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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 p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y o f B r i t i s h the L i b r a r y s h a l l I  f u r t h e r agree  for  scholarly  make i t f r e e l y a v a i l a b l e  that permission  this  thesis  written  It  for financial  thesis  i s understood that copying o r p u b l i c a t i o n gain s h a l l  PHYSIOLOGY  The U n i v e r s i t y o f B r i t i s h  2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  that  f o r reference and study.  f o r e x t e n s i v e copying o f t h i s  permission.  Department o f  I agree  purposes may be granted by the Head of my Department or  by h i s r e p r e s e n t a t i v e s . of  Columbia,  JANUARY i 1978  Columbia  not be allowed without my  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 D , i . e . 25-OH D , 24,25(0H) D 3  3  2  3  and 1,24,25(0H) D all showed some adaptation response when supplied 3  3  as dietary supplements; however, 1,25(0H)2D caused an increased Ca-45 3  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) D is the mechanism which governs the intestinal 2  3  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)2D causing a 6% calcium uptake within 10 minutes from the 3  duodenum into the blood.  A low phosphorus diet was also shown to  regulate calcium adaptation, presumably via synthesis of 1,25(0H)2D , 3  and this synthesis was shown to be independent of parathyroid hormone stimulation.  Research Supervisor  ii.  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, l a .  The Effect of Vitamin D Metabolites on Body and Bone Ash Weight.  Figure 2.  18, 18a,  Comparison of the Effects of Vitamin D Deficiency and Supplementation on Plasma Ca-45 from Li gated Gut Loops.  Figure 3.  20.  Comparison of the Effects of 25-0H D ,  24,25(0H) D  3  2  3  and 1,24,25(0H) D on Duodenal Calcium Uptake. 3  Figure 4.  3  22.  Comparison of the Effects of 1,25(0H) D 2  3  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 understanding 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^ , Kodicek \ Norman , therefore only a summary 8  4  63  will be given to introduce the metabolites used in this thesis. Vitamin D was discovered by Mellanby vitamin which could cure rickets.  57  in 1919 as a fat soluble  The role of sunlight in preventing  this disease was f i r s t recognized by Huldshinsky * and related to the 3  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 Rosen , Wassermann et a l . 80  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* , who used radioactive vitamin D3, and found 0  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 D was f i r s t used by Neville and DeLuca  59  3  and DeLuca et a l .  2 0  which allowed physiological tracing and revealed  the presence of other polar metabolites. The f i r s t metabolite to be discovered was 25-hydroxycholecalciferol (25-0H D ) by Blunt et a l . * . 3  It was found to be forty percent more  potent than the parent vitamin D in promoting calcium absorption from 3  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 D was 3  shown to accumulate in the liver 60 minutes after intravenous injection,  2.  Ponchon and DeLuca"; and here i t is converted to 25-OH D ; a process 3  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 DeLuca . 3  The next observation was that there was a loss of H at the C-l 3  position of 25-OH D in rachitic animals, Lawson et a l . ^ . 4  3  product was isolated from Intestine by Holick et a l . was elucidated by Semmler et a l . (1 ,25(0H) D ). 2  3  8 2  The resulting  and the structure  3 3  as 1,25-dihydroxycholecalciferol  This metabolite was found to be 10-15 times as potent  as vitamin D and 100 times as active as 25-OH 3  cultured bone calcium resorption.  in stimulating  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 D  3  at its biological sites in bone, intestine and kidney. The interest in l,25(0H) D.j was increased when i t was discovered 2  by Fraser and Kodicek  24  that this metabolite was produced solely in the  kidney (this was before the structure of 1,25(0H) D was confirmed) and 2  3  that nephrectomy abolished the conversion of 25-0H D to 1,25(0H) D , 3  Gray et a l . . 3 0  2  3  These nephrectomized animals showed no intestinal calcium  transport response to 25-OH D or bone calcium mobilization, and i t was 3  concluded that 25-0H D must be converted to 1,25(0H) D or a further 3  2  metabolite before i t is metabolically active.  3  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 s i t e , Ghazarian and DeLuca . 29  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 .  Two of these metabolites which were used 1n  32  the present research are 24,25-dihydroxycholecalciferol (24,25(0H) D ) 2  and 1,24,25-trihydroxycholecalciferol (1,24,25(0H) D ). 3  24,25(0H) D  3  2  was originally identified as 21,25(0H) D by Suda et a l . 2  3  subsequently reidentified as 24,25(0H) D by Holick . 32  2  3  3  8 5  3  and  This metabolite  has been shown to enhance calcium transport in the intestine, but shows no effect on bone calcium mobilization, Boyle et a l . , Henry et a l . ^ . 8  3  The 25-0H-D -24-hydroxylase system has been found in kidney, Maclntyre 3  et a l . ^ and probably exists in extra renal sites where f t accounts for 5  25 percent of 1,24,25(0H) D production, DeLuca^ . 9  3  3  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 D to 3  24,25(0H) D or 1,25(0H) D to 1,24,25(0H) D , Kleiner-Bossaller and 2  DeLuca . 39  3  2  3  3  3  Like the 25-0H-D -l-hydroxylase, the 24-hydroxylase is also 3  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 D  3  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 r s t observed by Fairbanks and Mitchell  22  and Rottenstein .  The f i r s t major study of this phenomenon  77  was performed by N i c o l a y s e n ' . 60  He utilized both in vivo and in vitro  62  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, Kane . 36  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 I n r i g , fatty acids, Nicolaysen *, 93  and oxalic acid, Lovelace et a l .  4 9  .  6  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 i n i t i a l rapid uptake followed by a slower step, both of which required the presence of 7Q  vitamin D to operate at maximum capacity, Schacter et a l .  .  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 c e l l s , Urban and Schedl , Papworth and Patri c k 92  57  and the brush border, Martin and  DeLuca , Walling and Rothman . 