-45 from Ligated Gut Loops. Doses for the vitamin D supplementation are shown in Table II. Graph shows the percent of init ial dose of Ca-45 appearing in plasma after uptake from proximal duodenal or terminal ileal gut loops, x + SEM with N=6. Duodenal and ileal values reflect low and high calcium with no vitamin D supplement. Ileal values for low and high calcium diets with vitamin D supplements are plotted on the same line with no statistical difference (p>0.05). 21. greater duodenal Ca-45 transport between low and high calcium diets (p<0.02 for 25-OH D3, p<0.05 for 24,25(0H)2D3 and p<0.05 for the 10, 20, 30 minute intervals with 1,24,25(0H)3D3). No statistical significance is apparent (p>0.05) between the high calcium diet values of plasma Ca-45 for all three metabolites, or vitamin D3 supplemented duodenal high calcium values of Fig. 1. 25-OH D3 supplemented low calcium diet values are significantly higher than control or vitamin D supplemented values of Fig. 1 (p-^O.Ol - p<0.02), and significantly greater than 24,25(0H)2D3 (p<0.001); no difference was observed with 1,24,25(0H)3D3 (p>0.05). 1,24,25(0H)3D3 low calcium values showed a significant increase (p<0.02) over control values of Fig. 1 and 24,25(0H)2D3 low calcium values. It is noted that rats fed low calcium diet which were administered 24,25(0H)2D3 took up significantly less Ca-45 from duodenal loops than from duodenum of rats fed high calcium diet and administered the same metabolite (p<0.05). This 1s the only metabolite studied in which this effect was observed. With the exception of low calcium diet values for 24,25(0H)2D3, all three metabolites showed values significantly greater than corresponding ileal values (see Fig. 1). The time course of transfer for all three metabolites is consistent with values obtained for duodenum in Fig. 1. Fig. 4 shows the effect of 1,25(0H)2D3 supplements on duodenal calcium uptake. Neither i.p. nor oral vitamin D metabolite supple-mentation resulted in any significant difference between plasma Ca-45 of low and high calcium regimens (p>0.05). Intraperitoneal admini-stration of 1,25(0H)2D3 caused significantly greater plasma Ca-45 uptake (p<0.001 - p<0.02), than any administration of other metabolites tested (both low and high calcium regimens), and was significantly more 22. 5.0 4.0 4.0 • 1,24,25 (0H)3 D 3 Supplemented 1 1 1 1— 10 20 30 Time in Minutes 24,25(0H) 2 D 3 Supplemented 1 1 1 1 -1 40 Fig. 3 Comparison of the Effects of 25-OH D 3 > 24,25(0H)2D3 and  1t24,25(0H)3D3 on Duodenal Calcium Uptake. Doses for the supplementation shown in Table II. Parameters of the graph are identical with Graph 2 with x + SEM and N-6. 23. Time in Minutes Fig. 4 Comparison of the Effects of 1,25(0^^3 Administration on  Duodenal Ca-45 Transport. I.P. or oral administration (doses shown in Table II). Parameters are identical with Fig. 2 with x + SEM and N=6. 24. effective (p<:0.05) when administered i.p. than when administered orally. The oral supplement, low calcium values, while larger, were not significantly greater (p>0.05) than 25-OH D3 pr the f i rst two time values of 1 ,24,25(0H)3D3 (all other metabolites were significantly lower). The high calcium diet values for both i.p. and oral doses of 1,25(0H)2D3 were significantly higher than all other metabolites (p<0.01 - p<0.02). The time course for 1,25(0H)2D3 is the same as those described for the previous duodenal values. The control rats for the i.p. injections, which received only ethanol as the vehicle substance as shown in Table II, were not shown in Figs. 1 or 4 because in all categories (i.e. body weight increase, bone ash weight increase and calcium uptake from duodenum on low calcium diets), there was no statistical difference (p>0.05) from control rats which received the vehicle substance propylene glycol as an oral dose. The control values given can therefore apply to either method of administration. Ca-45 uptake into tibia bone over the 40 minute total assay period was 0.3% dose Ca-45/mg. bone + 0.15% mg. (% Ca-45 is percent of init ial dose placed in the gut loop lumen). This measurement did not show any statistical variation between vitamin D3 or vitamin D metabolite supplemented groups when Ca-45 was placed in either duodenal or ileal regions. Endogenous secretion of Ca-45 into ligated gut loops showed that 1-2% of the i.p. administered dose of Ca-45 appeared 1n the lumen at 40 minutes, with most of the secretion occurring within the f irst 10 minutes. A further 3% dose + 0.8% was recovered in the ashed gut loops, with an insignificantly larger (p>0.05) fraction found in the ileal tissues. 