UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Intestinal calcium transport in the chicken Bhatti, Mohammad Suleman 1998

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

Item Metadata

Download

Media
831-ubc_1998-271056.pdf [ 7.27MB ]
Metadata
JSON: 831-1.0088740.json
JSON-LD: 831-1.0088740-ld.json
RDF/XML (Pretty): 831-1.0088740-rdf.xml
RDF/JSON: 831-1.0088740-rdf.json
Turtle: 831-1.0088740-turtle.txt
N-Triples: 831-1.0088740-rdf-ntriples.txt
Original Record: 831-1.0088740-source.json
Full Text
831-1.0088740-fulltext.txt
Citation
831-1.0088740.ris

Full Text

INTESTINAL CALCIUM TRANSPORT IN THE CHICKEN by M O H A M M A D STJLEMAN B H A T T I D . V . M . , University of Agriculture, Faisalabad, Pakistan, 1980 M . S . , University of Arkansas, Fayetteville, U . S . A . , 1990 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department of Animal Science) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A A p r i l , 1998 ®Mohammad Suleman Bhatti, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) A B S T R A C T The regulation of intestinal calcium transport is a subject of continuing controversy. Most investigations on this subject have involved use of the laboratory rat or layer-type chicken. The research reported in this thesis was conducted in 0- to 21-d-old rapidly growing broiler cockerels, mainly using in situ intestinal loop preparations. Intralumenal test solutions typically containing 75 m M each of 4 5 C a C k and 3H-mannitol were used to delineate the cellular and paracellular fractions of in situ calcium transport. A t this concentration, about 75 to 85 % of calcium transport from duodenal loops was shown to occur via the paracellular pathway. In contrast, calcium transport from distal ileal loops occurred entirely via the paracellular pathway and was not influenced by l ,25-(OH )2D3 administration. Further, mannitol disappearance from in situ intestinal loops was not affected by vitamin D-dependent mechanisms. In balance studies, calcium absorption in intact birds occurred primarily by a nonsaturable process when solubility was not a limiting factor, thereby supporting the in situ data that showed the predominance of paracellular calcium transport. Mannitol absorption and secretion were demonstrated to be nonsaturable suggesting both occur by paracellular mechanisms. The efficiency of paracellular transport tended to remain unchanged with age, in the duodenal, distal jejunal, and distal ileal loops of 0- to 14-d-old broiler chicks. The efficiency was 1.5 to 2 fold greater in the duodenal compared to the distal jejunal or distal ileal loops. The capacity of paracellular transport in these intestinal regions increased with age. It is concluded that when intralumenal calcium concentration is high and the solubility is not a limiting factor, intestinal calcium transport, both in in situ loops and in intact rapidly growing young broiler cockerels occurs largely by a paracellular process, which can take place in the absence of vitamin D . i i T A B L E O F C O N T E N T S A B S T R A C T i i T A B L E O F C O N T E N T S i i i LIST O F F I G U R E S x i LIST O F T A B L E S xv i A B B R E V I A T I O N S X v i i i A C K N O W L E D G M E N T S xx D E D I C A T I O N xxi C H A P T E R 1. General Introduction 1 1.1 Research Objectives 4 C H A P T E R 2. Review of Literature 6 2.1. Calc ium Homeostasis 6 2.2. Vitamin D in Calcium Metabolism 10 2.3. Calcium Transport Across the Intestinal Barrier 13 2.3.1. Physiological Anatomy of the Intestinal Wal l 13 2.3.2. Mechanisms Involved in Intestinal Calcium Transport 15 2.3.2.1. The Cellular Pathway 18 2.3.2.1.1. Classical Model of the Cellular Pathway 18 2.3.2.1.1.1. Calcium Entry 18 2.3.2.1.1.2. Transmural Calcium Transport 20 2.3.2.1.1.3. Calcium Extrusion 22 i i i 2.3.2.1.2. The Vesicular Model of the Cellular Pathway 23 2.3.2.2. The Paracellular Pathway 23 2.3.3. Kinetics of Intestinal Calcium Transport 26 2.3.4. Factors Regulating Intestinal Calcium Transport 28 2.3.4.1. Regulation by Vitamin D 28 2.3.4.2. Vitamin D-Independent Regulation 31 2.4. Experimental Systems Used in Intestinal Calcium Transport Research 33 2.4.1. Intestinal Preparations for Transport Studies 33 2.4.2. Techniques Used in Transport Studies 35 2.4.2.1. Saturation Technique 35 2.4.2.2. Molecular Marker Technique 36 2.4.2.3. Voltage Clamp Technique 37 C H A P T E R 3. Development of an Experimental System 38 3.1. Introduction 38 3.2. Animals and Housing 38 3.3. Feeding and Lighting Systems 39 3.4. Fasting Procedures 39 3.5. Preparation of In Situ Intestinal Loops 40 3.6. Intralumenal Test Solutions 41 3.7. In Situ Intestinal Loop Experimental Procedures 41 3.8. Parameters Used for Calcium and Mannitol Transport from In Situ Intestinal Loop Preparations 43 iv 3.9. In Vitro Recovery of Radioactive Tracers from the Intestinal Lumen 44 3.10. Disappearance of Calcium and Mannitol From In Situ Duodenal Loops, as a Function of Time 46 3.11. Collection of Plasma and Tibiae 49 3.12. Balance Experiments 50 3.13. Analytical Procedures 51 3.14. Animal Care 51 3.15. Statistical Analyses 51 3.16. Effect of Short-Term Fasting on Intestinal Calcium and Mannitol Disappearance from In Situ Duodenal Loop Preparations 52 3.17. Factors that May Influence In Situ Intestinal Loop Transport Transport 56 C H A P T E R 4. Regulation of Intestinal Calcium Transport in Rapidly Growing Young Broiler Cockerels by Vitamin D-Dependent Mechanisms 64 4.1. Introduction 64 4.2. Methods 67 4.2.1. Effects of High Calcium Intake on Calcium Transport in In Situ Duodenal and Ileal Loop Preparations 67 4.2.1.1. Diets 67 4.2.1.2. Duodenal Calcium Transport in Response to High Calcium Intake 68 4.2.1.3. Ileal Calcium Transport in Response to High Calcium Intake 69 v 4.2.2. Effects of Vitamin D on Intestinal Calcium Transport 69 4.2.2.1. Diets 69 4.2.2.2. Rachitogenesis 71 4.2.2.3. Effects of Vi tamin D-Deficiency Rickets and 1,25-(OH)2D3 Treatment on Calcium Transport in In Situ Duodenal and Ileal Loop Preparations 71 4.2.2.3.1. General Procedures 71 4.2.2.3.2. Duodenal Calcium Transport i n Rachitic and l,25-(OH)2D 3-Treated Chicks 72 4.2.2.3.3. Ileal Calcium Transport in Rachitic and l,25-(OH)2D 3-Treated Chicks 73 4.2.2.3.4. Intestinal Calcium Transport in Intact Rachitic Chicks 74 4.3. Results 75 4.3.1. Responses to High Calcium Intake 75 4.3.1.1. Body Weight, and Plasma and Excreta Calcium Concentrations in Response to High Calcium Intake 75 4.3.1.2. Duodenal Calcium Transport in Response to High Calcium Intake 75 4.3.1.3. Ileal Calcium Transport in Response to H i g h Calcium Intake 80 4.3.2. Responses to Dietary Vitamin D and l,25-(OH)2D3 Treatment 80 4.3.2.1. Rachitogenesis 80 vi 4.3.2.2. Body Weight, Tibia Fat-Free Dry Weight, Tibia A s h Weight, and Tibia Length in Rachitic and Control Chicks 83 4.3.2.3. Plasma Total-Calcium Concentration and Hematocrit Values in Rachitic and Control Chicks 85 4.3.2.4. Plasma Total-Calcium Concentration in Response to l,25-(OH)2D 3 Treatment in Rachitic and Control Chicks 87 4.3.2.5. Duodenal Calcium Transport in Rachitic and Control Chicks With or Without l,25-(OH)2D 3 Treatment 89 4.3.2.6. Ileal Calcium Transport in Rachitic and Control Chicks With or Without l,25-(OH)2D 3 Treatment 91 4.3.2.7. Calcium Retention by Duodenal and Ileal loop Tissues in Rachitic and Control Chicks Wi th or Without l,25-(OH)2D 3 Treatment 93 4.3.2.8. Calcium Transport in Intact Rachitic Chicks 93 4.4. Discussion 97 4.4.1. Relative Contribution of Vitamin D-Dependent Mechanisms to Duodenal Calcium Transport 97 4.4.2. Ileal Calcium Transport Largely Occurs Independent of Vitamin D 98 4.4.3. Paracellular Transport is Not Regulated by Vitamin D 100 4.4.4. Calcium Absorption in Intact Rachitic Chicks 101 4.4.5. Calcium Metabolism and Vitamin D 103 v i i 4.4.6. Vitamin D-Dependent Intestinal Calcium Transport: Are Juvenile Chickens Different from Neonatal Rats? 104 4.4.7. Mannitol as a Marker of Paracellular Calcium Transport 104 4.5. Conclusions 106 C H A P T E R 5. Paracellular Absorption of Dietary Calcium in Rapidly Growing Young Broiler Cockerels 107 5.1. Introduction 107 5.2. Methods 110 5.2.1. Characteristics of Intestinal Mannitol and Calcium Transport from In Situ Duodenal Loop Preparations 110 5.2.2. Characteristics of Intestinal Calcium Transport in Intact Birds with Two Different Regimens of Increasing Calcium Intake I l l 5.3 Results 113 5.3.1. Characteristics of Intestinal Mannitol and Calcium Transport from In Situ Duodenal Loop Preparations 113 5.3.2. Feed, Water, and Calcium Intakes in Intact Birds Fed a High Calcium Diet or Provided 40 m M C a C h 117 5.3.3. Characteristics of Intestinal Calcium Transport in Intact Birds with Two Different Regimens of Increasing Calcium Intake 120 5.4. Discussion 123 5.5. Conclusions 130 v i i i C H A P T E R 6. Age-Related Changes and Regional Differences in Paracellular Absorption, Regional Differences in Paracellular Secretion, and Regional Differences in Calcium Absorption in the Small Intestine of Rapidly Growing Young Broiler Cockerels 131 6.1. Introduction 131 6.2. Methods 134 6.2.1. Age-Related Changes and Regional Differences in Paracellular Absorption in In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels 134 6.2.2. Regional Differences in Paracellular Secretion from Blood into the Lumen of In Situ Duodenal, Jejunal, 6.3. Results 136 6.3.1. Age-Related Changes and Regional Differences in Paracellular Absorption in In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels 136 6.3.2. Regional Differences in Paracellular Secretion from Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels 141 6.4. Discussion 144 6.4.1. Age-Related Changes in Paracellular Absorption in In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels 144 6.4.2. Regional Differences in the Small Intestine of Broiler ix Cockerels for Paracellular Absorption 145 6.4.3. Regional Differences in Paracellular Secretion from the Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels 146 6.5. Conclusions 148 C H A P T E R 7. General Discussion and Conclusions 149 7.1. General Discussion 149 7.2. Conclusions 152 7.3. Further Research 155 R E F E R E N C E S 156 A P P E N D I C E S 171 x LIST O F FIGURES Figure 2.1 Schematic representation of the epithelial barrier of the intestinal absorptive surface 16 Figure 2.2. Schematic diagram showing some of the possible pathways involved in the transmucosal intestinal calcium transport 17 Figure 2.3. A typical diagram to illustrate kinetics of intestinal calcium transport, and the relative contribution of the cellular and paracellular pathways 29 Figure 3.1. Recovery of 4 5 C a C l 2 and 3 H-mannitol radionuclides (Mean ± SE) from the lumen of duodenal, distal jejunal, and distal ileal loops in vitro (n=5 to 7) in 13-d-old broiler cockerels, 2 min after an intralumenal injection of a solution containing 75 m M each of 4 5 C a C l 2 and 3H-mannitol (S .A . , 1.33 n C i pmol 1 ) 45 Figure 3.2. Disappearance of calcium and mannitol (Mean ± SE) from the lumen of in situ duodenal loop preparations at 2, 4, 6, 8, 10, and 12 min after injection of an intralumenal test solution containing 75 m M each of 4 5CaCl 2 and 3H-mannitol (S .A . , 1.33 n C i pmol 1 ) , in 14-d-old broiler cockerels (n=6) 47 Figure 3.3. Disapperance of calcium (Mean ± SE) from the in situ duodenal loop preparations at 2, 4, 6, 8, and 10 min after injection of an intralumenal test solution containing 75 m M each of 4 5 C a C l 2 and 3 H-mannitol (S .A . , 1.33 n C i pmol" 1), in 14-d-old broiler cockerels (n=6) 48 xi Figure 3.4. Ingesta calcium concentration (Mean ± SE) in the duodenum, proximal jejunum, distal jejunum, proximal ileum, and distal ileum from 12-d-old unfasted broiler cockerels (n=6 or 7) fed a commercial broiler starter diet (1.09 % Ca of D M ) 53 Figure 3.5. Intralumenal calcium concentration (Mean + SE) at different times after a 100 m M 4 5 C a C h solution was injected into the lumen of in situ duodenal loop preparations in 21-d-old broiler cockerels fasted for 12 h (n=3) 57 Figure 3.6. Calcium concentration of test solutions, either 10 min (left panel; n = l ) , or 1 h (right panel; Mean + SE ; n = 2 to 5) after an intralumenal injection into the in situ duodenal loop preparation in 10-d-old broiler cockerels 58 Figure 3.7. Plasma mannitol concentration at different times after a 300 m M 3 H -Mannitol solution (S .A . , 0.33 n C i pmol"1) was injected @ 1.5 m L kg"1 into wing veins of 18-d-old normally fed broiler cockerels (n=4) 60 Figure 3.8. Plasma total-calcium concentration (Mean ± SE) during a 24 h period in 18-d-old broiler cockerels raised on a 24 h lighting program and provided continuous access to feed and water 61 45 Figure 3.9. Specific radioactivity (S.A.) of 100 m M C a C l 2 test solution (Mean + SE) at 2, 4, 8, 12, 16, and 20 min after intralumenal injection into the in situ duodenal loop preparation in 10-d-old broiler cockerels (n=5) 63 Figure 4.1. The effects of a high calcium intake on calcium and mannitol disappearance (Mean + SE) from in situ duodenal loop preparations in 4- (n=10), 7- (n= 11 to 12), 14- (n=12), and 21-d-old (n=12) broiler x i i cockerels fasted for 12 h 77 Figure 4.2. The effects of a high calcium intake on calcium and mannitol disappearance (Mean + SE) from in situ duodenal loop preparations in 10-d-old broiler cockerels fasted for 12 h, in a cross-over experiment (n=9 or 10) 79 Figure 4.3. The effects of a high calcium intake on calcium and mannitol disappearance (Mean ± SE) from in situ ileal loop preparations in 4- (n=7 to 9), 7- (n= 10 to 12), and 14-d-old (n=9 to 10) broiler cockerels fasted for 12 h 81 Figure 4.4. The effect of l , 2 5 - ( O H ) 2 D 3 treatment (i .m. injection @10 pg /kg) on plasma total-calcium concentration (Mean + SE) in (rachitic and control) 13-d-old fed (left panel), and 16-d-old fasted (right panel) broiler cockerels, 6 h after the treatment (n=7 to 9) 88 Figure 4.5. The effects of vitamin D-deficiency rickets, and l , 25 - (OH) 2 D 3 treatment (i .m. injection @ 10 pg/kg) on calcium and mannitol disappearance (Mean + SE) from in situ duodenal loop preparations in 12-, and 13-d-old broiler cockerels 90 Figure 4.6. The effects of vitamin D-deficiency rickets, and l , 25 - (OH) 2 D 3 treatment (i .m. injection @10 pg/kg) on calcium and mannitol disappearance (Mean + SE) from in situ ileal loop preparations in 15-, (n=9), and 16-d-old (n= 7 to 9) broiler cockerels fasted for a 12 h 92 Figure 4.7. Calcium retention by duodenal and ileal loop tissues (Mean + SE) x i i i after in situ loop calcium and mannitol transport experiments in rachitic and nonrachitic-control broiler cockerels with or without 1,25-(OH) 2D 3 treatment (n=7 to 10) 94 Figure 4.8. The effects of vitamin D-deficiency rickets on feed intake, body weight gain, and net calcium absorption (Mean + SE) in intact broiler cockerels during a 24 h balance experiment conducted between 11 and 12 d of age 95 Figure 5.1. Mannitol disappearance (Mean ± SE) from in situ duodenal loop preparations in 0-, 4-, and 21-d-old broiler cockerels (n=4 or 5), as a function of intralumenal mannitol concentration 114 Figure 5.2. The effect of intralumenal mannitol concentration on the percentage of 3H-mannitol that disappears (Mean + SE) from the lumen of in situ duodenal loop preparations in 0-, 4-, and 21-d-old broiler cockerels ( n = 4 o r 5) 115 Figure 5.3. The amount of calcium (umol g-110 min ' ) (left panel), and the percentage of calcium (% loss of dose in 10 min) (right panel) that disappears (Mean ± SE) from in situ duodenal loop preparations in 21-d-old broiler cockerels as a function of intralumenal calcium concentration 116 Figure 5.4. The effect of high calcium diet ( H C D ) on a 24 h feed intake (Mean + SE) of 4-, 7-, and 14-d-old broiler cockerels fed the diet from hatch ...118 Figure 5.5. The effect of drinking 40 m M C a C l 2 for a 24 h period on feed xiv (left panel) and water (right panel) intakes (Mean + SE) in 4-, 7-, and 21-d-old broiler cockerels fed a commercial broiler starter diet 119 Figure 5.6. The percentage of calcium retained (Mean + SE) by 4-, 7-, and 14-d-old intact broiler cockerels, in two different regimens (indicated in titles of left and right panels) used to increase calcium intake 122 Figure 6.1. Weight to length ratios (Mean + SE) of the duodenum (n= 10 to 30), distal jejunum(n= 10 to 13), and distal ileum (n=13 to 23) from 0-, 2-, 4-, 7-, and 14-d-old broiler cockerels fasted for 12 h 137 Figure 6.2. The efficiency of mannitol absorption (Mean + SE) from the lumen of the in situ duodenal (n = 10 to 30), distal jejunal (n= 10 to 13), and distal ileal (n = 13 to 23) loop preparations in 0-, 2-, 4-, 7-, and 14-d-old broiler cockerels fasted for 12 h 139 Figure 6.3. The capacity of mannitol absorption (Mean ± SE) in the in situ duodenal (n= 10 to 30), distal jejunal (n= 10 to 13), and distal ileal (n=13 to 23) loop preparation in 0-, 2-, 4-, 7-, and 14-d-old broiler cockerels fasted for 12 h 140 Figure 6.4. Mannitol secretion into the lumen of the in situ duodenal (n=20), distal jejunal (n= 11), and distal ileal (n=9) loop preparations, as a function of plasma mannitol concentration in 18-d-old broiler cockerels fasted for 12 h 142 Figure 6.5. Mannitol secretion (normalized for plasma mannitol concentration) from the blood into the lumen (Mean ± SE) of the in situ duodenal (n=20), distal jejunal ( n = l l ) and distal ileal (n=9) loop preparations from 18-d-old broiler cockerels fasted for 12 h 143 xv LIST O F T A B L E S Table 3.1. Calcium and mannitol disappearance from in situ duodenal loops, calcium retention by the duodenal tissue, relative weight of the duodenal tissue, and plasma calcium concentration (Mean ± SE) in 7-d-old broiler cockerels either continuously fed or fasted for 12 h before the experiment (n=12) 55 Table 4.1. Calcium and phosphorus concentrations in the experimental diets 67 Table 4.2. Composition of the rachitogenic and normal diets 70 Table 4.3. Body weight (n=10 to 14), and plasma total-calcium (n=7 to 12), and excreta calcium concentrations (Mean ± SE) of broiler cockerels fed the control diet (1.09 % Ca of D M ) or high calcium diet ( H C D ) (1.65% Ca of D M ) from hatch 76 Table 4.4. Body weight, and plasma total-calcium and tibia calcium concentrations (Mean ± SE) in 7- (n=18), 12- (n=7), and 21-d-old (n=6) broiler cockerels fed control or rachitogenic diets 82 Table 4.5. Body weight, tibia fat-free dry weight, tibia ash weight, and tibia length (Mean ± SE) in 12-, 13-, 15-, and 16-d-old broiler cockerels (n=9 or 10) fed the control diet or the rachitogenic diet (R. diet) 84 Table 4.6. Plasma total-calcium and hematocrit values (Mean ± SE) in 12-, 13-, 15-, and 16-d-old broiler cockerels (n= 9 or 10) fed control or rachitogenic diets 86 xvi Table 5.1. Calcium intake and retention (Mean ± SE) in 4-, 7-, and 14-d-old intact broiler cockerels, when supplemental calcium was provide as gluconate plus lactate salts in the diet, or as 40 m M C a C b in drinking water xvi i A B B R E V I A T I O N S l , 2 5 - ( O H ) 2 D 3 A P A T P c A M P c G M P CIF Da D M H C D I . U . i .m. i .v . kcal Kev M . W . n n C i N R C P P E G P T H 1,25-dihydroxyvitamin D 3 alkaline phosphatase adenosine triphosphate cyclic adenosine monophosphate cyclic guanosine monophosphate calcium influx factor dalton(s) dry matter high calcium diet international unit intramuscular intravenous kilocalorie kilo-electron-volt molecular weight sample size nanocurie(s) National Research Council probability polyethylene glycol parathyroid hormone xv i i i standard error (of mean) specific radioactivity statistical analysis system volume xix A C K N O W L E D G M E N T S Special thanks go to my research supervisor Dr . Thompson for his very valuable instruction, guidance and support. He is a good man. Dr . Thompson accepted me as a graduate student after Dr . Hart's departure for California. Thanks go to Dr . Hart for originally accepting me as her student and for stimulating my interest in the subject. Members of my research supervisory committee took special interest in my research and progress. Among them, Dr . March has been very concerned and encouraging, Dr . Blair has been very supportive and helpful, and Dr . Kitts, besides being very stimulating and helpful, became involved to the extent of providing financial assistance to this barely-funded research project. I thank my committee members for their help and suggestions. I am very thankful to Western Hatcheries, Clearbrook, B . C . for providing me with chicks for this research. I thank Dr . Youngblut, and M s . Linda Jane of Roche Canada for sending me a gift of 1,25-dihydroxyvitamin D 3 . Dr . Norman from the University of California Riverside was very kind to send me antibody for calcium binding protein. Thanks go to my friend Miche l in the Department of Biochemistry, U . B . C . , for assisting me during my attempts to develop a calcium binding protein assay. Thanks also go to Pacific Egg and Poultry Association, California for providing a financial grant for this research. I am thankful to The University of British Columbia for the awards of Graduate Fellowship, The British Columbia Egg Marketing Board for their annual scholarship awards, and the Department of Animal Science for providing the research and teaching assistantships and for the Professor B . E . March Travel Award . I thank D r . Leichter and Dr . Samuels for being helpful in my comprehensive examination, and Drs. Cheng, Shackleton, and Shelford for their help in academic matters. I pay thanks to all faculty, staff, and graduate students for providing great company and being accommodative. Sylvia Leung's help was remarkable. Thanks go to Canada for being a land of opportunity. xx DEDICATION This thesis is dedicated to my teachers in Canada, Pakistan, and the United States. xxi C H A P T E R 1 General Introduction The importance of calcium in human and animal nutrition is well recognized. Numerous disorders such as osteoporosis in humans and skeletal abnormalities in rapidly growing broiler chickens have been linked to calcium malnutrition. Calcium nutrition has a significant role in public and animal health, and animal production. The intestine is the sole port of calcium entry into the animal body under normal conditions and, therefore, is an organ of major importance in calcium nutrition. In terms of understanding and further developing physiological and clinical aspects of improving calcium nutrition, information pertaining to regulation mechanisms of intestinal calcium transport is of great significance. Intestinal calcium transport involves translocation of calcium ions from the intestinal lumen to the lateral space occupied by the lamina propria. There are two possible pathways for this translocation, the cellular pathway and the paracellular pathway. In the cellular pathway, calcium ions move via the cytosolic compartment whereas in the paracellular pathway, calcium ions move via the tight junctions between adjacent cells lining the lumenal surface of the intestine. The cellular pathway has been shown to be saturable and active whereas the paracellular pathway is generally considered to be nonsaturable and passive. The l well established effects of vitamin D on the regulation of intestinal calcium transport are generally considered to apply only to the cellular pathway (Wasserman and Fullmer, 1995). Most of our information pertaining to the relative contribution of the cellular and paracellular components of intestinal calcium transport has been obtained