54  97  The mechanism of calcium uptake at these sites is usually associated with a specific calcium binding protein discovered by Wassermann and Taylor  99  and calcium sensitive adenosine triphosphatase  carriers (Ca-ATPase), Melancon and DeLuca . 56  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 i r s t macromolecular constituent of the intestine associated with intestinal calcium transport shown to respond to changes in dietary calcium, Wassermann and T a y l o r  100  , Omdahl and Thornton . 66  This effect has been  localized at the brush border and shown to be vitamin D dependent, Taylor and Wassermann . 88  The Ca-ATPase activity, while related to  vitamin D, has not shown the same degree of adaptation to low calcium diets, Taylor and Wassermann , but this is disputed by Patrick . 89  68  The brush border has also been cited as the location for increased calcium uptake following dietary calcium restriction in isolated duodenal c e l l s , and operates independent of these other components of the transport system, Krawitt and K a t a g i r i , Krawitt , Spencer et a l . 43  42  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, Cramer , Corradino , Lorenc et a l . 16  14  4 8  .  Its role in adaptation is  therefore considered to be minimal or restricted to returning hypercalcemia 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 Larsson  1  using in vivo techniques, Kemm , Shah and Draper , who actually showed 37  83  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) D 2  3  which accumulated in the intestine, while a high calcium diet repressed synthesis of 1,25(0H) D and stimulated the synthesis of 24,25(0H) D . 2  3  2  3  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 . , Omdahl and DeLuca , Malm . 6  65  52  The current status of calcium homeostasis and the adaptation phenomenon can be summarized into the following system. levels are maintained at approximately 10 mg./lOO ml.  Serum calcium 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) D , and has been shown to exert an inhibitory effect on the 2  3  secretion of PTH, Care et a l .  1 0 , 1 1  .  When calcium levels f a l l below  this set point value, PTH is secreted.  PTH acts directly on bone in a  synergistic fashion with 1,25(0H) D~, Garabedian et a l . 9  2 8  to cause bone  8.  calcium mobilization and elevation of serum calcium.  The production  of 1,25(0H) D acts directly on intestine to increase calcium 2  3  absorption and is believed to mediate this response by stimulation of calcium binding protein production at the brush border, Freund and Bronner , but not at the mitochondrial site, Krawitt et a l . 25  feedback system exists for l,25(0H)  n 2  3  A  4 4  , so that when 1,25(0H) D 2  accumulates, the synthesis of 24,25(0H)2D  3  is activated, Tanaka et a l .  3  8 7  and presumably the existing 1,25(0H) D is converted to 1,24,25(0H) D . 2  3  3  3  The last major area of regulation is the possible role of PTH in stimulating the production of 1,25(0H) D . 2  In contrast to the  3  literature already cited, there are several reports which state that PTH is the prime regulator of the production of 1,25(0H) D in the 2  kidney.  Garabedian  27  3  showed that parathyroidectomy decreased the  synthesis of 1,25(0H) D , and that injection of PTH restored this 2  ability.  3  Garabedian et a l .  2 8  also demonstrated that the adaptation  response was equal in the intestine when 1,25(0H) D was supplied in 2  both TPTX and control animals.  This hypothesis would supply another  feedback loop, in which l,25(0H) as shown by Chertow et a l J . 2  3  n 2  3  would decrease secretion of PTH  The opposite effect, however, has also  been demonstrated, Oldham et a l . * , Dominguez 5  21  and therefore no firm  conclusion can be stated. To test the hypothesis that 1,25(0H) D was the prime regulator 2  3  of the adaptation response, Ribovitch and DeLuca  74  proposed that i f an  animal was supplied with an exogenous source of 1,25(0H) D , then this 2  3  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 DeLuca . 76  and DeLuca  75  Ribovitch  have also demonstrated that PTH 1s required in this  adaptation process and acts via the stimulation of 1,25(0H) D . 2  3  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 and DeLuca . 55  as modified by Martin  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 u t i l i z e 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 i n i t i a l l y 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. of 12.  After one week on this diet, rats were divided into groups 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., with 3-0 silk.  3 him. O.D.) was inserted Into the lumen and Hgated  Flanges at the end of the cannula prevented leakage.  11. Table I Composition of Diet Mix (per 1 Kg. of diet) Starch Casein Alpha-Cell (Cellulose) Vegetable o i l + 2000 I.U. Vitamin A Trace Mix Vitamin Mix NaCI KC1 MgS0 „ • 7H 0 4  2  Na HP0 2  620.5 200 37 80 10 10 10 15 3  g g g g g g g g g  13.5 g  4  Choline,Chloride  1  g  Fe Citrate CuS0 • 5H 0  100 13  g g  MnS0 • 4H 0  4  g  ZnS0  Trace Mix  4  4  CoCl  (per 1 Kg. mix)  2  2  4  • 7H 0  2  g  2  • 6H 0  1  g  1 879  g g  2  2  Kl Starch Vitamin Mix  (per 1 Kg. mix)  Thiamine HC1 Riboflavin Alpha Tocopherol Folic Acid Pyridoxine Niacin Calcium Pantothenate I-Inositol Ascorbic Acid Vitamin B-j  1.0 1.0 1.0 1.0 1.4 3.0 3.0 10.0 20.0 0.01  2  Starch  g g g g g g g g g g  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. gut loop.  The body wall was closed around the  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 l e f t 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. fluortoluene 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. 24 hours.  Samples were then ashed in a muffle furnace at 550° C. for 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 i n i t i a l l y 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 Group  Supplement and Administration  control 1  P.G.  ; oral  control 2  Ethanol  ; i.p.  1  P.G. + Vitamin D  2  P.G. + 25-OH D  3  P.G. + 24,25(0H) D  4  Dose of Vi tami n or Metabolite/rat  -  ; oral  625.0 ng.  ; oral  25.0 ng.  ; oral  25.0 ng.  P.G. + 1,24,25(0H) D  ; oral  25.0 ng.  5  P.G. + 1,25(0H) D  ; oral  12.5 ng.  6  Ethanol + 1,25(0H) D  ; i.p.  12.5 ng.  NX  P.G. + Vitamin D  3  ; oral  250.0 ng.  TPTX  P.G. + Vitamin D  3  ; oral  250.0 ng.  3  3  2  3  3  2  3  3  2  3  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. then dissected free and removed.  