25. Nephrectomized Rats Fig. 5 shows the effect of nephrectomy (NX) on calcium uptake from the duodenum 10 hours after metabolite administration. The graph of control rats demonstrates statistically significant difference (p<0.05) between plasma Ca-45 uptake of rats fed high and low calcium diets for all time intervals. The values for the low calcium diet were significantly lower (p<0.05) than duodenal vitamin D3 treated low calcium diet values in Fig. 2 for the 20, 30 and 40 minute interval; however, all other values for both low and high calcium diets demonstrated no difference from Fig. 2. 25-OH D3 treated rats (i.e. rats receiving vitamin D3 administrations plus one pulse dose of 25-OH D3 10 hours prior to surgery) showed significantly higher results (p<0.05) for low calcium diet compared with the high calcium diet for all time intervals. There was no significant difference (p>0.05) between 25-OH D3 treated and control values for any time interval on either diet. Comparison of data from 25-OH D3 treated rats in Fig. 3 and comparable values in Fig. 5 show that there was a significant decrease in duodenal calcium transport (p<0.001 for low calcium diet and p<0.05 for high calcium diet) in nephrectomized rats. Plasma Ca-45 from 1,25(0H)2D3 treated rats showed no significant difference (p>0.05) whether fed high or low calcium diets. All values were significantly greater (p<0.01) than either control or 25-OH D3 treated rats. A comparison with values obtained in Fig. 4 demonstrated that while the NX rats showed lower values than i.p. treated rats and higher values than the oral treated rats, neither of these differences was statistically significant (p>0.05) at most intervals, with the exception of the 10 and 20 minute values in the i.p. supplemented low calcium diet where p<0.05. 26. A.O-i Time in Minutes F1g. 5 Effect of Nephrectomy on Duodenal Calcium Uptake. All rats received a daily vitamin D3 supplement until 48 hours prior to nephrectomy (Table II). Immediately following nephrectomy, a pulse dose of the indicated metabolite 1n doses outlined in the text, was administered i.p. Control rats received the vehicle propylene glycol. All parameters are identical to those of Fig. 2 with x + SEM and N=6. 27. 6.0 n Time in Minutes Fig. 6 Effect of Dietary Phosphorus Levels on Duodenal Calcium Uptake  in Thyroidparathyroidectomized Rats. All rats received a daily vitamin D3 supplement until 48 hours prior to surgery when a pulse dose of 25-OH D3 (25 ng./rat) was administered i.p. Both low and high calcium values are shown on the same line with no statistical difference (p>0.05). All parameters are identical with Graph 2 with x + SEM and N=6. 28. Thyroidparathyroidectomized (TPTX) Rats Plasma phosphorus and calcium of rats represented in Fig. 6 were analysed after 3 weeks on their respective diets (i.e. before rats were TPTX). Results were as follows: rats on 0.3% phosphorus diet— 6.8 mg./lOO ml. + 0.3 plasma phosphorus and 10.5 mg./lOO ml. + 0.1 plasma calcium. Rats on 0.01% phosphorus diet—2.1 mg./lOO ml. + 0.2 plasma phosphorus and 13.5 mg./lOO ml. + 0.4 plasma calcium (all values N=10). Fig. 6 shows the effect of the phosphorus content in the diet on duodenal calcium absorption in TPTX rats. Neither the 0.01% or the 0.3% phosphorus diet results demonstrated a significant difference (p>0.05) between high and low calcium diet plasma Ca-45 values. There was a significant increase (p<0.01) of both the high and low calcium diet plasma Ca-45 uptake by rats fed phosphate deficient diet. The 0.3% (adequate) phosphorus diet promoted plasma Ca-45 values similar to the values obtained from duodenal in a vitamin deficiency state in Fig. 2, with the exception of the 10 minute values which were greater in the TPTX rats (p<0.05). The 0.01% phosphorus diet values of plasma Ca-45 coincided with duodenal values obtained in l^fOH^Dg oral supplemented rats of Fig. 4. 