with the laboratory rat. Unfortunately, the nature of information provided by different research groups has been determined to a large extent by the particular experimental model used. Using the saturation technique, Pansu, Bronner and associates (Pansu et a l . , 1981, 1983b) concluded that the cellular pathway of calcium transport from in situ loops is restricted to the proximal intestine, in the laboratory rat. In addition, these researchers demonstrated that the paracellular component of calcium transport, determined by slope of the absorption curve, did not change along the entire length of the rat small intestine. In contrast, Nellans and Kimberg (1978) demonstrated with isolated intestine mounted on Ussing chambers that a low dietary intake of calcium markedly increases the cellular component of calcium transport in the ileum. More recently, Karbach (1991, 1992) was able to show that intestinal calcium transport in all segments of the rat small intestine mounted on Ussing chambers has a significant cellular component. With the same method, Karbach and Feldmeier (1993) reported that nearly 45 % of total calcium absorption in the rat cecum is cellular and that the cecum is the site with the highest efficiency of calcium absorption in the rat intestine. The regulation of intestinal calcium transport in avian species has largely been studied with layer-type chickens. Wasserman (1962) was able to stimulate calcium transport from in situ duodenal, jejunal, and ileal loop preparations, after vitamin D treatment of young layer-2 type chickens. These results indicate that a cellular component is present throughout the length of the chicken small intestine. Hurwitz et al . (1995) showed that boiler-type young chickens develop hypercalcemia when fed a diet containing 1.5 to 2% calcium whereas layer-type young chickens remained normocalcemic. It is possible that mechanisms of intestinal calcium transport vary between these two types of chickens, to match their growth needs. It may be noted that the incidence of skeletal abnormalities is primarily associated with rapidly growing broiler-type young chickens. To the best of the author's knowledge, the relative contribution of the vitamin D-dependent and vitamin D-independent components of intestinal calcium transport in rapidly growing young broiler chickens has not been reported in the literature. Although the well established effects of vitamin D are accounted for by its regulation of the cellular pathway, it has also been hypothesized that vitamin D stimulates calcium transport via the paracellular pathway. This hypothesis is controversial. Findings by Wasserman et al. (1966) that vitamin D stimulates both calcium absorption and calcium secretion in in situ ileal loops in the layer-type chicks, provided the first indication that vitamin D may increase the diffusional permeability of the intestine to calcium by altering paracellular mechanisms. Karbach (1991, 1992) accumulated evidence that showed vitamin D treatment to increase the paracellular permeability in all regions of the rat small intestine as demonstrated by its effects on mannitol transport, a commonly used marker molecule restricted to the intercellular space. On the other hand are data which show that vitamin D does not regulate calcium transport via the paracellular pathway. Pansu et al. (1983b) demonstrated, that calcium transport in the rat distal small intestine is entirely paracellular and that it cannot be stimulated in response to 3 vitamin D treatment. Similarly, Bronner (1992), and Bronner and Stein (1995) maintained the view that paracellular calcium transport is not under acute regulation by vitamin D . A large body of research related to regulation mechanisms of the cellular pathway has eclipsed a relatively much smaller body of research related to the significance of calcium transport via the paracellular pathway. Since the permeability of the mucosal epithelial lining of the intestine is high (Fromter and Diamond, 1972), large quantities of water and solutes are expected to cross the mucosal barrier via the paracellular pathway. Pappenheimer (1990) demonstrated that a major portion of nutrients such as glucose and some amino acids are absorbed from the intestinal lumen of laboratory rodents via the paracellular pathway. The significance of the paracellular pathway in intestinal calcium transport needs to be studied. The present research is the first to explore the mechanism and regulation of intestinal calcium transport in rapidly growing young broiler cockerels. 1.1. Research Objectives 1. To develop an in situ intestinal loop experimental system to study the cellular and paracellular components of intestinal calcium transport in rapidly growing young broiler chickens. 2. To determine the fractional contribution of the vitamin D-dependent and vitamin D-independent components of calcium transport in the small intestine of rapidly growing young broiler chickens. 3. To determine the significance of paracellular calcium transport in intact young broiler chickens. 4. To determine: a) the age-related and regional differences in paracellular absorption, and b) the regional differences in paracellular secretion in the small intestine of young broiler chickens. 5 C H A P T E R 2 Review of Literature 2.1. Calcium Homeostasis The electropositivity and ionic radius of calcium determine its high protein-binding affinity that enables it to perform a plethora of vital regulatory functions in biological systems. A s an intracellular messenger in the signal transduction cascade, calcium ions are ubiquitously involved in regulating functions such as cellular metabolism, exocrine and endocrine function and secretion, neuromuscular activity, growth, and mitosis. The intracellular environment is highly sensitive to calcium ion concentration, since phosphate esters are highly abundant in the cytosol and calcium phosphates, being very insoluble, may precipitate in the cytosol when the calcium ion concentration is high. The cytosolic concentration of calcium ions is typically 0.1 p M , several orders of magnitude less than its concentration in the extracellular space. (Fullmer, 1992). Persistently subnormal plasma calcium concentrations may lead to the development of pathological states such as tetany, rickets, or osteomalacia. In effect, intracellular calcium concentration is one of the most tightly regulated parameters in land vertebrates (Hurwitz, 1996; Bronner and Stein, 1995). Regulation of the calcium concentration in body fluids is achieved through the action of a complex feedback-control system that includes several subsystems and regulating hormones. Most of total body calcium is associated with the skeleton in the form of hydroxyapatite crystals, while the rest resides in the intracellular and extracellular fluid compartments of the 6 soft tissues. Calcium in the extracellular compartment is in constant exchange with calcium in the intracellular compartment, certain compartments of the bone, and the glomerular filtrate (Norman, 1990). Calcium in body fluids is present in three forms: ionized, complexed with organic acids such as citrate, and bound to proteins (Robertson, 1976). The ionized calcium is diffusible, that complexed with citrate is not ionized but is diffusible, and the protein bound form is neither ionized nor diffusible. Nearly 50% of the plasma calcium is in the free ionized form, and 40% is protein bound. Calcium in the urine and cerebrospinal fluid is either in the free ionized or complexed form but is not protein bound. The plasma calcium concentration in normal animals rarely varies more than 10% from its mean 2.5 m M value. Most of the intracellular calcium is normally kept sequestered by cellular organelles such as the mitochondrion, endoplasmic reticulum, or Golgi apparatus, and is released in response to stimuli destined to regulate cellular functions. The resulting increase in cytosolic calcium concentration is transitory, as calcium is rapidly re-sequestered by the organelles, or extruded from the cell . Apart from cellular organelles, three organ systems, namely the intestine, bone, and kidney play primary roles to establish calcium homeostasis, since these organs constitute the major portals of calcium entry into and exit from the blood. Calcium that enters the blood after absorption from the gut, or after a parenteral injection has four essential fates. These are, 1) it may be used for growth and production, 2) it may be sequestered by bone and cellular organelles, 3) it may be excreted via the urine, or 4) it may be secreted from plasma into the intestinal lumen for excretion via the feces. Calcium from the plasma ultrafiltrate that enters 7 the lumen of the nephron is largely reabsorbed in the distal convoluted tubule, the remaining quantities are excreted in the urine. Bone is subject to continuous remodeling and in this process replenishes or sequesters plasma calcium. After an increase by a bolus intravenous injection of calcium, the plasma calcium concentration returns to normal in about 40 min in growing birds (Hurwitz et a l . , 1983), and in about 1 h in rats (Bronner and Stein, 1992). The shortness of the period involved in normalizing the experimentally perturbed plasma calcium concentration has led to an evaluation of the concept that surfaces of the mineral in bone act as a calcium buffer (McLean and Urist, 1968). Bronner and Stein (1995) have calculated that bone, not the intestine or kidneys can account for such a normalization of plasma calcium concentration. Since, the sequestration of calcium by or release of calcium from the bone mineral surface appears to be a noncellular process (Bronner and Stein, 1995), the explanation pertaining to the physiological basis of regulation of calcium homeostasis by this process is pending. Parathyroid hormone (PTH), calcitonin, and vitamin D are the major molecules involved in regulating calcium homeostasis. The initial response of P T H , and calcitonin is within minutes after a perturbation of plasma calcium ion concentration (Hurwitz, 1996). In terms of the magnitude of response, P T H is of relatively greater importance (Parfitt, 1994) compared to calcitonin. Parathyroid hormone is a peptide hormone that stimulates bone resorption, causes augmentation of renal tubular calcium absorption, and is involved in biological activation of 8 the vitamin D molecule. Indirectly important in calcium homeostasis is the phosphaturea induced by P T H (Kinoshita et a l . , 1986). Collectively all these actions of P T H increase calcium flow from bone to the circulation. Dietary calcium deficiency in the chicken leads within a few days to large increases in parathyroid size (Hurwitz and Grimmer, 1961). Growing chicks can hardly survive parathyroidectomy due to reduction of plasma calcium to lower than 1 m M (Bar et a l . , 1972). Receptors of P T H have been identified in rat osteoblast-like cells, opossum kidney cells, and human osteoblast-like cells (Abou-Samra et a l . , 1992). The P T H receptor and receptor binding are down regulated in kidney cells (Abou-Samra et a l . , 1994) , and osteoblast-like cells (Okano et a l . , 1994) by continuous exposure to P T H . Calcitonin is also a peptide hormone that is secreted in response to an elevated calcium ion concentration (Care and Bates, 1972). Tauber (1967) identified the ultimobranchial origin of calcitonin i n the chicken. In contrast to P T H , calcitonin lowers plasma calcium ion concentration, by inhibiting bone resorption (Raisz and Niemann, 1967). The importance of calcitonin in calcium homeostasis has not been established, since thyroidectomy results in a transient and small hypercalcemic response (Kalu et a l . , 1975). Munson and Hirsch (1992) concluded that calcitonin could protect against hypercalcemia under extreme conditions, but under ordinary conditions this protection may not be required. Calcitonin receptors have been identified in the kidney (Marx et a l . , 1973), and osteoclasts (Nicholson et a l . , 1986). A s with P T H , and other peptide hormones, continuous exposure to calcitonin leads to down-regulation of receptor binding by suppression of the calcitonin receptor gene expression (Wada et a l . , 1995) . 