Both kidneys were  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) D or 25.0 ng. 25-0H D in 0.1 ml. propylene glycol 2  3  3  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 D as  shown in Table II.  3  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 D supplements for both groups were 3  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 t e s t i n a l absorption.  Although the plasma calcium l e v e l i s a  measurement of a l l f a c t o r s which e f f e c t calcium metabolism i n c l u d i n g release of calcium by bone and excretion by kidney, i t i s s t i l l assumed to be a v a l i d i n d i c a t i o n 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 f a c t o r s to influence t h i s l e v e l and the analysis of the Ca-45 leaving the gut lumen (appendix) shows c o r r e l a t i o n with the Ca-45 appearing on the serosal side f o r both the low and high calcium diets.  17. Results Normal Rats Fig. 1 shows the effect of vitamin D metabolites on both bone ash 3  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) D 2  p<0.001 for 1,25(0H) D as an i.p. dose). 2  3  -  There was no statistically  3  significant difference (p>0.05) between the body weight increases of 24,25(0H) D 2  and 1,24,25(0H) D .  3  3  3  Body weight in those receiving 25-OH D  3  was significantly greater (p<0.02) than those receiving 24,25(0H) D , 2  3  but showed no significant difference over those receiving 1,24,25(0H) D 3  (p>0.05).  3  Body weight of rats administered 1,25(0H) D was significantly 2  3  greater (p<0.01 - p<0.05) than that of rats receiving the other metabolites, and 1,25(0H) D as in i.p. dose was significantly greater 2  3  (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) D 2  3  or 1,24,25(0H) D . 3  Tibia ash weight of rats receiving 25-OH D was significantly increased 3  (p<0.05) over ash weights of either the previous two metabolites or the control.  Ash of 1,25(0H) D , whether administered i.p. or orally, 2  3  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 D on intestinal Ca-45 uptake. 3  Part A of the graph shows the control rat results which received no  3  .18.  Effect of Vitamin D Metabolites on Bone and Body Weight  6.0  -t  I H  CO  <  4.0  H  2.0  H  c o CD  E  I  Low Ca Diet High "  0.0  ' —,—' *—„—'  *  v  24,25 1,24,25 (0H)  o o  D  3  2  (0H)  3  25  1.25  OH  (0H) 3 Oral D  2  J  >.  1.25 (0H)  2  3 I.P D  6.0 n 03 a a > \— o c  4.0  f  2.0  H  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. following 10 days.  High calcium diet (2.0%) values are calculated for the 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.  lower half shows % body weight increase per ng. metabolite.  The  18a.  Effect of Vitamin D Metabolites on Bone and Body Weight.  Fig. la  n  Low Ca Diet  •  High  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. therefore identical with Figure 1 with x + SEM.  All parameters are  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 D supplementation on 3  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 on duodenal Ca-45 transfer.  3>  24,25(0H) D and 1,24,25(0H) D 2  3  3  3  All three metabolites caused a significantly  I  20.  Effects of Vitamin D x  High  "  o a. = B. Supplementation ifi 4.0-\  Fig. 2 Comparison of the Effects of Vitamin D Deficiency and Supplementation on Plasma Ca-45 from Ligated Gut Loops. Doses for the vitamin D supplementation are shown in Table II.  Graph shows the  percent of i n i t i a l 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).  j  21.  greater duodenal Ca-45 transport between low and high calcium diets (p<0.02 for 25-OH D , p<0.05 for 24,25(0H) D 3  2  and p<0.05 for the  3  10, 20, 30 minute intervals with 1,24,25(0H) D ). 3  No statistical  3  significance is apparent (p>0.05) between the high calcium diet values of plasma Ca-45 for all three metabolites, or vitamin D supplemented 3  duodenal high calcium values of Fig. 1.  25-OH D supplemented low 3  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) D 2  3  1,24,25(0H) D (p>0.05). 3  3  (p<0.001); no difference was observed with 1,24,25(0H) D low calcium values showed a 3  3  significant increase (p<0.02) over control values of Fig. 1 and 24,25(0H) D 2  3  low calcium values.  It is noted that rats fed low calcium  diet which were administered 24,25(0H) D 2  took up significantly less  3  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) D , a l l three 2  3  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) D supplements on duodenal 2  calcium uptake.  3  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) D caused significantly greater plasma Ca-45 uptake 2  3  (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 •  24,25(0H)  2  D  3  Supplemented  1  1,24,25 (0H)  1 10  3  D  3  1  1  1  1  Supplemented  1  20 Time in Minutes  1— 30  -1 40  Fig. 3 Comparison of the Effects of 25-OH D  3>  1 24,25(0H) D on Duodenal Calcium Uptake.  Doses for the supplementation  t  3  3  shown in Table II.  24,25(0H) D and 2  3  Parameters of the graph are identical with Graph 2  with x + SEM and N-6.  23.  Time  Fig. 4  in Minutes  Comparison of the Effects of 1,25(0^^3 Administration on  Duodenal Ca-45 Transport. Table II).  I.P.  or oral administration (doses shown in  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 D pr the f i r s t two time 3  values of 1 ,24,25(0H) D (all other metabolites were significantly 3  lower).  3  The high calcium diet values for both i.p. and oral doses of  1,25(0H) D were significantly higher than all other metabolites 2  3  (p<0.01 - p<0.02).  The time course for 1,25(0H) D is the same as 2  3  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 i n i t i a l dose placed in the gut loop lumen).  This measurement did not show any  statistical variation between vitamin D or vitamin D metabolite 3  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 i r s t 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 D 10 hours prior to surgery) 3  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 D treated rats in Fig. 3 and comparable values in Fig. 5 show 3  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)2D treated rats showed 3  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 D treated rats. 3  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  D supplement until 48 hours prior to surgery when a pulse dose of 3  25-OH D  3  (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 ^ f O H ^ D g 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  D and its metabolites were therefore selected from these authors* work 3  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 D /rat/day and 12.5 ng. 1,25(0H) D /rat/day were 3  maximum at this same time interval.  