29. Discussion The purpose of this thesis was to develop an in vivo assay, based upon the work of Rlbovitch and DeLuca, which could be used to examine the calcium adaptation response and its regulation. The dosages of vitamin D3 and its metabolites were therefore selected from these authors* work on the basis of the vitamin's dose which would be physiologically effective 74 in producing an adaptation response. Ribovitch and DeLuca demonstrated that 625 ng. vitamin D^/rat/day was optimal in producing a prolonged effect which was maximum at 48 hours after administration. They also found that 25 ng. 25-OH D3/rat/day and 12.5 ng. 1,25(0H)2D3/rat/day were maximum at this same time interval. The 250 ng. dose of vitamin D3/rat/day as a supplement for NX and TPTX rats is used by most authors, and by Ribovitch and DeLuca75. Doses of 24,25(0H)2D3 and 1,24,25(0H)3D3 for adaptation were not found in the literature; therefore, doses were selected to equal (by weight) the 25-OH D3 supplement. Fig. 1 shows that the dosage of metabolites chosen produce significant growth and increase of bone ash as compared with vitamin D deficient controls. The metabolites produced a 70% gain above control (1,25(0H)2D3 i .p.), and in Fig. 1 is expressed per ng. of metabolite. Rats of this age, sex and strain should increase 100-,120% in body weight over a 3-4 week period on a normal diet. Nutritionalists use weight gain as a general index of a nutrient deficiency in the diet. Decreases in weight gain are attributed to a combination of the lack of metabolic reactions, specific to the deficient nutrient, and the condition of anorexia in the rat. From our data, i t is observed that all groups of rats treated by one of the vitamin D metabolites showed mean weight gain below normal as expected; probably due to the calcium deficiency. When rats were transferred to a high calcium diet for 10 days, the mean 30. weight gain increased in each case, and i t is possible that i f the experimental period were longer, these rats could return to normal weight gains. The administration of vitamin D metabolites served to minimize the effects of a calcium deficiency, as indicated by increasing weight gain over control values. 1,25(0H)2D3 increased body weight to the greatest extent and demonstrated its greater potency per ng. compared to the other metabolites. It is noted that 1,25(0H)2D3 as an i.p. dose produced significantly greater results than the oral dose of the same metabolite. Because both control values were identical, this difference cannot be due to the different vehicle substance used. It would therefore appear that 1,25(0H)2D3 is more potent as an i.p. dose in producing a biological response. This is a confirmation of the work by Omdahl65, who showed that 1,25(0H)2D3 as an I.p. dose was 1.5-2.0 times as effective as the oral administration method and recommended that this metabolite be used intravenously as a therapeutic agent. It might be expected that the potency of the oral administration would be Increased proportionately with dosage; however, the results for the same doses show the i.p. administration as the most effective. Omdahl suggested the reason for this decreased oral response was that either less of the metabolite was absorbed or was more rapidly metabolized at the brush border. The graph of the effect of the metabolites on bone ash weight shows an Index of the mineralization 1n the bones of these groups. In general, i t is observed that the shape of the graph corresponds to the percent weight increase graph. As previously Indicated, this shows the specific effect of the metabolite or nutrient deficiency on body weight; i.e. acting via bone mineralization. It is noted that both 31. 24,25(0H)2D3 and 1,24,25(0H)3D3 are the least effective 1n stimulating bone mineralization and do not show any significant increase over control rats. 25-OH D3 and 1,25(0H)2D3 supplements both show significant increases in bone mineralization and confirm their biological action on bone as outlined by DeLuca17. The results here demonstrate that an in vivo assay can be used to show the adaptation response and constitute the f irst presentation of this approach using vitamin D metabolites. The method of collecting the unabsorbed gut loop contents after 40 minutes provided a suitable method of detecting any error in the system. All rats from a particular group had a similar fraction of Ca-45 remaining 1n the lumen after 40 minutes. If any rat had produced data which were radically different from the average of the same group, i t would have indicated either a different uptake process, or more probable, a leakage around the cannula tube. This disappearance of Ca-45 from the lumen could be used as another index of absorption provided that care was taken to