9 Vitamin D acts in multiple ways to regulates calcium homeostasis. The effects of vitamin D on calcium homeostasis and the effects of P T H and calcitonin on vitamin D metabolism are outlined in the following section. 2.2. Vitamin D in Calcium Metabolism Vitamin D designates a group of closely related compounds that possess the property of preventing the occurrence of rickets. The two most prominent forms of vitamin D are ergocalciferol (vitamin D2) and cholecalciferol (vitamin D 3 ) . Ergocalciferol is derived from a common plant steroid precursor, ergosterol. Cholecalciferol is produced from its precursor 7-dehydrocholesterol primarily in the Malpighian layer of the epidermis, by a nonenzymatic photolysis reaction (Esvelt et a l . , 1978). 7-Dehydrocholesterol is derived from cholesterol or squalene, which is produced in the body. Precursors of vitamin D have no antirachitic property (anonymous). The precursors of vitamin D are widely distributed in nature. O n the other hand, the level of vitamin D in precursor sources is dependent upon irradiation of the source. Vitamin D2 has relatively limited bioavailability for birds compared to that of vitamin D 3 . Since commercial poultry are raised under confined conditions, where birds are not exposed to ultraviolet radiation, vitamin D supplementation of rations is essential since most feed ingredients provide negligible amounts of vitamin D . Dietary vitamin D is primarily absorbed with fat from the distal intestine through the lacteal system into the chylomicrons (Norman and DeLuca, 1963). Bile represents the major excretory route for vitamin D (DeLuca and Schnoes, 1976). 10 Since a time lag of 6 to 8 h was observed between vitamin D 3 administration and its biological response, the possibility that vitamin D 3 undergoes a biological transformation to elicit activity was predicted by Wasserman (1962). To become fully active, vitamin D 3 undergoes two hydroxylations in the body. The first hydroxylation takes place in the microsomes of the liver (Ponchon and DeLuca, 1969) to convert vitamin D 3 to 25-hydroxy vitamin D 3 . The second hydroxy lation takes place in the kidney mitochondria (Fraser and Kodicek, 1970), to convert vitamin D 3 into its metabolically most active form 1,25-dihydroxyvitamin D 3 ( l ,25-(OH )2D3) . Although other metabolites of vitamin D have been identified, Brommage and DeLuca (1985) provided strong evidence that the only active form was l ,25-(OH )2D3. This form of vitamin D is also linked to the autosomal recessive disorder vitamin D-dependency rickets type I, which is likely a defect in the enzyme la-hydroxylase (Fraser e ta l . , 1973). l,25-(OH)2D3 biosynthesis is primarily regulated at the la-hydroxylase level. Parathyroid hormone stimulates la-hydroxylase activity and thereby acutely regulates the biosynthesis of l,25-(OH)2D 3 (DeLuca and Schnoes, 1976; Fraser, 1980). The effects of high calcium intake on l,25-(OH)2D 3 biosynthesis appear to be secondary to P T H since P T H concentration varies in a reciprocal manner to serum calcium and phosphorus concentrations (Fraser, 1980). Hypocalcemia in intact animals results in a marked elevation of the l a -hydroxylase both in vivo (Boyle et a l . , 1971) and in vitro (Tanaka and DeLuca, 1981). 11 The vitamin D metabolite, l ,25-(OH )2D3, is now widely considered to be a hormone since its mechanism of action in some tissues is similar to the mechanism of action of steroid hormones (Norman, 1990). Stumpf et al . (1979) provided evidence that l , 2 5 - ( O H ) 2 D 3 localizes in the nucleus of target tissues. Vitamin D-dependency rickets type II has been shown to be caused by a series of mutations in the vitamin D receptor gene (Brooks et a l . , 1978). In most target tissues, l ,25-(OH )2D3 binds with selected genes to effect production of m R N A s coding for calcium-binding proteins (CaBP) and other proteins. Some effects of 1,25-(OH)2D3 have been attributed to these proteins. l , 2 5 - ( O H ) 2 D 3 has versatile physiological activity and is reported to produce effects in multiple systems of the animal body (DeLuca, 1988). There exists some evidence that l ,25-(OH )2D3 may act in a non-genomic manner (deBoland and Norman, 1990) which remains to be characterized. The presence of a specific high-affinity vitamin D intracellular receptor in the intestinal absorptive cell has been identified by Brumbaugh and Haussler (1974). Furthermore, 1,25-(OH)2D3 was localized in the nucleus of crypt and villus cells of the intestine after injecting radiolabelled l , 2 5 - ( O H ) 2 D 3 in vivo (Stumpf et a l . , 1979). Intestinal calcium transport is stimulated in response to l ,25-(OH )2D3 treatment. This effect is considered to be primarily mediated by C a B P . Wasserman and Taylor (1966), isolated a C a B P with a molecular weight of approximately 28,000 Da (calbindin-28k) from the mucosa of the chicken intestine. A C a B P of 9,000 Da molecular weight (calbindin-9k) was discovered soon after in the rat intestine (Kallfelz et a l . , 1967). Both of these proteins have a high binding affinity for calcium. Calbindin-9k has two domains for binding calcium and has so far been detected only in mammalian tissues, whereas calbindin-28k has four such domains and has been isolated from a 12 variety of avian and mammalian tissues, but not in the mammalian intestine (Christakos et a l . , 1989). It must be pointed out that the expression of some of the C a B P is vitamin D-dependent only in some tissues (Christakos et a l . , 1989). It is well established that 1,25-(OH)2D3 is indirectly required for normal skeletal growth and remodeling. Osteoblasts represent a key target for vitamin D . Vitamin D causes the synthesis and secretion of a number of bone-specific proteins in the osteoblasts, such as osteocalcin (Lian and Gundberg, 1988), osteopontin (Butler, 1989), and alkaline phosphatase (Kyeyune-Nyombi et a l . , 1989). Normal biosynthesis of collagen in the bone organic matrix is promoted by vitamin D (Dickson et a l . , 1979). There is, however, evidence that vitamin D is not directly involved in bone mineralization. Underwood and DeLuca (1984) demonstrated that bone development and mineralization are normal in vitamin D-deficient rats when intravenously infused with calcium and phosphorus solutions. Interestingly, these researchers were able to further show that vitamin D-deficient rats infused with calcium and phosphorus solutions had greater amounts of mineral and matrix in their bones than did those rats only treated with vitamin D , pointing out that bone mineralization was dependent on availability of minerals and not on vitamin D . 2.3. Calcium Transport Across the Intestinal Barrier 2.3.1. Physiological Anatomy of the Intestinal Wall The epithelium on the lumenal surface of the intestine is organized into two morphologically and functionally distinct cellular compartments: the crypts of Lieberkuhn and 13 the v i l l i . The crypts lie at the base of the v i l l i and constitute a repertoire of stem cells with immense proliferation potential. The crypt cells divide, differentiate into four categories, and move along the crypt villus axis. Three of them, the enterocytes, goblet cells, and enteroendocrine cells move towards the apex whereas the fourth type, the Paneth cells, migrate towards the crypt base. The absorptive surface of the intestine is, therefore, a monolayer of terminally differentiated epithelial cells. The four cell types of the intestinal epithelium have specific functional roles. The enterocytes are the absorptive cells with a directionality of nutrient flow from the brush border-bearing lumenal membrane to the basolateral membrane. A t the basolateral membrane, the nutrients exit into the extracellular fluid in the lamina propria and ultimately diffuse into the general circulation. Goblet cells produce mucin which provides protection against chemical damage to the intestinal absorptive surface. The production by the enteroendocrine cells of hormonal substances such as the serotonin, gastrin, and vasoactive intestinal peptides is well known. These hormonal substances are considered to contribute to several intestinal functions. Paneth cells produce lysozyme which help prevent microbial invasion through the gut wall . Among the four general types of membrane contacts between adjacent epithelial cells namely, tight junctions, adherens or intermediate junctions, desmosomes, and gap junctions, the tight junctions are important in terms of solute passage across the tissue (Ballard et a l . , 1995). Tight junctions are protein complexes that are part of the actin cytoskeleton (Madara et a l . , 1987). A n extensive actin network supports the apical microvi l l i in the intestinal epithelial cells (Matsudaira and Burgess, 1982) and is linked to the tight junctions (Drenckhahn and 14 Dermietzel, 1988) (Figure 2.1). Tight junctions appear in electron micrographs as complex strands of fibrils embedded in the cell membrane (Staehelin, 1973). Adherens junctions are located immediately below the tight junctions and are tightly coupled to a circumfrential actin-myosin II ring (Madara and Pappenheimer, 1987). This peri-junctional actin ring has been demonstrated to be a dynamic structure which may transmit cytoskeletal changes to the tight junctions (Madara, 1987). The complexity and number of tight-junction strands generally correlate with the permeability of the epithelia (Claude, 1978). A high permeability of the intestinal absorptive epithelium is attributed to the relatively little resistance of passage offered by the intercellular membrane contact provided by the tight junctions (Madara, 1989). 2.3.2. Mechanisms Involved in Intestinal Calcium Transport The intestinal epithelial barrier is the gateway for dietary nutrients to reach the peripheral circulation. Intestinal calcium transport is essentially a transmucosal transport process, as the mucosal layer of the intestine is the only barrier that calcium has to cross to reach the portal circulation. The different mechanisms involved in intestinal calcium transport and discussed in the following text are shown in Figure 2.2. There are two possible pathways by which calcium crosses the intestinal epithelial barrier; the cellular pathway and the paracellular pathway. In the cellular pathway, calcium ions move via the cytosolic compartment of the enterocytes. In the paracellular pathway, the movement of calcium ions takes place via the extracellular spaces bounded by the tight junction between adjacent enterocytes. 15 a S3 u .a E au u X5 H co •5 s & 3 03 CD o3 J3 5 © CO 03 o '£ 03 X> 13 03 o Xi c "3 ^ o c _o *4-t 03 •*-» c 1/3 tu l-l U i o3 s O GO .6.2 .3 £ '3, 4-> CJ CO et< co IS O 03 ^ s ° H u 53 X3 CI, 4-> " 2 U ° ^ co 2 « 3 •2 -5 " B •2, 3 £ -2 .2? o 4-> £3 <^> 3. .2 ob > <u o o <D j3 ON ^1 -3 ON 03 ^ 4-" CO 2 § d 6 I ts CO - H _ _ U M » +->+-> 03 c u ^ ^ c £ o • - g o « Jo ^ i 16 17 2.3.2.1. The Cellular Pathway The general scheme of events for the cellular pathway of intestinal calcium transport involves three sequential steps: 1) calcium entry into the cytosol via the apical membrane, 2) transmural calcium movement from the apical membrane to the basolateral membrane of the enterocyte, and 3) calcium extrusion at the basolateral membrane into the lateral space occupied by the lamina propria. From the lateral space, calcium diffuses into the portal circulation. Details of the mechanisms involved in these steps has been recently reviewed (Fullmer, 1992; Stein, 1992; Wasserman et a l . , 1992a). Two models have been described to explain intestinal calcium transport via the cellular pathway. The first model may be called the classical model based on its wider recognition. The second model is referred as the vesicular model, since intracytoplasmic vesicles have been shown by Norman and his colleagues (Norman, 1990, Nemere, 1992; Nemere and Norman, 1989) to play a significant role in intestinal calcium transport via the cellular pathway. 