2  3  The 250 ng. dose of vitamin D /rat/day 3  as a supplement for NX and TPTX rats is used by most authors, and by Ribovitch and DeLuca . 75  Doses of 24,25(0H) D 2  3  and 1,24,25(0H) D for 3  3  adaptation were not found in the literature; therefore, doses were selected to equal (by weight) the 25-OH D supplement. 3  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) D i . p . ) , and in Fig. 1 is expressed per ng. of metabolite. 2  3  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) D increased body weight to 2  3  the greatest extent and demonstrated its greater potency per ng. compared to the other metabolites.  It is noted that 1,25(0H) D as 2  3  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) D is more potent as an i.p. 2  dose in producing a biological response.  3  This is a confirmation of  the work by Omdahl , who showed that 1,25(0H) D as an I.p. 65  2  3  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) D and 1,24,25(0H) D are the least effective 1n stimulating 2  3  3  3  bone mineralization and do not show any significant increase over control rats.  25-OH D and 1,25(0H) D supplements both show significant 3  2  3  increases in bone mineralization and confirm their biological action on bone as outlined by DeLuca . 17  The results here demonstrate that an in  vivo assay can be used to show the adaptation response and constitute the f i r s t 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 a l l contents and the gut loop i t s e l f 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 i r s t 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 i r s t 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. Kimberg » 94  Walling and  have shown that active secretion of calcium occurs  95  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 D on bone tissue and absorption. 3  Fig. 2 demonstrates that no adaptation response is observed when vitamin D is absent. 3  This confirms the work of Nicolaysen  62  who showed  that a minimal dose of vitamin D was required, and Ribovitch and 3  DeLuca  74  who demonstrated that at least 250 ng. vitamin D /rat/day was 3  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  70  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 S c h e d 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 l u i d ; 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 Kerstein . 2  Urban and Schedl  91  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 a l .  .  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 Lengemann , Cramer . 53  Although its absorptive rate is slower, the  15  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. in all three experiments.  It is observed that adaptation occurred  25-OH D showed the greatest degree of 3  adaptation, and reached a maximum net absorption of Ca-45 at 10 minutes which was significantly greater than the maximum rate in the vitamin D treated group.  The effect of 24,25(0H) D was unique in that i t 2  3  appeared to significantly suppress Ca-45 absorption in rats fed a low calcium diet.  This may suggest that 24,25(0H)2D is not merely a 3  degradation product of 25-OH D but has a specific function. When 3  serum calcium levels are low; i . e . as induced by a low calcium diet, 24,25(0H) D levels in the body would be low. 2  If this metabolite is  3  given exogenously, the absorption 1s further decreased and this suggests that 24,25(0H)2D may actively suppress absorption by either 3  a direct action, or by inhibiting 1,25(0H) D . 2  3  This would imply that  there was either a two-receptor site for these metabolites (inhibition by 24,25(0H) D and stimulation by l,25(0H) D- ) or a one-receptor site 2  3  2  3  3  35.  for 1,25(0H) D which is modulated by the presence of 24,25(0H) D . 2  3  2  3  This effect is presumably due to the specific action of the 24-OH moiety because the 1,24,25(0H) D did not produce these results. 3  is noted that 24,25(0H) D 2  3  It  3  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 a l . Miravet et a l . .  and  Both of these papers showed that 24,25(0H) D  5 8  2  3  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) D 2  mobilization.  3  does not stimulate bone  The graph reflecting 1,24,25(0H) D3 treatment showed 3  adaptation at most of the time intervals studied and was similar in general outline to the 25-OH D treated group, except that plasma Ca-45 3  values for the 1,24,25(0H) D low calcium diet group were consistently 3  3  lower than the 25-OH D low calcium diet group.  It is also observed  3  that all graphs reflecting a high calcium diet are results which are statistically identical.  If the theory of calcium adaptation via  specific vitamin D metabolites is correct, then the most probable 3  explanation is that when the rats were adapted to a high calcium diet, the predominant metabolites would be 24,25(0H) D 2  compared to the production of 1,25(0H) D . 2  3  and 1,24,25(0H) , n  3  3 3  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) D given e i t h e r by i . p . or oral 2  3  administration abolishes the adaptation response.  These r e s u l t s  i n d i c a t e that t h 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 t h i s metabolite i s less potent when administered by oral than by the i . p . route.  Comparison  with the 1 ,24,25(0H) D treated r a t plasma Ca-45 of F i g . 3 shows that 3  the 1,24,25(0H) D 3  3  3  i s s i g n i f i c a n t l y less potent than 1,25(0H) D 2  3  in  stimulating i n t e s t i n a l absorption and because adaptation occurred in the presence of 1 , 2 4 , 2 5 ( O H ) »  t h i s metabolite may well represent a  degradation product of the 1 , 2 5 ( O H ) The r e s u 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 III.  The 10 minute value for  the low calcium d i e t group from each f i g u r e was selected as the reference of adaptation because t h i s represents the time i n t e r v a l of maximum plasma Ca-45 l e v e l s .  The table shows the d i f f e r e n c e between  the plasma Ca-45 l e v e l s from rats fed low and high calcium d i e t s (low high) and the maximum plasma Ca-45 l e v e l of the low calcium d i e 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 i e t / the d i f f e r e n c e i n plasma Ca-45 between the high and low d i e t s . and 1,25(0H) D 2  3  Values which are high (>20)  adaptation.  