collect all contents and the gut loop itself was analysed for Ca-45. The analysis of the calcium secretion data confirmed that the results we obtained reflected changes in absorption data and that calcium secretion 1s a minor component of a bi-directional calcium flux. The secretion values show that over 40 minutes, a maximum of only 2.0 percent of an 1.p. dose was secreted into the gut loop. This indicates that while some of the Ca-45 absorbed from the lumen in our data could be secreted and re-absorbed, the amount being secreted 1s negligible compared to the percent uptake. Our uptake values obtained for the f irst 10 minutes are therefore considered to reflect the true absorption from the gut loop. Each graph shows a slight decrease in plasma Ca-45 32. as a function of time after the maximum value has been obtained. This probably represents the net result of a number of factors affecting the body calcium levels; i.e. uptake Into bone and soft tissue, a decreasing calcium concentration gradient from mucosal to serosal borders 1n the gut as more is absorbed and the possibility of increased secretion into the lumen. For these reasons, absorption was dominant for only approximately the f irst 10 minutes, when maximal plasma Ca-45 uptake was observed. The role of the secretion component of calcium movement in the intestine has been studied in relation to adaptation. Walling and Kimberg 9 4 » 9 5 have shown that active secretion of calcium occurs throughout the intestine, and may reflect dietary calcium levels. Their in vitro studies demonstrated that the ileum is the preferential site of intestinal secretion, and while our data showed a higher secretion Into this region, no statistical difference from the duodenum rates was observed. The uptake of Ca-45 from the gut loop into tibia bone during the 40 minute assay period did not produce any significant difference between groups and individual results were quite variable. It is assumed that our method of assay is too short to reflect a significant bone uptake of calcium, and hence bone ash data which represents a long period of calcium uptake are presented for discussion of the Interaction of vitamin D3 on bone tissue and absorption. Fig. 2 demonstrates that no adaptation response is observed when vitamin D3 is absent. This confirms the work of Nicolaysen62 who showed that a minimal dose of vitamin D3 was required, and Ribovitch and DeLuca74 who demonstrated that at least 250 ng. vitamin D3/rat/day was 33. needed to produce adaptation. When vitamin D3 treatment was used in Part B, the duodenum shows the adaptation response with a difference occurring between low and high calcium diet values. The Ileum in both graphs shows no adaptation and demonstrates a slower rate of absorption in comparison with the duodenum. The ileum has been shown to be quite variable in its ability to adapt to calcium absorption. The original data by Klmberg et a l . 3 8 showed that the ileum was second to the duodenum in its ability to adapt, although this was reversed in the golden hamster. Most of DeLuca's work has Indicated that the duodenum is the major site of the adaptive response and our data confirm these findings. This does not mean that the ileum does not have a role in adaptation. Petlth and Schedl 7 0 noted that both secretion and absorption of calcium change in the ileum during calcium restriction to allow a net Increase in calcium uptake, and that this property of adaptation continues Into the cecum and colon, Petlth and Sched l 6 9 , 7 1 . The absorption of calcium during adaptation appears to vary with the intestinal site. Calcium absorption in the duodenum in vivo appears to be Independent of the composition of the Intraluminal f luid; whereas, in the ileum, i t is Influenced by the concentration of sodium, actively transported sugars, and by the rates of net water absorption, Behar and Kerstein2. Urban and Schedl 9 1 showed that vitamin D3 does not affect the ileal absorption or secretion process, but the duodenum is dependent upon vitamin D3 for active transport of calcium. In summary, i t would appear that the ileal region is the site of a passive relatively constant calcium absorption. The ileum's ability to adapt is quite variable in normal physiological states depending on age, strain and diet of the animal, and can also 34. adapt under surgically induced resections, Teitelbaum et al. . Existence of this passive uptake