2.3.2.1.1. Classical Model of the Cellular Pathway 2.3.2.1.1.1. Calcium Entry Calcium entry at the brush border is the least understood phenomenon (Bronner et a l . , 1986). Direct evidence for the mechanism of calcium entry into the enterocyte through the brush border membrane is lacking. Based on the difference in the intralumenal free calcium concentration (-10 m M ) and the intracellular free calcium concentration (10" 7-10 6 m M ) , it is generally believed that calcium entry is a process of simple diffusion proceeding down an 1 8 electrochemical gradient which does not require metabolic energy. The fact that calcium entry is saturable at high concentrations of lumenal calcium (Rasmussen et a l . , 1979), suggests the existence of calcium channels or a transporter, although direct evidence for either is lacking. Rasmussen et al . (1979) reported that calcium entry is energy independent, although saturable. Alkaline phosphatase (AP) activity associated with the brush border has been implicated to be involved in calcium entry into enterocytes (Holdsworth, 1970; Norman et a l . , 1970). A possible mechanism involving A P is through increasing the concentration of intralumenal free calcium by hydrolyzing organic pyro- and orthophosphates, to which calcium may be bound. A P activity in enterocytes is enhanced in response to vitamin D treatment (Norman et a l . , 1970). Nasr et al . (1988) suggest a temporal coincidence between enhanced A P activity and calcium entry, though others disagree. Pansu et a l . (1989) demonstrated that theophylline significantly inhibits A P activity but not calcium transport in isolated brush border membrane vesicles suggesting that A P is not involved in calcium entry. The role of A P in calcium entry into the cell , remains unclear. A n increase in the fluid state of the brush border membrane is likely to enhance calcium permeation. Matsumoto et al . (1981) demonstrated an increased biosynthesis of phosphatidylcholine in isolated enterocytes in response to l ,25-(OH )2D3 administration to vitamin D-deficient chicks. They also noted an increased incorporation of arachidonic acid into the phosphatidylcholine fraction of the brush border, and showed that the time of change in the incorporation of choline and ethanolamine into the brush border l ipid fraction closely correlates with the time course of change in calcium uptake by the brush border membrane 19 vesicles. Similar observations were made by Wasserman et al. (1982). It appears that the l ipid composition of cell membranes may be a factor involved in regulating calcium entry into the cell . Calcium-dependent modulation of the brush border cytoskeleton may regulate calcium entry into the enterocyte. Mooseker et al . (1991) speculated that calcium-dependent binding of the microvillar calmodulin (Howe et a l . , 1982) to myosin-I, an ATPase with mechanoenzyme properties (Bikle et a l . , 1991), might effect permeability of the microvillar membrane for calcium. The vitamin D dependence of these effects has been recently demonstrated by Kaune et. al . (1994). In the vitamin D-deficient state, calcium readily enters the intestinal cell but remains associated with the microvillar region (Chandra et a l . , 1990; Sampson et a l . , 1970). The plasma membrane-bound fraction of calbindin-28k (Feher and Wasserman, 1978) has also been hypothesized to play a role in internalization of calcium by the cell (Norman, 1990). Purified intestinal brush border preparations from vitamin D-deficient chicks contain essentially no calbindin-28k whereas calbindin-28k amounts to - 1 2 % of the membrane protein in preparations isolated from vitamin D-replete chicks (Shimura and Wasserman, 1984). 2.3.2.1.1.2. Transmural Calcium Transport Facilitated diffusion of intracellular calcium from the brush border membrane to the basolateral membrane of the enterocyte is the most popular model of transmural calcium transport. Using a flow dialysis model, Feher (1983) suggested facilitated diffusional transport 20 of calcium by cytosolic calbindin-28k. Pansu et al . (1989) reported that theophylline inhibits calcium binding by calbindin-9k and decreases calcium transport in the rat. These observations and those by others (Fullmer, 1992) implicate a transmural calcium transport role for calbindins. Stein (1992) hypothesized that free calcium and calbindin are in a state of dynamic equilibrium inside the cell so that calbindin does not function as a transporting vehicle but increases the partitioning coefficient of calcium within the cytosol. Since calbindin concentration in the enterocyte (100 p M ) is approximately 1000 fold the concentration of intracellular free C a + + (Bronner et a l . , 1986), and since the diffusion coefficient of calcium bound to calbindin compared with the diffusion coefficient of hydrated calcium is very high (Feher et a l . , 1989; Feher, 1983), Stein (1992) predicted that calbindin-9K in rat enterocytes may augment the transmural calcium flux 75 fold compared to when calbindin is absent. This value of augmentation conforms to that predicted by Bronner et al . (1986). Wasserman and Fullmer (1995) have proposed, that diffusional transmural C a + + transport is accomplished by a cascade of events. In this cascade, the Ca + + -b ind ing affinity of carriers increases according to their relative location from the brush border pole to the basolateral pole of the enterocyte. Calcium, first binds to calcium-binding sites associated with brush border membrane (Wilson and Lawson, 1980) and is subsequently released from these sites to bind to calmodulin (Glenney and Glenney, 1985), followed by its release from calmodulin, calcium binds to calbindin. In the last step, calcium is released by calbindin and binds to the calcium extrusion pump located at the basolateral side of the plasma membrane. Calcium is extruded from the cell by the extrusion pump in an energy dependent manner (Wasserman et a l . , 1992a). The calcium-binding affinities increase at each step as calcium 21 moves from the brush border membrane to the calcium extrusion pump during the proposed cascade of transmural diffusional transport (Bredderman and Wasserman, 1974; Glenney and Glenney, 1985; Wilson and Lawson, 1980). This proposed mechanism makes calcium transport through the cytosolic compartment a thermodynamically favorable process. 2.3.2.1.1.3. Calcium Extrusion To ensure cellular viability, intracellular free calcium, which cannot be sequestered or buffered must be immediately extruded against an electrochemical gradient. Extrusion of calcium from the basolateral side of enterocytes into the extracellular space in the lamina propria takes place against a steep electrochemical gradient. In addition to a positive electropotential difference of 58 m V , an approximately 50,000 fold greater concentration of free calcium exists in the extracellular fluid compartment compared to the cytosolic compartment (Wasserman and Fullmer, 1995). Two systems capable to extrude C a + + from the enterocyte against this considerable electrochemical potential differential have been isolated and characterized. The first is an ATP-dependent calcium pump (calcium-ATPase), and the second is an N a + / C a + + exchanger. Penniston and Enyedi (1994) have recently reviewed the properties of calcium ATPase. Calcium ATPase transports one mole calcium per mole A T P hydrolyzed with a half saturation constant (Km) of 0.2 p M in the presence of calmodulin. A net increase in the synthesis of calcium ATPase (Wasserman et a l . , 1992b) and an increase in the gene expression of calcium ATPase (Cai et a l . , 1993) have been reported in the duodenum of layer-type chicks in response to l,25-(OH)2D3 treatment. The N a + / C a + + exchanger, on the other hand is reported to be independent of vitamin D and accounts for nearly 20% of calcium 22 extrusion from the rat duodenum (Ghijsen et a l . , 1983). The capacity of the calcium ATPase exceeds the rates of intracellular diffusive movement of calcium (Bronner, 1992). 2.3.2.1.2. The Vesicular Model of the Cellular Pathway Mitochondria (Sampson et a l . , 1970), Golg i apparatus (Freedman et a l . , 1977), and more recently microsomes (Rubinoff and Nellans, 1985) are involved in intracellular calcium sequestration in many tissues. The role of these organelles during enterocyte transmural calcium transport is not clear. Davis and Jones (1981) and Nemere and Szego (1981) suggested the involvement of lysosomes in vitamin D mediated calcium transport responses in the intestine. Nemere et al. (1986) provided experimental evidence that lysosomes act as a carrier for transmural movement of calcium and proposed an endocytotic-exocytotic model for intestinal calcium transport. The role of calbindin was also implicated in this model because it was found associated with lysosomes. Norman (1990) proposed a pathway by which calcium is recognized by a specific moiety at the brush border membrane, probably calbindin, and internalized by endocytosis. The endocytic vesicles are then conveyed along the microtubules and coalesce with lysosomes, and move along the microtubules to the basolateral border where exocytosis of calcium with calbindin (Lee et a l . , 1988) completes the transport process. 2.3.2.2. The Paracellular Pathway In the paracellular pathway of nutrient transport, the solute moves between adjacent cells via the tight junctions. This pathway is known to be nonsaturable, energy independent, and therefore passive (Bronner, 1992). A solvent drag effect is frequently proposed as a 23 mechanism that facilitates calcium absorption via the paracellular pathway which implies convective transfer of lumenal calcium to the extracellular space (Munk and Rasmussen, 1977; Nellans and Kimberg, 1979; Karbach, 1992). Active extrusion of calcium by the transmembrane calcium extrusion pump located in the basolateral cell membrane (Wasserman et al., 1992b) results in increased concentrations of calcium in the extracellular space occupied by the lamina propria, creating an osmotic gradient between the intestinal lumen and the extracellular space. This osmotic gradient attracts lumenal water along with the solutes, thereby, leading to net solute absorption via the paracellular pathway; the mechanism is called solvent drag. It is important to note the paradox that emerges between the concept that paracellular transport is passive and energy independent and the concept that paracellular transport is driven by solvent drag which depends upon cellular mechanisms. There is evidence in the literature that paracellular transport is also subject to regulatory control (Pappenheimer, 1990; Madara and Pappenheimer, 1987). This evidence challenges the conventional theories of solute and water absorption from the intestine (Ballard et al., 1995). Pappenheimer and associates noted that an increase in the permeability of the tight junctions was a squeal to intestinal transport of glucose or amino acids such as alanine (Pappenheimer, 1990). Based on the fact that glucose and amino acid transport is coupled to that of sodium transport, it was proposed