3  (-D)  supplemented r a t s show the l e a s t adaptation; while D ,  1 ,24,25(0H) D and 25-OH D 3  such as control  3  3  show low values and therefore demonstrate  The negative value f o r 24,25 (0H) D^ i s due to the e f f e c t ?  37. Table  III  Measurement of Adaptation from 10 Minute Ca-45 Plasma Values (Duodenum) Difference i n % Ca-45 plasma l e v e l s between low and high calcium diets = A  Supplement  Control  0.1  N.S.  2.1 + 0.3  21 .0  1.2  p< 0.01  3.4 + 0.3  2.8  1.6  p<r 0.05  4.4 + 0.4  2.8  1.6  p< 0;05  1.3 + 0.5  1.9  p< 0.02  5.1 + 0.4  2.7  Oral  0.2  N.S.  5.3 + 0.5  27.0  i.p.  0.3  N.S.  6.7 + 0.5  22.0  (-D)  Vitamin 1,24,25(0H) D 3  24,25(0H) D 2  25-OH D  3  3  3  1,25(0H) D  3  1,25(0H) D  3  2  2  Index ratio (B/A)  %Ca-45 plasma l e v e l on low calcium d i e t = B  -  0.8  x + S.E.M. N = 6  of the low calcium d i e t values being lower than the high calcium values as previously described.  While t h 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 q u a n t i t a t i v e d i s c r i m i n a t i o n between the metabolites which show adaptation, i . e . D , 25-OH D , 24,25(0H) D 3  3  2  3  and 1,24,25(0H) D . 3  3  To confirm that the adaptation in the duodenum was p r i m a r i l y due to the presence, or absence, of 1,25(0H) D , nephrectomies were 2  3  performed to remove the s i t e of the 1-hydroxylase system. had received a d a i l y dose of vitamin D as o u t l i n e d in Table II.  3  A l l rats  u n t i l 48 hours p r i o r to surgery  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 i r s t signs of a decreased duodenal response in the uremic state occurring at 24 hours, Walling et a l . These effects have been confirmed in humans by Recker and S a v i l l e  9 6  .  73  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 a r t i f i c i a l l y 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 5 4 + 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 D supplemented rats in 3  Fig. 2.  The significant decrease in low calcium diet values in 25-OH D  3  supplemented rats is noted after nephrectomy as compared with Fig. 3. 1,25(0H) D treated rats, while slightly lower, showed no similar 2  3  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 D to 1,25(0H) D 3  is essential to produce the increased intestinal absorption.  2  3  When  this step is blocked by nephrectomy, the 25-OH D cannot be converted 3  into 1,25(0H) D and hence l i t t l e adaptation occurs; whereas, 1,25(0H) D 2  3  2  3  supplementation can bypass this regulation point and produce a response which is independent of adaptation.  This provides the f i r s t 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 D rats is presumably due to residual vitamin D 3  3  (and therefore 1,25(0H) D ) which the rat received as a daily supplement 2  3  until 48 hours prior to surgery.  This is confirmed by the fact that  control rats on vitamin D which received only a pulse dose of the 3  vehicle after nephrectomy also showed the same level of adaptation.  The  other explanation is that 10 hours is insufficient time for 25-OH D to 3  be converted to 1,25(0H) D* . 2  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) D is the prime regulator of calcium absorption in the duodenum. 2  3  , 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 .  demonstrated that TPTX rats on a  2 7  low calcium diet were dependent on PTH to synthesize 1,25(0H) D , these 2  results were questioned by Galante et a l . . 2 6  and Larsson et a l .  3  Both Maclntyre et a l .  5 0  suggested that Garabedian's results could be  4 6  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) D . 2  3  This is supported by the fact  that glucose, which lowers serum phosphorus, reverses the Inhibitory effect of TPTX on l,25(0H)  n 2  3  production.  This would suggest that low  plasma phosphorus levels can Independently stimulate the synthesis of 1,25(0H) D3 in the absence of PTH as shown by Tanaka and DeLuca . 86  2  To test this hypothesis, we designed an experiment using essentially the same rationale for studying TPTX rats as Ribovitch and DeLuca . 75  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 . 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. suggestion by Maclntyre et a l . synthesis.  5 1  This supports the  that PTH is required for 1,25(0H) D 2  3  When PTH is absent, l i t t l e or no conversion of the  administered 25-OH D can occur, hence no increased absorption to a low 3  calcium diet due to 1,25(0H) D was observed. 2  3  When plasma phosphorus  levels are low, 1,25(0H) D production is stimulated by an independent 2  3  2  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) D . 2  3  When hypophosphatemia is the cause of 1,25(0H) D 2  3  production, in the absence of PTH, 1t would appear that there is no control of 1,25(0H) D production via stimulation of high plasma calcium 2  3  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) D , PTH system in addition to calcium. 2  DeLuca  75  3  Ribovitch and  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) D production and exogenous sources of the 2  3  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) D production directly, 2  3  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 D . 3  Vitamin D was required before any adaptation response was 3  observed and both vitamin D and 25-OH D produced an adaptation 3  3  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)2D  3  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)2D is the prime factor in regulating calcium 3  adaptation in duodenal absorption.  If this control point is bypassed  by supplying exogenous 1,25(0H)2D , then the duodenum absorbs calcium 3  at a constant rate independent of dietary calcium intake. Both 24,25(0H) D and 1,24,25(0H) D produced adaptation, but in 2  3  3  3  all categories their responses were less than the parent compound (I.e. 25-OH D and 1,25(0H) D respectively). 3  2  3  While these data may  suggest that 1,24,25(0H) D represents a degradation product of 1,25(0H) D , 3  3  2  i t also shows that 24,25(0H) D may have an additional metabolic role. 2  3  3  43.  On the basis of the 24,25(0H) D decreased response to a low calcium 2  3  diet, this metabolite may represent an active inhibitor of calcium absorption in the duodenum, either by its direct action on the intestinal c e l l , or by interacting with 1,25(0H) D to decrease its 2  3  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) D ). 