could explain the slower rate of calcium absorption observed in Fig. 2 which is constant in both vitamin D deficient and supplemented rats compared to the rapid uptake in duodenum which is known to utilize more active transport mechanisms. Net absorption from the ileum has been shown by some workers to contribute more calcium to the body than the duodenal sites of absorption, Marcus and Lengemann53, Cramer15. Although its absorptive rate is slower, the lumen contents remain longer in the ileal region and assuming that the calcium exists in an absorbable form, the ileum would therefore constitute the site of a significant proportion of the calcium absorbed. Fig. 3 shows the effect of three metabolites of vitamin D3 on duodenal calcium absorption. It is observed that adaptation occurred in all three experiments. 25-OH D3 showed the greatest degree of adaptation, and reached a maximum net absorption of Ca-45 at 10 minutes which was significantly greater than the maximum rate in the vitamin D3 treated group. The effect of 24,25(0H)2D3 was unique in that i t appeared to significantly suppress Ca-45 absorption in rats fed a low calcium diet. This may suggest that 24,25(0H)2D3 is not merely a degradation product of 25-OH D3 but has a specific function. When serum calcium levels are low; i.e. as induced by a low calcium diet, 24,25(0H)2D3 levels in the body would be low. If this metabolite is given exogenously, the absorption 1s further decreased and this suggests that 24,25(0H)2D3 may actively suppress absorption by either a direct action, or by inhibiting 1,25(0H)2D3. This would imply that there was either a two-receptor site for these metabolites (inhibition by 24,25(0H)2D3 and stimulation by l,25(0H)2D-3) or a one-receptor site 35. for 1,25(0H)2D3 which is modulated by the presence of 24,25(0H)2D3. This effect is presumably due to the specific action of the 24-OH moiety because the 1,24,25(0H)3D3 did not produce these results. It is noted that 24,25(0H)2D3 can s t i l l stimulate duodenal calcium absorption in rats fed a low calcium diet at a rate which 1s comparable to ileal rates, but had no effect on bone mobilization of calcium as 31 shown by Graph 1; which has also been reported by Henry et al. and Miravet et a l . 5 8 . Both of these papers showed that 24,25(0H)2D3 decreased calcium absorption of rats on a normal diet, but actually increased calcium absorption of rats on a calcium deficient diet. These workers used an in vitro assay and this may explain the difference between their data and results presented In this paper; however, all laboratories concur that 24,25(0H)2D3 does not stimulate bone mobilization. The graph reflecting 1,24,25(0H)3D3 treatment showed adaptation at most of the time intervals studied and was similar in general outline to the 25-OH D3 treated group, except that plasma Ca-45 values for the 1,24,25(0H)3D3 low calcium diet group were consistently lower than the 25-OH D3 low calcium diet group. It is also observed that all graphs reflecting a high calcium diet are results which are statistically identical. If the theory of calcium adaptation via specific vitamin D3 metabolites is correct, then the most probable explanation is that when the rats were adapted to a high calcium diet, the predominant metabolites would be 24,25(0H)2D3 and 1,24,25(0H)3n3, compared to the production of 1,25(0H)2D3. These metabolites acting individually or synerglstically would therefore be present in all groups and be responsible for the particular absorption response to the high calcium diet. 36. -Fig. 4 shows that 1 ,25(0H) 2D 3 given either by i . p . or oral administration abolishes the adaptation response. These results indicate that th i s metabolite i s the control point of the system, and i t s presence allows maximum calcium absorption from the duodenum as shown by the values obtained in the i .p . treated group. The data from the oral treated group demonstrates that th i s metabolite i s less potent when administered by oral than by the i . p . route. Comparison with the 1 ,24,25(0H) 3D 3 treated rat plasma Ca-45 of F ig. 3 shows that the 1,24,25(0H) 3D 3 i s s i g n i f i c an t l y less potent than 1,25(0H) 2D 3 in st imulating in tes t ina l absorption and because adaptation occurred in the presence of 1 , 2 4 , 2 5 ( O H ) » th i s metabolite may well represent a degradation product of the 1 , 2 5 ( O H ) -The resu l t s of Figs. 2-4 are used to estimate