that intracellular sodium modulates tight junction permeability through specific changes in the cytoskeleton (Pappenheimer and Volpp, 1992). Since hypertonicity in the space occupied by the lamina propria dilates the lateral intercellular spaces and decreases transepithelial resistance for convective flow, whereas 24 hypertonicity on the lumenal side has the opposite effect (Madara, 1983; Reuss and Finn, 1977), the question arises whether a physical change in the lateral intercellular space is subsequent to l iquid absorption or is independent of this effect. Pappenheimer and Reiss (1987) demonstrated that dilation of the lateral space was independent of liquid movement, because the dilation was observed even when ferrocyanide, an impermeable osmolyte, was used as a solute. From this observation, Madara and Pappenheimer (1987) concluded that a concomitant contraction of the cytoskeleton and not a solvent drag effect was responsible for dilation of the lateral intercellular spaces. These conclusions are tenuous because the ferrocyanide-associated decrease in liquid flow was reduced by only 20% (Pappenheimer and Reiss, 1987). Whereas chemical modulation of the cytoskeleton is well known, the inductive effect of cytoskeletal changes in the tight junction complex is an area of emerging information. A change in the actin cytoskeleton could induce a functional alteration in the tight junction by varying the tension applied to the junctional complex. A n elevation in intracellular c A M P levels has been reported to directly correlate with paracellular permeability (Duffey et a l . , 1981). Similar effects have been noted following addition of phosphodiesterase inhibitors (Bakker and Groot, 1984). Phorbol dibutyrate, a potent stimulator of protein kinase C , increases tight junction permeability in hepatocyte couplets (Nathanson et a l . , 1992). Al so , an increase in intracellular calcium has been associated with a decrease in tight junction permeability (Palant et a l . , 1983). Whether or not the intracellular free calcium concentration changes in response to calcium uptake from the intestine is not known. Such an effect would 25 possibly influence the contribution of the paracellular mode of calcium transport. The Pappenheimer hypothesis, therefore, has a theoretical basis and awaits further evidence. 2.3.3. Kinetics of Intestinal Calcium Transport When the rate of intestinal calcium transport is plotted against a wide range of intralumenal calcium concentration, the fit obtained is curvilinear in shape (Pansu et a l . , 1981; 1993). This type of fit is suggestive of the coexistence of saturable nonsaturable components of the intestinal calcium transport process. Based on kinetic considerations, the rate of net calcium transport from the mucosal side of the intestine to the serosal side of the intestine, Jm - * , can be mathematically expressed as a composite function of the saturable plus nonsaturable components: Jm^s= A + P[Ca;+] where A is the net saturable C a + + flux, P is the slope (or rate) of the net nonsaturable calcium flux as a function of [Caf+], the intralumenal calcium concentration. Wasserman and Taylor (1969) proposed an analogy of the saturable component of intestinal calcium transport kinetics to the Michaelis-Menten type of enzyme kinetics. The mathematical expression of the saturable component, A , therefore is: ~ K, + [Ca;+] 26 where Fmax is the maximum rate of the saturable component of calcium transport, K, is the apparent half saturation constant of the saturable component, that is, the intralumenal free calcium concentration for which the half-maximum rate of the saturable component is achieved, and [Ca, + + ] i s the intralumenal free calcium concentration. If the rate of net C a + + transport is plotted as function of [Ca*+], the slope of the regression line, P, obtained with high calcium concentrations only, represents the rate of the nonsaturable component for a given [Caf* ] and the y-intercept represents V m a x . The mechanisms of the cellular pathway involves proteins, the biosynthesis or activity of which may be rate limiting. Since there must be an upper limit to the capacity of calcium transport via the enterocytes, the saturable component is hypothesized to represent transport via the cellular pathway. By contrast the nonsaturable component represents transport via the paracellular pathway (Pansu et a l . , 1981). The very nature of the intestinal calcium transport curve is used to separate the saturable component from the nonsaturable component of this process. A regression line drawn through the data points obtained at high intralumenal calcium concentrations wi l l create an intercept representing the maximum rate of calcium transport via the cellular pathway, and a slope representing the rate of calcium transport via the paracellular pathway (Pansu et a l . , 1981). Using this model, it is clear that the rate of calcium transport via the saturable route 27 attains a constant value beyond a certain intralumenal calcium concentration, whereas the rate via the nonsaturable route increases as a linear function of the concentration (Figure 2.3). The nature of cellular mechanisms involved in vesicular calcium transport are different from those used in the classical model of intestinal calcium transport via the cellular pathway (Wasserman and Fullmer, 1995; Pansu et a l . , 1993). It is, therefore, difficult to predict the kinetic characteristics of intestinal calcium transport proceeding via the vesicular pathway. 2.3.4. Factors Regulating Intestinal Calcium Transport 2.3.4.1. Regulation by Vitamin D The intestinal calcium transport process occurs in a biphasic fashion following 1,25-(OH)2D3 administration with an initial rapid stimulation of calcium transport, reaching a peak after 6 h. This response is followed by a decline before increasing to a new higher level by 24 h post-administration, with the new level maintained for at least 96 h (Halloran and DeLuca, 1980). The initial rapid response can be re-induced by a booster injection of l,25-(OH)2D3, but the second phase response cannot be re-induced by a booster injection. It has been argued that the initial rapid response is generated by the existing villus cells and is nuclear mediated, whereas the second phase response is generated by the crypt cells which apparently are programmed for calcium transport by l,25-(OH)2D3. This capacity of calcium transport is retained as progress along the villus occurs (DeLuca and Shnoes, 1983). 28 ,1 O CO CV, " co cu it w n 3 to 2 « CN C3 oo TJ S cu j j o d s u B j ; u m p r e D jo 3 ; B ^ IS aJ . CJ CO — a a co "J3 CU cS M ? C+H cd o 3 CO cu 03 ^ a , co H o "3 o S 2 a3 • £ . 2 ° o S 3 < 8 03 is "cu WD cu cu c cu E 7 3 cu 03 i .3 03 O X ! >> „ .9 W 2 3 g DC T5 .2 e cS co 2 S 2 & T3 2 M ^  3 co CO -S CU £ cu T 3 H CU o3 co co 3 O co \j3 cu cu 3 c 7 3 •f co a cu c a 03 -3 29 l,25-(OH)2D3 receptor occupancy is correlated with the expression of calbindin in the cytosol, and the calbindin levels are correlated with the rate of intestinal calcium transport (Hunzikar et a l . , 1982). Morrissey and Wasserman (1971) found a significant correlation between the rate of calcium absorption in the in situ duodenal loops of layer-type chicks, and cytosolic calbindin concentration. The expression of calbindin, therefore, appears to serve as a good marker for quantitation of vitamin D-mediated stimulation of intestinal calcium transport via the cellular pathway. The role of vitamin D in regulating intestinal calcium transport, however, may not be limited to mechanisms involving biosynthesis of calcium binding proteins. Spencer et al . (1978) reported that following l,25-(OH)2D3 treatment of vitamin D-deficient chickens, intestinal calcium transport was stimulated before adequate induction of calbindin occurred. Wasserman et al . (1982) reported an early increase in calcium transport without a significant increase in calbindin biosynthesis after 1,25-(OH)2D3 repletion of partially vitamin D-deficient chicks. There appears to exist a role of vitamin D action on intestinal calcium transport beyond the genomic pathway induction of calbindin biosynthesis. A non-genomic mechanism of vitamin D action on intestinal calcium transport has been demonstrated and termed transcaltachia (Nemere et a l . , 1984). Transcaltachia is manifested as an acute increase (within minutes) in intestinal calcium transport in response to 1,25-(OH)2D3 perfusion of the intestinal vasculature. The effects have been demonstrated in vitamin D -normal but not in vitamin D-deficient chickens. More recently, deBoland and Norman (1990) reported activation of protein kinase C and cAMP-dependent protein kinase in enterocytes in 30 response to l,25-(OH)2D3 perfusion of the intestinal vasculature. These researchers proposed that activation of these proteins results in a transient increase in the intracellular calcium concentration which in turn stimulates calcium extrusion from the basolateral cell membrane. 1,25-(OH)2D3-induced rapid activation of second messenger systems such as the c G M P (Barsony and Marx , 1991), inositol triphosphate, and diacylglycerol (Lieberherr et a l . , 1991), and adenylate cyclase systems (Corradino, 1974) supports the findings of deBoland and Norman (1990). While confirmation is awaited from other laboratories, contradictory evidence for the existence of transcaltachia has begun to appear in the literature. Wasserman and Fullmer (1995) questioned the physiological relevance of the transcaltachia response. They noted an initial pulse in calcium transport but did not observe a significant increase until at least 4 to 8 h post-injection with l,25-(OH)2D3. It is important to note, however, that they used an intravenous route for l,25-(OH)2D3 repletion instead of perfusing the arterial supply of the intestine. The absence of transcaltachia in vitamin D-deficient animals may lead to proposing underlying genomic effects of vitamin D prior to transcaltachia responses. Although a cellular pathway has been suggested for transcaltachia (deBoland and Norman, 1990), the possibility exists that a paracellular pathway (Karbach, 1992) is involved. 2.3.4.2. Vitamin D-Independent Regulation Casein phosphopeptides have also been reported to stimulate intestinal calcium absorption (Lee et a l . , 1983) in the rat and chick. Mykkanen and Wasserman (1980) demonstrated that purified casein phosphopeptides may stimulate intestinal calcium transport 31 both in normal and rachitic chicks. The effect of casein phosphopeptides on intestinal calcium transport, therefore, appears to be independent of molecular changes induced by vitamin D . Kitts and Yuan (1992) have suggested that casein phosphopeptide mediated enhancement of intestinal calcium transport involves enhancement of calcium bioavailability. Webling and Holds worth (1966) were first to report the stimulatory effects of bile salts on intestinal calcium absorption. Subsequently, H u et al . (1993) demonstrated that bile salts stimulate intestinal calcium transport in the rat ileum. Sanyal et al . (1994), similarly, reported that premicellar taurocholate enhanced calcium uptake in all regions of the rat small intestine. Since the physicochemical environment in the ileum favors calcium precipitation (Allen, 1982), the process of micellar solubilization of calcium may explain the effects of bile salts on intestinal calcium transport. The stimulatory effects of lactose on intestinal calcium transport have been widely reported. Lactose increases serum and bone calcium concentration and restores responses to parathyroid hormone in vitamin D-deficient rats (Au and Raisz, 1967). Mi l l e r et al. (1988) reported that dietary lactose improved endochondral bone growth and bone mineralization in rats fed a vitamin D-deficient diet. Rats fed a diet containing 30% lactose and 0.4% calcium have been shown to absorb as much calcium as i f their diet contained 0.7% calcium and no lactose (Pansu et a l . , 1981). The mechanism by which lactose helps maintain calcium homeostasis is not known. Bronner (1987) considers that the effects of lactose are associated with hyperosmotic conditions in the intestinal lumen caused by high concentrations of lactose, and that the effects of lactose are restricted to the paracellular pathway. Favus and Backman 32 (1984) showed that lactose increases net calcium absorption in the rat ileum in the absence of transepithelial electrochemical or osmotic gradients. They proposed that since lactose induced hyperpolarization of the brush border, a cellular pathway was involved in calcium transport. Prolactin is known to increase intestinal calcium absorption (Mainoya, 1975). It was suggested that prolactin was involved in regulating the biosynthesis of l,25-(OH)2D3 (Sponos et al . (1981). Halloran and DeLuca (1980), however, reported earlier that intestinal calcium transport increases during pregnancy and lactation in vitamin D-deficient mothers suggesting that the effects of prolactin may not be mediated through l,25-(OH)2D3. It is apparent that regulation of intestinal calcium transport is not restricted to vitamin D-dependent mechanisms. 