2  3  When the phosphorus content is deficient, 1,25(0H) D production can 2  3  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) D production is modulated in 2  3  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.  It is the regulation of the synthesis and interconversion  of these metabolites which is affected by PTH and plasma levels of calcium and phosphorus.  Once these factors determine the level of the  metabolites in the body, these in turn regulate the duodenal calcium response.  The net effect of the metabolites on the total control of  calcium absorption throughout the entire intestine remains to be investigated.  44. Bibliography 1.  Ahlgren, 0., Larsson, S. "The Role of the Parathyroids for the Adaptation to a Low Calcium Intake", Acta Pathologica Microbiologica Scandinavia, A83 (1975), 13-24. —  2.  Behar, J . , Kerstein, M.D. "Intestinal Calcium Absorption: Differences in Transport Between Duodenum and Ileum", American Journal of Physiology, 230 (1976), 1255-1260.  3.  Bhattacharyya, M., DeLuca, H.F. "Subcellular Location of Rat Liver Calciferol - 25- Hydroxylase", Archives of Biochemistry and Biophysics, 160 (1970), 58-62.  4.  Blunt, J.W., DeLuca, H.F. "The Synthesis of 25- Hydroxychol ecal ci ferol , a Biologically Active Metabolite of Vitamin D-3", Biochemistry, 8 (1969), 671-674.  5.  Boyle, I.T., Gray, R.W., DeLuca, H.F. "Regulation by Calcium of in Vivo Synthesis of 1,25 01hydroxycholecalciferol and 21,25 DIhydroxycholecalciferol", Proceedings of the National Academy of Science (U.S.A.), 68 (197lf! 2l3l.  6.  Boyle, I.T., Gray, R.W., Omdahl, J . L . , DeLuca, H.F. "Calcium Control of the in Vivo Biosynthesis of 1,25 (0H)2 D-3: Nicolaysen's Endogenous Factor", Proceedings of the International Symposium on Endocrinology, ed. S. Taylor, Helneman, London (1972), 486-496.  7.  Boyle, I.T., Mlravet, L., Gray, R.W., Holick, M.F., DeLuca, H.F. "The Response of Intestinal Calcium Transport to 25- Hydroxy and 1,25- Dihydroxyv1taminD-3 1n Nephrectomized Rats", Endocrinology, 90 (1972), 605-608.  8.  Boyle, I.T., Omdahl, J . L . , Gray, R.W., DeLuca, H.F. "The Biological Activity and Metabolism of 24,25- Dihydroxyv1tam1n D-3", Journal of Biological Chemistry, 248 (1973), 4174-4180.  9.  Brickman, A.S., Masry, S.G., Norman, A.W., Coburn, J.W; "On the Mechanism and Nature of the Defect 1n Intestinal Absorption of Calcium 1n Uremia", Kidney International, 7 supplement 2 (1975), S113-S117.  10.  Care, A.D., Bates, R.F.L., Plckard, D.W., Peacock, M., Tomlinson, S., 0'Riordan, J.L.H., Mawer, E.B., Taylor, C M . , DeLuca, H.F., Norman, A.W. "The Effects of Vitamin D Metabolites and Their Analogues on the Secretion of Parathyroid Hormone", Calcified Tissue Research, 21 supplement (1976), 142-146.  45.  11.  Care, A.D., Pickard, D.W., Peacock, M., Mawer, E.B., Taylor, CM., Redel, J . , Norman, A.W. "Reduction of Parathyroid Hormone by 24,25- Dihydroxycholecalciferol", Abstracts: Third Workshop on Vitamin D; Pacific Grove California, (1977), 31.  12.  Chertow, B.S., Baylink, D.J., Wergedal, J . E . , Su, M.H.H., Norman, A.W. "Decrease in Serum Immunoreactive Parathyroid Hormone Secretion in Vitro by 1,25Di hydroxycholecalci f e r o l " , Journal of Clinical Investigation, 56 (1975), 668-677.  13.  Chu, L.L.H., MacGregor, R.R., Anact, C.S., Hamilton, J.H., Cohn, D.V. "Studies on the Biosynthesis of Rat Parathyroid Hormone and Proparathyroid Hormone: Adaptation of the Parathyroid Gland to Dietary Restriction of Calcium", Endocrinology, 93 (1973) 915-924.  14.  Corradino, R.A. "Parathyroid Hormone and Calcitonin: No Direct Effect on Vitamin D-3 Mediated, Intestinal Calcium Absorptive Mechanisms", Hormone and Metabolic Research, 8 (1976), 485-488. ~  15.  Cramer, C.F. "Sites of Calcium Absorption and the Calcium Concentration of Gut Contents in the Dog", Canadian Journal of Physiology and Pharmacology, 43 (1965), 75-78.  16.  Cramer, C.F. "Effects of Salmon Calcitonin on in Vivo Calcium Absorption in Rats", Calcified Tissue Research, 13 (1973), 169-172.  17.  DeLuca, H.F. "Vitamin D; the Vitamin and the Hormone", Federation Proceedings, 33 (1974), 2211-2219.  18.  DeLuca, H.F. "Recent Advances 1n Our Understanding of the Vitamin D Endocrine System", Journal of Laboratory Clinical Medicine, 87 (1976), 7-26.  19.  DeLuca, H.F. "Recent Developments 1n the Metabolism of Vitamin D and its Regulation", Abstracts: Third Workshop on Vitamin D; Pacific Grove California, (1977), 33.  20.  DeLuca, H.F., Weiler, M., Blunt, J.W., Neville, P.F. "Synthesis, Biological Activity and Metabolism of 22,23 3-H Vitamin D-4", Archives of Biochemistry and Biophysics, 124 (1968), 122-128.  Domlnguez, J.H., Gray, R.W., Lemann, J . "Dietary Phosphate Deprivation 1n Women and Men: Effects on Mineral and Acid Balances, Parathyroid Hormone and the Metabolism of 25-OH Vitamin D-3", Journal of Clinical Endocrinology and Metabolism, 43 (1976), 1056-1068. Fairbanks, B.W., Mitchell, H.H. "Relation Between Calcium Retention and Store of Calcium 1n the Body with Particular Reference to the Determination of Calcium Requirements", Journal of Nutrition, 11 (1936), 551>5 . 72  Favus, M.J., Walling, M.W., Kimberg, D.V. "Effects of Dietary Calcium Restriction and Chronic Thyroldparathyroidectomy on the Metabolism of 3-H 25-Hydroxyvitamin D-3 and the Active Transport of Calcium by Rat Intestine", Journal of Clinical Investigation, 53 (1974), 1139-1148. ~ Fraser, D.R., Kodicek, E. "Unique Biosynthesis by Kidney of a Biologically Active Vitamin D Metabolite", Nature, 228 (1970), 764-766. Freund, T., Bronner, F. "Stimulation 1n Vitro by 1,25Dihydroxyvitamin D-3 of Intestinal Cell Calcium Uptake and Calcium Binding Protein", Science, 190 (1975), 4221-4224. Galante, L., Colston, K.W., Evans, I.M.A., Byfield, P.G.H., Matthews, E.W., Maclntyre, I. "The Regulation of Vitamin D Metabolism", Nature, 244 (1973), 438-440. Garabedian, M., Holick, M.F., DeLuca, H.F. "Control of 25-Hydroxycholecalciferol Metabolism by the Parathyroid Glands", Proceedings of the National Academy of Science (U.S.A.), 69 (1972), 1673-1676. Garabedian, M., Tanaka, Y., Holick, M.F., DeLuca, H.F. "Response of Intestinal Calcium Transport and Bone Calcium Mobilization to 1,25 Dihydroxyvitamin D-3 in Thyroidparathyroidectomized Rats", Endocrinology, 94 (1974), 1022-1027. Ghazarian, J.G., DeLuca, H.F. "25-Hydroxycholecalciferol1-Hydroxylase: A Specific Requirement for NADPH and a Hemoprotein Component in Chick Kidney Mitochondria", Archives of Biochemistry and Biophysics, 160 (1974), 63-65. Gray, R., Boyle, I.T., DeLuca, H.F. "Vitamin D Metabolism: The Role of Kidney Tissue", Science, 172 (1971), 1232-1234.  