an index r a t i o of adaptation for the metabolites in Table I I I. The 10 minute value for the low calcium diet group from each f igure was selected as the reference of adaptation because th i s represents the time interval of maximum plasma Ca-45 l e ve l s . The table shows the difference between the plasma Ca-45 leve l s from rats fed low and high calcium diets (low -high) and the maximum plasma Ca-45 level of the low calcium d ie t . Both of these values are then combined into an "absorption index value" which i s a r a t i o of the two parameters; i . e . r a t i o = Maximum %Ca-45 in plasma on low calcium d iet / the difference in plasma Ca-45 between the high and low d ie t s . Values which are high (>20) such as control (-D) and 1,25(0H) 2D 3 supplemented rats show the least adaptation; while D 3, 1 ,24,25(0H) 3D 3 and 25-OH D3 show low values and therefore demonstrate adaptation. The negative value for 24,25 (0H) ?D^ i s due to the ef fect 37. Table III Measurement of Adaptation from 10 Minute Ca-45 Plasma Values (Duodenum) Supplement Difference in % Ca-45 plasma leve l s between low and high calcium diets = A %Ca-45 plasma level on low calcium d iet = B Index ra t i o (B/A) Control (-D) 0.1 N.S. 2.1 + 0.3 21 .0 Vitamin 1.2 p< 0.01 3.4 + 0.3 2.8 1,24,25(0H)3D3 1.6 p<r 0.05 4.4 + 0.4 2.8 24,25(0H) 2D 3 1.6 p< 0;05 1.3 + 0.5 - 0.8 25-OH D 3 1.9 p< 0.02 5.1 + 0.4 2.7 1,25(0H) 2D 3 Oral 0.2 N.S. 5.3 + 0.5 27.0 1,25(0H) 2D 3 i . p . 0.3 N.S. 6.7 + 0.5 22.0 x + S.E.M. N = 6 of the low calcium d iet values being lower than the high calcium values as previously described. While th i s index shows presence or absence of adaptation, i t may not be s u f f i c i e n t l y accurate to allow a quant i tat ive discr iminat ion between the metabolites which show adaptation, i . e . D 3, 25-OH D 3, 24,25(0H) 2D 3 and 1,24,25(0H) 3D 3. To confirm that the adaptation in the duodenum was pr imar i ly due to the presence, or absence, of 1,25(0H) 2D 3, nephrectomies were performed to remove the s i t e of the 1-hydroxylase system. A l l rats had received a da i l y dose of vitamin D 3 unt i l 48 hours pr ior to surgery as outl ined in Table II. This schedule was selected to provide the minimal dose of vitamin D^  which would produce the adaptation response 38. and yet not interfere with the pulse dose of metabolite given after nephrectomy. Boyle et a l . 7 showed that no impairment of intestinal calcium transport occurred 12 hours after nephrectomy in response to 1,25(0H)2D3 stimulation, with the f irst signs of a decreased duodenal response in the uremic state occurring at 24 hours, Walling et a l . 9 6 . These effects have been confirmed in humans by Recker and Savi l le 7 3 who demonstrated that most of the abnormal calcium absorption that existed in advanced renal failure was associated with the proximal small intestine and was relatively independent of dietary calcium intake. 1,25(0H)2D3 administration was also found to improve calcium absorption under these conditions. Brickman et a l . 9 showed that the decrease in intestinal absorption only occurred in advanced renal failure and was not improved by dialysis which was consistent with the idea that this effect was more of a metabolic than excretory function per se. This effect has also been shown to be independent of chronic metabolic acidosis when this conition is art i f ic ial ly induced. Whether the same variables are present when acidosis is combined with renal failure is not known, Weber et a l . 1 0 1 . On the basis of this literature, our schedule of surgery which tested calcium uptake from the duodenum 10 hours after nephrectomy allows experiments to be conducted before major changes occur in calcium absorption. Nephrectomized rats lived 54+8 hours (N=6) and therefore data obtained after 10 hours should s t i l l reflect a physiological condition. Fig. 5 shows the effect of the pulse doses of metabolites given immediately following nephrectomy. Both control and 25-OH D3 rats show adaptation, and produced graphs which were statistically equal. With the exception of three points in the control graph, both graphs have 39. similar results to those obtained for vitamin D3 supplemented rats in Fig. 2. The significant decrease in low calcium diet values in 25-OH D3 supplemented rats is noted after nephrectomy as compared with Fig. 3. 