2.4. Experimental Systems Used in Intestinal Calcium Transport Research 2.4.1. Intestinal Preparations for Transport Studies For determining the rate of calcium transport across the small intestine, three types of preparations have been commonly used: 1) in situ intestinal loops, 2) everted gut sacs incubated in vitro, and 3) isolated intestinal tissue mounted on a Ussing chamber (Ussing and Zerahn, 1951). In situ intestinal loops are prepared surgically under general anesthesia (Pansu et a l . , 1993). A test solution is injected into the lumen of the loop and the apparent rate of transport is determined by the difference in the amount of solute injected and the amount of solute recovered after a given period of time. This model may closely represent the physiological 33 state as the circulation of the loop is intact and the intestinal tissue is viable and placed in its natural surroundings. In the everted gut sac preparation, a segment of intestine is excised out of the animal body, turned inside out and a loop is prepared so that a cavity lined by the serosal layer is formed (Boass and Toverud, 1996). With mucosal surface out, the loop is incubated in a test solution (Halloran and DeLuca (1980). The rate of solute transport from the mucosal side to the serosal side is usually determined by measuring the rate of appearance of the solute in the serosal cavity. In an analogous manner, the rate of solute transport from the serosal side to the mucosal side may be determined. The transport capacity of the everted gut sacs is much smaller than that of the in situ loop since the circulation of the everted gut sacs is not intact. A serious shortcoming of the everted gut sac is, that to complete the transport process in either direction, unlike the physiological state, the solute is required to move across all four histological layers of the intestine. Al so , the viability of the intestinal tissue that has been removed from the animal is compromised which may also result in a change in the permeability of the intestine. Transport measurements with Ussing chambers involve the use of an isolated piece of intestinal tissue mounted on a Ussing chamber so that the mucosal and the serosal sides are kept bathed in test solution(s). Whole intestinal tissue (Nellans and Goldsmith, 1981) or the mucosal lining of the intestine (Karbach and Feldmeier, 1993) may be mounted. General shortcomings associated with use of whole intestinal tissue in these procedures are the same as described for the everted gut sacs. The use of the mucosal lining of the intestine in the Ussing 34 chamber may more closely represent the intestinal tissue in situ in that the solute movement is only transmucosal. However, the possibility of a solvent drag effect is nearly eliminated as the lateral space is filled with test solution contained in a reservoir of infinite size compared to the lateral space itself. 2.4.2. Techniques Used in Transport Studies Three techniques have been developed to determine the relative contributions of the cellular and paracellular pathways to intestinal calcium transport process. A l l three techniques aim at determining transport via the paracellular pathway, transport via the cellular pathway is then estimated by difference. Each technique has its own advantages and valuable information has been obtained with its use. 2.4.2.1. Saturation Technique The capacity of cellular mechanisms of calcium transport is expected to be finite. Therefore, these mechanisms wi l l become saturated at a certain intralumenal calcium concentration. Use of the saturation technique is based on the assumption that with increasing calcium concentrations, the rate of calcium transport via the cellular pathway wi l l achieve a constant value whereas the rate via the paracellular pathway w i l l increase in a linear manner since it is assumed to offer little resistance. Based on Michaelis-Menten enzyme kinetics concepts, the calcium absorption curve can be resolved into a saturable or cellular, and a nonsaturable or paracellular components. In this manner, the y-intercept of the absorption 35 curve represents the maximum rate of calcium transport via the cellular pathway whereas the slope represents the rate via the paracellular pathway (see section 2.3.3). To obtain a reliable value of the slope, intralumenal test solutions containing calcium in excess of the 200 m M range have been typically used by Pansu et al . (1981, 1983a,b, 1993) since transport via the saturable pathway contributes a negligible portion of total calcium transport, at high concentrations. A very high calcium concentration in the test solutions may: 1) impede transport via the tight junctional passage which has been suggested to be lined with titrable negative charges (Moreno, 1974), and 2) compromise the viability of the absorptive cell (Wasserman, 1962). A l so , since calculation of the slope value includes only the high calcium concentrations, the transport at low concentrations appears to be taking place largely via the cellular pathway, (Bronner et a l . , 1986) leaving the question that why would calcium take an energy requiring cellular pathway over the availability of the thermodynamically suitable paracellular pathway. In addition, the use of hyper-osmolar test solutions may be questioned since an exchange of fluid between the lumen and body compartments w i l l rapidly render the solution iso-osmolar. In this case, the significance of concentration in slope calculations is misleading. The saturation technique may be used in in situ or in in vitro intestinal preparations, as well as in preparations such as the plasma membrane vesicles. 2.4.2.2. Molecular Marker Technique A second technique involves use of marker molecules that, do not penetrate the cell and are restricted to the extracellular space (Peeters et a l . , 1994). Intestinal transport of these molecules is presumed to closely reflect calcium transport via the paracellular pathway. 36 Mannitol, P E G , and creatinine are examples of such molecules (K im, 1996). This technique is more suitable for tissues with high permeability so that any spontaneous change in tissue permeability may not significantly alter a reliable correlation between the transport rates of the marker molecule and calcium (Nellans, 1990). The fact that marker molecules are inert and have different chemical properties than calcium may be a significant limitation of this technique. The technique may seem, therefore, to provide an indirect estimate of calcium transport via the paracellular pathway based on the assumption that transport of equivalent molar amounts of calcium and marker molecule proceed via the paracellular pathway. This assumption may not be completely valid. 2.4.2.3. Voltage Clamp Technique A third technique used to distinguish calcium transport via the cellular and the paracellular pathways is the voltage clamp technique. Typically this technique is carried out with the intestinal tissue mounted on a Ussing chamber. The potential difference across the tissue is kept at a predetermined level with constant tissue conductance. In this manner, transport from the mucosal side to the serosal side or from the serosal side to the mucosal side represents absorption and secretion, respectively. The voltage independent component of transport is presumed to represent transport via the cellular pathway whereas the voltage dependent component represent transport via the paracellular pathway. This presumption is compromised because the cellular pathway may have both voltage dependent and voltage independent components. The concomitant use of marker molecules such as mannitol with the voltage clamp technique is a usual practice (Karbach; 1991, 1992). 37 C H A P T E R 3 Development of an Experimental System 3.1. Introduction A n experimental system was developed to determine the cellular and paracellular components of intestinal calcium transport using in situ intestinal loop preparations. Preliminary experiments were conducted with the saturation technique following the general procedures described by Bronner, Pansu and associates to determine the cellular and paracellular components of calcium transport in in situ intestinal preparations in laboratory rats (Pansu et a l . , 1981, 1983a, 1993). These procedures involve the use of intralumenal calcium concentrations in the range of 10 to 250 m M , and an experimental duration of up to 2 h. The data from these preliminary experiments show that a dramatic and rapid decrease in test solution calcium concentration occurs with time in young broiler cockerels (Section 3.17). Therefore, the use of the initial intralumenal calcium concentration in calculations of the rate of calcium transport would be misleading. The marker molecule technique was, therefore, chosen for the present research to delineate the cellular and paracellular components of calcium transport. 3.2. Animals and Housing Newly hatched male broiler chicks (Arbor Acres) were provided by Western Hatcheries, Clearbrook B . C . In a complete random manner, seven to thirteen chicks were allocated to each of 12 battery brooder pens located in an environmentally controlled animal 38 housing facility in the Department of Animal Science at The University of British Columbia. In each pen, 63 linear cm of feeder space and 66 linear cm of drinker space were available. 3.3. Feeding and Lighting Systems Unless described otherwise, the birds were raised on a commercially obtained broiler starter diet obtained from a local manufacturer. The nutrient composition of this diet according to manufacturer's specification was; protein, 23%; calcium, 1%; total phosphorus, 0.8%; vitamin D 3 , 2200 I .U . /kg on an air dry basis. Calcium and phosphorus concentrations in the feed were determined analytically and found to be consistent with the manufacturers' specifications. Feed and water were provided to the birds ad libitum. The birds were raised on a 24 h light system provided by incandescent source. 3.4. Fasting Procedures Unless described otherwise, the birds were fasted for approximately 12 h prior to use in the in situ loop experiments. The objective of fasting was to minimize the amount of ingesta in the intestinal lumen. The quantity of ingesta in the duodenum of broiler chickens with continuous access to feed was found to be usually low, but was high in the jejunum and ileum. After fasting, the quantity of ingesta recovered from the small intestine was insignificant. Free access to drinking water was maintained during fasting. 