47.  31.  Henry, H.L., Norman, A.W., Taylor, A.N., Hartenbower, D.L., Coburn, J.W. "Biological Activity of 24,25 Dihydroxycholecalciferol in Chicks and Rats", Journal of Nutrition, 106 (1976), 724-734.  32.  Holick, M.F., DeLuca, H.F. "A New Chromatographic System for Vitamin D-3 and its Metabolites: Resolution of a New Vitamin D-3 Metabolite", Journal of Lipid Research, 12 (1971), 460-465.  33.  Holick, M.F., Schnoes, H.K., DeLuca, H.F. "Identification of 1,25- Dihydroxycholecalciferol, a Form of Vitamin D-3 Metabolically Active in the Intestine", Proceedings of the National Academy of Science (U.S.A.), 68 (1971), 803-804.  34.  Holick, M.F., Schnoes, H.K., DeLuca, H.F., Gray, R.W., Boyle, I.T., Suda, T. "Isolation and Identification of 24,25- Dihydroxycholecalciferol; a Metabolite of Vitamin D-3 Made in the Kidney", Biochemistry, 11 (1972), 4251-4255.  35.  Huldshinsky, A. "Heilung von Rechitis durch Kuenstliche Lochensonne", Deutsche Medizinische Wochenschrift, 45 (1919), 712^713:  36.  Kane, G.G. "Dietary Fat and Calcium Wastage in Old Age", Journal of Gerontology, 4 (1949), 185-192.  37.  Kemrn, J.R. "The Effect of Parathyroidectomy and Large Doses of Cholecalciferol on the Ability of Rats to Adapt to Changes in Dietary Intake of Calcium", Journal of Physiology, 256 (1976), 103-115.  38.  Kimberg, D.V., Schacter, D., Schenker, H. "Active Transport of Calcium by Intestine: Effects of Dietary Calcium", American Journal of Physiology, 200 (1961), 1256-1262.  39.  Kleiner-Bossaller, A., DeLuca, H.F. "Formation of 1,24,25Trihydroxyvitamin D-3 from 1,25- Dihydroxyvitamin D-3", Biochemica et Biophysica Acta, 338 (1974), 489-495.  40.  Kodicek, E. Bone Structure and Metabolism: A CIBA Foundation Symposium, ed. G.E.W. Wolstenholme, L i t t l e Brown, Boston (1956), 161.  41.  Kodicek, E. "The Story of Vitamin D, From Vitamin To Hormone", Lancet, 1 (1974), 325-329.  42.  Krawitt, E.L. "Calcium Uptake by Isolated Intestinal Brush Border Membranes Following Dietary Calcium Restriction", Life Sciences, 19 (19769, 543-548.  48.  43.  Krawitt, E.L., Katagiri, C.A. "Calcium Uptake by Isolated Small Intestinal Brush Border Membranes Following Dietary Calcium Restriction", Gastroenterology, 68A73 (1975), 930.  44.  Krawitt, E.L., Kunin, A.S., Bacon, B.F. "Intestinal Mitochondrial Calcium Uptake During Adaptation to Dietary Calcium Restriction", Calcified Tissue Research, 21 (1976), 129-133.  45.  Krawitt, E.L., Kunin, A.S., Sampson, H.W. "Effect of Hypophysectomy on Calcium Transport by Rat Duodenum", American Journal of Physiology, 1 (1977), E229-E233.  46.  Larsson, S., Ahlgren, 0., Lorentzon, R. "Calcium Deficiency Osteoporosis and the Role of the Parathyroids for Adaptation to a Low Calcium Intake", Calcified Tissue Research, 21 supplement (1976), 166-171.  47.  Lawson, D.E.M., Fraser, D.R., Kodicek, E., Morrison, H.R., William, D.H. "Identification of 1,25 Dihydroxycholecalciferol; a New Kidney Hormone Controlling Calcium Metabolism", Nature, 230 (1971), 228-230.  48.  Lorenc, R., Tanaka, Y., DeLuca, H.F., Jones, G. "Lack of Effect of Calcitonin on the Regulation of Vitamin D Metabolism in the Rat", Endocrinology, 100 (1977), 468-472.  49.  Lovelace, F.C., Liu, C.H., McKay, CM. "Age of Animals in Relation to the Utilization of Calcium and Magnesium in the Presence of Oxalates", Archives of Biochemistry and Biophysics, 27 (1950), 48-56.  50.  Maclntyre, I., Colston, K.W., Evans, I.M.A. "The Regulation of Vitamin D Metabolism", Calcified Tissue Research, 21 supplement (1976), 136-T4T!  51.  Maclntyre, I., Imogen, M., Evans, I.M.A., Larkin, R.G. "Vitamin D", Clinical Endocrinology, 6 (1977), 65-80.  52.  Malm, O.J. "The Endogenous Factor in Intestinal Calcium Absorption", Scandinavian Journal of Clinical and Laboratory Investigation, 33 (1974), 95-96.  53.  Marcus, C.S., Lengemann, F.W. "Absorption of Ca-45 and Sr-85 from Solid and Liquid Food at Various Levels of the Alimentary Tract of the Rat", Journal of Nutrition, (1962), 155-160.  54.  Martin, D.L., DeLuca, H.F. "Calcium Transport and the Role of Vitamin D", Archives of Biochemistry and Biophysics, 134 (1969), 139-148.  49.  55.  Martin, D.L., DeLuca, H.F. "Influence of Sodium on Calcium Transport by the Rat Small Intestine", American Journal of Physiology, 216 (1969), 1351-1359.  56.  Melancon, M.J., DeLuca, H.F. "Vitamin D Stimulation of Calcium Dependent Adenosine Triphosphatase in Chick Intestinal Brush Borders", Biochemistry, 9 (1970), 1658-1664.  57.  Mellanby, E. "The Part Played by Accessory Food Factors in Etiology of Rickets", Journal of Physiology, 52 (1919), Llll.  58.  Miravet, L., Redel, J . , Carre, M., Queille, M.L., Bordier, P. "The Biological Activity of Synthetic 25,26 (0H)2 and 24,25 (0H)2 D-3 in Vitamin D Deficient Rats", Calcified Tissue Research, 21 (1976), 145-152.  59.  Neville, P.F., DeLuca, H.F. "The Synthesis of 1,2 3-H Vitamin D and the Tissue Localization of a 0.25 micro g. (10 I.U.) Dose per Rat", Biochemistry, 5 (1966), 2201-2207.  60.  Nicolaysen, R. "The Absorption of Calcium as a Function of the Body Saturation with Calcium", Acta Physiologica Scandinavia, 5 (1943), 200-211.  61.  Nicolaysen, R. "The Utilization of Calcium Soaps in Rats", Acta Physiologica Scandinavia, 5 (1943), 215-218.  62.  Nicolaysen, R., Eeg-Larsen, N., Malm, O.J. "Physiology of Calcium Metabolism", Physiological Reviews, 33 (1953), 424-444.  63.  Norman, A.W., Henry, H.L. "1,25-Dihydroxyvitamin D-3; a Hormonally Active Form of Vitamin D-3", Recent Progress in Hormone Research, 30 (1974), 431-480.  64.  Oldham, S.B., Smith, R., Hartenbower, D.L., Henry, H.L. "Effects of 1,25- Dihydroxyvitamin D-3 on Serum Calcium, Immunoreactive Parathyroid Hormone and Intestinal and Parathyroid Calcium Binding Protein in the Dog", Federation Proceedings, 35 (1976), 302.  65.  Omdahl, J . L . , DeLuca, H.F. "Regulation of Vitamin D Metabolism and Function", Physiological Reviews, 53 (1973), 327-372.  66.  Omdahl, J . L . , Thornton, P.A. "Intestinal Calcium Absorption and Calcium Binding Protein: Influence of Dietary Calcium", Proceedings of the Society for Experimental Biology and Medicine, 139 (1972), 975-980.  50.  67.  Papworth, D.G., Patrick, G. "The Kinetics of Influx of Calcium and Strontium into Rat Intestine in Vitro", Journal of Physiology, 210 (1970), 999-1020.  68.  Patrick, G. "Regulation of Intestinal Calcium Transport by Vitamin D", Nature, 243 (1974), 89-90.  69.  Petith, M.M., Schedl, H.P. "Cecal and Colonic Adaptation to Dietary Calcium Restriction: in Vivo Studies in the Rat", Clinical Research, 23 (1975), 519A.  70.  