1,25(0H)2D3 treated rats, while slightly lower, showed no similar statistical decrease compared with non-nephrectomized values. These results are consistent with the idea that the kidney is the sole site of 1-hydroxylation, and that the conversion of 25-OH D3 to 1,25(0H)2D3 is essential to produce the increased intestinal absorption. When this step is blocked by nephrectomy, the 25-OH D3 cannot be converted into 1,25(0H)2D3 and hence l i t t le adaptation occurs; whereas, 1,25(0H)2D3 supplementation can bypass this regulation point and produce a response which is independent of adaptation. This provides the f irst experimental basis of the adaptation theory due to the presence of a humoral agent in which nephrectomized results have been included. The small adaptation which occurred in 25-OH D3 rats is presumably due to residual vitamin D3 (and therefore 1,25(0H)2D3) which the rat received as a daily supplement until 48 hours prior to surgery. This is confirmed by the fact that control rats on vitamin D3 which received only a pulse dose of the vehicle after nephrectomy also showed the same level of adaptation. The other explanation is that 10 hours is insufficient time for 25-OH D3 to be converted to 1,25(0H)2D*3. All literature surveyed stated that 25-OH D produced a biological response within 4-6 hours and therefore i t is, assumed that some effect should have been observed at 10 hours. As a result of data obtained with and without nephrectomy, i t appears that 1,25(0H)2D3 is the prime regulator of calcium absorption in the duodenum. , The controversy which surrounds the role of parathyroid hormone (PTH) in adaptation already has been discussed in the introduction and 40. only literature which has a direct bearing on our data is presented here. Although Garabedian et a l . 2 7 demonstrated that TPTX rats on a low calcium diet were dependent on PTH to synthesize 1,25(0H)2D3, these results were questioned by Galante et a l . 2 6 . Both Maclntyre et a l . 5 0 and Larsson et a l . 4 6 suggested that Garabedian's results could be explained by assuming that operatively Induced hyperphosphatemia, rather than absence of parathyroid hormone per se, may have caused the decreased production of 1,25(0H)2D3. This is supported by the fact that glucose, which lowers serum phosphorus, reverses the Inhibitory effect of TPTX on l,25(0H) 2 n 3 production. This would suggest that low plasma phosphorus levels can Independently stimulate the synthesis of 1,25(0H)2D3 in the absence of PTH as shown by Tanaka and DeLuca86. To test this hypothesis, we designed an experiment using essentially the same rationale for studying TPTX rats as Ribovitch and DeLuca75. This method overcomes the high mortality rate due to tetany of TPTX rats which were previously on a low calcium diet as used by Favus et a l . 2 by adapting the rats after TPTX surgery and administering a calcium supplement 1n the drinking water for 2 days following surgery. The results 1n Fig. 6 indicate that the phosphorus content of the diet does affect the role of PTH in calcium adaptation. When plasma phosphorus levels are normal, or above normal, calcium adaptation is abolished in TPTX rats as shown by the 0.3% phosphorus graph. This supports the suggestion by Maclntyre et a l . 5 1 that PTH is required for 1,25(0H)2D3 synthesis. When PTH is absent, l i t t le or no conversion of the administered 25-OH D3 can occur, hence no increased absorption to a low calcium diet due to 1,25(0H)2D3 was observed. When plasma phosphorus levels are low, 1,25(0H)2D3 production is stimulated by an independent 41. pathway, and this production occurs without PTH and provides the Increased absorption response observed. This increased response was statistically equal to results previously obtained for an oral dose of 1,25(0H)2D3. When hypophosphatemia is the cause of 1,25(0H)2D3 production, in the absence of PTH, 1t would appear that there is no control of 1,25(0H)2D3 production via stimulation of high plasma calcium levels because no adaptation occurred on the high calcium diet values. If there had been sufficient time, i t would be interesting to conduct another experiment in which exogenous PTH is supplied to TPTX rats on a normal phosphorus diet, to observe 1f the increased absorption response with a low calcium diet could be restored. These results demonstrate that phosphorus 1s also an integral part of the 1,25(0H)2D3, PTH system in addition to calcium. Ribovitch and DeLuca75 drew certain conclusions which were opposite in part to those presented 1n this thesis. They stated that PTH was required for any adaptation via 1,25(0H)2D3 production and exogenous sources of the hormone counteracted the effect of TPTX surgery. We have shown that this is true for rats on an adequate phosphorus diet; however, a low phosphorus diet can stimulate 1,25(0H)2D3 production directly, independent of PTH, to produce an increased calcium absorption response, Maclntyre et a l . 