Petith, M.M., Schedl, H.P. "Duodenal and Ileal Adaptation to Dietary Calcium Restriction: in Vivo Studies in the Rat", American Journal of Physiology, 231 (1976), 865-871.  71.  Petith, M.M., Schedl, H.P. "Intestinal Adaptation to Dietary Calcium Restriction: in Vivo Cecal and Colonic Calcium Transport in the Rat", Gastroenterology, 71 (1976), 1039-1042.  72.  Ponchon, G., DeLuca, H.F. "The Role of the Liver in the Metabolism of Vitamin D", Journal of Clinical Investigation, 48 (1969), 1273-1279.  73.  Recker, R.R., Saville, P.D. "Calcium Absorption in Renal Failure: Its Relation to Blood Urea Nitrogen, Dietary Calcium Intake, Time on Dialysis and Other Variables", Journal of Laboratory and Clinical Medicine, 78 (1971), 380-388.  74.  Ribovitch, M.L., DeLuca, H.F. "The Influence of Dietary Calcium and Phosphorus on Intestinal Calcium Transport in Rats Given Vitamin D Metabolites", Archives of Biochemistry arid Biophysics, 170 (1975), 529-535.  75.  Ribovitch, M.L., DeLuca, H.F. "Intestinal Calcium Transport: Parathyroid Hormone and Adaptation to Dietary Calcium", Archives of Biochemistry and Biophysics, 175 (1976), 256-261.  76.  Ribovitch, M.L., DeLuca, H.F. "The Effect of Dietary Calcium and Phosphorus on Vitamin D-3 Metabolism", Federation Proceedings, 36 (1977), 1096.  77.  Rottenstein, K.V. "The Effect of Body Stores on the Efficiency of Calcium Utilization", Biochemical Journal, 32 (1938), 1285-1292.  78.  Sammon, P.J., Stacey, R.E., Bronner, F. "Role of Parathyroid Hormone in Calcium Homeostasis and Metabolism", American Journal of Physiology, 281 (1970), 479-485.  51.  79.  Schacter, D., Kimberg, D.V., Schenker, H. "Active Transport of Calcium by Intestine: Action and Bioassay of Vitamin D", American Journal of Physiology, 200 (1961), 1263-1271.  80.  Schacter, D., Rosen, S.M. "Active Transport of Ca-45 by the Small Intestine and its Dependence on Vitamin D", American Journal of Physiology, 196 (1959), 357-362.  81.  Schenck, F. "liber das Kristalbisierte Vitamin D-3", Naturwissenschaften, 25 (1937), 159-165.  82.  Semmler, E.J., Holick, M.F., Schnoes, H.K., DeLuca, H.F. "The Synthesis of 1 alpha-25- Dihydroxycholecalciferola Metabolically Active Form of Vitamin D-3", Tetrahedron Letters, 40 (1972), 4147-4150.  83.  Shah, B.G., Draper, H.H. "Depression of Calcium Absorption in Parathyroidectomized Rats", American Journal of Physiology, 211 (1966), 963-966.  84.  Spencer, R., Charman, M., Wilson, P., Lawson, E. "Vitamin D Stimulated Intestinal Calcium Absorption May Not Involve Calcium Binding Protein Directly", Nature, 263 (1976), 161-163.  85.  Suda, T., DeLuca, H.F., Schnoes, H.K., Ponchon, G., Tanaka, Y., Holick, M.F. "21,25 Dihydroxycholecalciferol; a Metabolite of Vitamin D-3 Preferentially Active on Bone", Biochemistry, 9 (1970), 2917-2922.  86.  Tanaka, Y., DeLuca, H.F. "The Control of 25-Hydroxyvitamin D Metabolism by Inorganic Phosphorus", Archives of Biochemistry and Biophysics, 154 (1973), 566-574.  87.'  Tanaka, Y., DeLuca, H.F. "Stimulation of 24,25 Dihydroxyvitamin D-3 Production by 1,25 Dihydroxyvitamin D-3", Science, 183 (1974), 1198-1200.  88.  Taylor, A.N., Wassermann, R.H. "Immunofluorescent Localization of Vitamin D Dependent Calcium Binding Protein", Journal of Histochemistry and Cytochemistry, 18 (1970), 107-115.  89.  Taylor, A.N., Wassermann, R.H. "Vitamin D Stimulation of Calcium Binding Protein and Brush Border Enzyme Activities", Federation Proceedings, 29 (1970), 368.  90.  Teitelbaum, S.L., Halverson, J.D., Bates, M., Wise,:L., Hoddad, J.G. "Abnormalities of Circulating 25 Hydroxyvitamin D After Jejunal Ileal Bypass for Obesity: Evidence of an Adaptation Response", Annals of Internal Medicine, 86 (1977), 289-293.  52.  91.  Urban, E., Schedl, H.P. "Comparison of in Vivo and in Vitro Effects of Vitamin D on Calcium Transport in the Rat", American Journal of Physiology, 217 (1969), 126-130.  92.  Urban, E., Schedl, H.P. "Vitamin D, Tissue Calcium, and Calcium Transport in the in Vivo Rat Small Intestine", American Journal of Physiology, 219 (1970), 944-951.  93.  Walker, A.R.P., Inrig, J.T. "Phytate and Calcium Metabolism in Man", Biochemistry Journal, 42 (1948), 452-462.  94.  Walling, M.W., Kimberg, D.V. "Calcium Absorption or Secretion by Ileum in Vitro", American Journal of Physiology, 226 (1974), 1124-lTlC\  95.  Walling, M.W., Kimberg, D.V. "Active Secretion of Calcium, Sodium and Chloride by Adult Rat Duodenum in Vitro", Biochemica et Biophysica Acta, 382 (1975), 213-217.  96.  Walling, M.W., Kimberg, D.V., Wassermann, R.H., Feinberg, R.R. "Duodenal Active Transport of Calcium and Phosphate in Vitamin D Deficient Rats: Effects of Nephrectomy, Cestrum Diurnum, and l-alpha-25-Dihydroxyvitamin D-3", Endocrinology, 98 (1976), 1130-1134.  97.  Walling, M.W., Rothman, S.S. "Adaptive Uptake of Calcium at the Duodenal Brush Border", American Journal of Physiology, 225 (1973), 618-623.  98.  Wassermann, R.H., Kallfelz, F.A., Comar, C L . "Active Transport of Calcium by Rat Duodenum in Vivo", Science, 133 (1961), 883-884.  99.  Wassermann, R.H., Taylor, A.N. "Vitamin D-3 Induced Calcium Binding Proteins in Chick Intestinal Mucosa", Science, 153 (1966), 791-793.  100.  Wassermann, R.H., Taylor, A.N. "Vitamin D Dependent Calcium Binding Protein: Response to Some Physiological Variables", Journal of Biological Chemistry, 243 (1968), 3987-3993.  101.  Weber, H.P., Gray, R.W., Dominguez, J.H., Lemann, J . "The Lack of Effect of Chronic Metabolic Acidosis on 25-OH Vitamin D Metabolism and Serum Parathyroid Hormone in Humans", Journal of Clinical Endocrinology and Metabolism, 43 (1976), 1047-1055.  102.  Windaus, A., Schenck, F., Von Werder, F. "Uber das Antirachitish Wirksame Bestrahlungs-Produkt aus 7-Dehydro-Cholesterin", Zeitschrift fur Physiologische Chemie, 241 (1936), 100-103.  53.  |  | Low Ca Diet High "  25 (OH) D  Appendix.  24,25 (OH) D2  ,24,25 (OH) 3  "  1,25 (OH) D IP  2  3  The graph shows the percent of i n i t i a l administered dose of  Ca-45 not recovered from the i s o l a t e d duodenal lumen at 40 minutes ( i . e . 100%-% flushed out of lumen).  This i s a c o r r e l a t i o n of absorption  as measured from the serosal side in Figs. 3 and 4.  There i s a  s i g n i f i c a n t d i f f e r e n c e between the low and high calcium d i e t values (p<0.05) f o r a l l supplemented metabolites except 1,25 (0H) D 2  Values are x + SEM with N=6.  3  (p>0.05).  


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