5 1 . Although neither phosphorus diet produced its own adaptation response on high and low calcium diets, a significant adaptation did occur when results from the two phosphorus diets are compared. Ribovitch and DeLuca used only one dietary phosphate level and did not report plasma phosphate levels. Their assay is also in vitro and all of these factors indicate that their conclusions are limited by these experimental methods. 42. Conclusions We have shown that our in vivo assay can be used to examine the calcium adaptation response quantitatively. This assay was shown to reflect calcium absorption and demonstrated that calcium adaptation occurred preferentially in the duodenum as compared with the ileum; however, the net effect which this duodenum response contributes to the total amount of calcium absorbed is not known. The ileum showed a slower rate of absorption and this rate was not affected by vitamin D3. Vitamin D3 was required before any adaptation response was observed and both vitamin D3 and 25-OH D3 produced an adaptation response which could be decreased by nephrectomy. Both of these compounds produced a significant increase in body weight gain and bone ash weight increase over vitamin D deficient controls. 1,25(0H)2D3 produced the greatest response in body weight increase, bone ash weight increase and absolute calcium absorption. This metabolite is more effective as an i.p. dose compared with oral administration, but neither method produces the adaptation response either before or following nephrectomy. It is concluded from these data that the production.of 1,25(0H)2D3 is the prime factor in regulating calcium adaptation in duodenal absorption. If this control point is bypassed by supplying exogenous 1,25(0H)2D3, then the duodenum absorbs calcium at a constant rate independent of dietary calcium intake. Both 24,25(0H)2D3 and 1,24,25(0H)3D3 produced adaptation, but in all categories their responses were less than the parent compound (I.e. 25-OH D3 and 1,25(0H)2D3 respectively). While these data may suggest that 1,24,25(0H)3D3 represents a degradation product of 1,25(0H)2D3, i t also shows that 24,25(0H)2D3 may have an additional metabolic role. 43. On the basis of the 24,25(0H)2D3 decreased response to a low calcium diet, this metabolite may represent an active inhibitor of calcium absorption in the duodenum, either by its direct action on the intestinal ce l l , or by interacting with 1,25(0H)2D3 to decrease its potency. From data obtained after 10 minutes of absorption, i t was possible to quantify each metabolite's effect on adaptation. This "adaptation index" can be used as a general guide to determine 1f adaptation occurred but is not sufficiently accurate to discriminate between individual metabolites. The data obtained from TPTX rats indicates that parathyroid hormone is required for calcium adaptation when the diet has a normal or above normal phosphorus content (presumably via stimulation of 1,25(0H)2D3). When the phosphorus content is deficient, 1,25(0H)2D3 production can be stimulated in the absence of PTH to produce an increased calcium absorption. It is therefore concluded that the extent to which PTH affects calcium adaptation via 1,25(0H)2D3 production is modulated in part by the plasma phosphorus level. In summary, i t is concluded that the overall control of calcium absorption in the duodenum is under the regulation of the vitamin D metabolites. 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The graph shows the percent of i n i t i a l administered dose of Ca-45 not recovered from the isolated duodenal lumen at 40 minutes ( i . e . 100%-% flushed out of lumen). This i s a corre lat ion of absorption as measured from the serosal side in Figs. 3 and 4. There i s a s i gn i f i can t difference between the low and high calcium diet values (p<0.05) for a l l supplemented metabolites except 1,25 (0H) 2D 3 (p>0.05). Values are x + SEM with N=6. 


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