39 3.5. Preparation of In Situ Intestinal Loops In situ loops were prepared under general anesthesia provided by intramuscular injection of a mixture of ketamine (25 mg/kg body weight) and xylazine (5 mg/kg body weight). Deep anesthesia was established in approximately 3 to 5 min, although individual variations existed. The incidence of an occasional xylazine-induced respiratory depression (Ludders et a l . , 1989) was found to increase with age, with older birds ( 7 to 14 d of age) appearing to be more susceptible to this effect than younger birds (0 to 7 d of age). When a respiratory depression was encountered, these birds were discarded from the experiment. The dose of anesthetics was, therefore, slightly decreased with increasing age. A single dose of anesthetics was found to be adequate to maintain the state of anesthesia for at least 15 min. In instances, where experiments were carried out for longer periods (1 h), half the original dose was administered at 20 min which appeared to be adequate to maintain deep anesthesia. The surgical procedures involved laparotomy on the left flank, using a pair of scissors. In situ duodenal loops were prepared by ligating the duodenum at the distal end immediately proximal to the openings of the pancreatic and bile ducts. Ligation at the proximal end was made close to the gizzard. In situ jejunal loops were made proximal to the site of yolk stalk. In situ ileal loops were made proximal to the ileo-cecal junction. A single ligation was made at each site maintaining continuity of the intestinal tissue, in these preparations. Within a given age group, the length of the jejunal or ileal preparations was kept approximately equal to the length of the in situ duodenal preparation. Waxed dental tape was used as ligation material to minimize trauma to the intestinal tissue. The laparotomy and the in situ loop preparation were completed within approximately 1 min in the case of the duodenum, and within 3 min in case 40 of the jejunum or ileum. Little bleeding occurred during these procedures. In the rare case of a large blood vessel rupture, the bird was discarded. 3 . 6 . Intralumenal Test Solutions Three types of test solutions were primarily used in this research: 1) 100 m M CaCh. ( B D H Inc. Toronto, Ontario, Canada), 2) 300 m M mannitol (Difco Laboratories; Detroit, Michigan, U . S . A . ) , 3) solution containing 75 m M C a C h + 75 m M mannitol. The approximate osmolarity of these test solutions was 300 m M . A l l these were iso-osmolar and were prepared in deionized autoclaved water. In a few instances when hypo-osmolar solutions 45 were used, they were made iso-osmolar with N a C l . Approximately 0.1 ptCi/mL of C a C h (Amersham Canada L t d . , Oakville Ontario) and/or 3H-mannitol (Dupont Canada Inc., Mississauga Ontario) were added to the test solutions as required. The solutions with equimolar concentrations of calcium and mannitol (75 m M of each) were used in experiments conducted with an objective to delineate cellular and paracellular components of calcium transport. This composition satisfies an equal access for calcium and mannitol for sites of paracellular absorption and, therefore, provides more reliable data in this respect. 3 . 7 . In situ Intestinal Loop Experimental Procedures The experiments were normally performed between 05:00 and 12:00 hours. The volume of the test solution was experimentally predetermined. The test solution was injected through a 27 gauge needle. Care was taken not to distend the intestinal wall with the solution. In case of the duodenum, half of the test solution was injected into the proximal end and the 41 other half into the distal end as the bend of the duodenum was found in some cases to partially impede the free flow of test solution through the duodenal lumen during injection. The volume injected was determined by the difference in the weight of the injection syringe before and after injection. Injection of test solution was completed within 5 to 10 sec. Completion of injection marked the beginning time of the intestinal calcium and/or mannitol transport experiments. The abdominal wall was clamped after the injection with hemostat(s). The incision site was kept moist with a gauze soaked in normal physiological saline solution. A t a predetermined time after the beginning of the experiment, the intestinal loop was removed from the body. The contents of the loop were collected in a plastic vial by flushing with excess volume of the 'cold ' test solution and the total volume of this collection was determined. When the test solution contained mannitol, the collection vial was kept on ice to avoid any bacterial degradation of mannitol. The amount of radioactivity present in the collection vial was determined by liquid scintillation counting procedures using a L6000 model Beckman liquid scintillation counter. For this determination, 0.5 m L of the loop contents were added into 5 m L of scintillation cocktail (Ready Safe; Beckman Instruments Inc., Fullerton, California, U . S . A . ) in a mini plastic vial of 7 m L capacity. When both 4 5 C a and 3 H radionuclides were present, a dual label quench curve was used to determine the amount of radioactivity in the test sample, where 3 H radioactivity was measured within the 0-8 Kev window, and 4 5 C a radioactivity was measured within the 8-256 Kev window. The empty intestinal tissue was put on a glass plate, opened along its length, blotted dry, and its length and/or wet weight were determined. 4 2 3.8. Parameters Used for Calcium and Mannitol Transport from In Situ Intestinal Loop Preparations Two parameters were used to describe intestinal calcium and/or mannitol transport in in situ intestinal loop preparations: 1) amount disappeared, or 2) percent disappearance. Both of these parameters were calculated based on the amount of 4 5 C a C h or 3H-mannitol tracer(s) disappeared (dpm injected - dpm recovered) during the in situ loop procedures. The amount of calcium and/or mannitol that disappeared from the intestinal lumen in situ was calculated as follows: pmol disappeared = (dpm injected - dpm recovered) -H dpm pmol" 1. This parameter also takes into account the amount (weight) or length of the intestinal tissue and duration of the in situ loop transport procedure and, therefore, expressed as pmol g"1 10 min" 1, or pmol cm 1 10 min" 1. O n the other hand, the percent disappearance of the radionuclides was calculated as follows: percent disappearance = [(dpm injected - dpm recovered) -f- dpm injected] x 100%. This calculation does not involve the amount or length of intestinal tissue. When treatment effects are to be compared between groups of animals of different ages, or between different intestinal regions, the results, i f expressed as the amount disappeared may be misleading as the weight to length ratio, or apparent absorptive surface areas may vary. In this case, therefore, a more reliable way of presenting the results is as percent disappearance. One condition that must be satisfied for presenting the results as percent disappearance, 43 however, is that the relative volume of test solution injected, m L / c m tissue, does not differ significantly. 3.9. In Vitro Recovery of Radioactive Tracers from the Intestinal Lumen It was necessary to know whether there was a difference between calcium and mannitol in terms of binding to the intestinal tissue and whether there were differences between the duodenum, jejunum, and ileum for binding of the two radionuclides. The existence of such differences would require appropriate corrections in the calculation of the intestinal transport. To determine recovery of radioactive tracers, 13-d-old birds were kil led and equal lengths of the duodenum, distal jejunum, and distal ileum were removed and ligated at both ends. The lumens were filled by injection of a solution containing 75 m M each of 4 5 C a C h (S .A . , 1.33 n C i pmol"1) and 3H-mannitol ( S . A . , 1.33 n C i pmol" 1). Two minutes after the injection, the lumenal contents were collected by flushing with excess volume of the "cold" test solution. The amount of radioactivity recovered from the lumens was determined as described in the preceding text. There were no significant differences in the percent recovery of radioactivity between 3H-mannitol and 4 5 C a C k in the duodenum (n=7), distal jejunum (n=5), or distal ileum (n=5). Nor were there a difference between these intestinal regions for percent recovery of either of the radionuclide. These recovery study results are shown by Figure 3.1. 44 o •a = 1 § •a •«? C/2 0> OS et-i O en G O '53D ( U I U I j je asop jo %) i(j3A033J He P U B B 3 s f CO O ID 1) 45 3.10. Disappearance of Calcium and Mannitol from In Situ Duodenal Loops, as a Function of Time Time-related changes in the percent disappearance of calcium and mannitol from the duodenal lumen were determined with 14-d-old birds. The test solution contained 75 m M each of 4 5 C a C k ( S . A . , 1.33 n C i pmol"1) and 3H-mannitol (S .A . , 1.33 n C i pmol" 1). The in situ loop experiments were terminated at 2, 4, 6, 8, 10, or 12 min after intralumenal injection (n=6). Time related changes in cumulative percent disappearance (% of dose in given time) of calcium and mannitol from the lumen of the in situ duodenal preparation in 14-d-old birds are presented in Figure 3.2. The disappearance (% of dose) of both the radionuclides from the duodenal lumen gradually increased with time. Although the disappearance of mannitol increased with time, its disappearance at 6 min was not significantly different from that at 12 min. Calcium disappearance from the duodenal lumen at 6, 8, 10, and 12 min was significantly greater (P < 0.05) than that of the mannitol, at each time point. The amount of calcium associated with the intestinal tissue (remained constant from 2 to 12 min) was subtracted from that which disappeared from the lumen in order to determine the amount of calcium that had entered the body beyond the intestinal tissue. The percentage of calcium that disappeared beyond the intestinal tissue was plotted as a function of time; this percentage appeared to be a direct function of time (r=0.976) (Figure 3.3). Regression analysis revealed a relationship between time and calcium disappearance that is described by the equation y = 3.09 + 2.11x, with the y-intercept not significantly (P < 0.148) different 46 1 -3 1 * (3UIIJ U 3 A l § J B 3S0p J O % ) 3DUBJB3ddBSip JOJIUUBUI pUB U i n p | B 3 a © c CD E i 60 c5 3 Vi 03 O .a o 47 u 6 a g a JO u 0> OS a u u CO s H v ' l-l CD ° __ to • £ 2 U i l (9uip u9AiS ye asop jo %) CT3 60 <u fcl OH 48 from zero. A 10 min incubation time was chosen for this research and considered appropriate since the percent disappearance of calcium across the intestinal wall occurred as a direct function of time over this experimental period. The amount of calcium associated with the tissue did not change from 2 to 12 min. Al so , the results of the in vitro recovery study (Section 3.9) indicated that the amounts of 4 5 C a C b and 3H-mannitol associated with the intestinal tissue do not differ. Therefore, the amounts of calcium and mannitol that remain associated with the tissue were not routinely determined in in situ intestinal loop experiments conducted for the research presented in this thesis. It is worth noting, however, that a moderate to high degree of correlation was found to exist between the amount of 4 5 C a disappeared (pmol g"1 10 min ' ) from the lumen of in situ intestinal loops, and the plasma 4 5 C a concentration (dpm m L 1 10 5dpm injected"1 kg"1) at termination of the experiment (see Appendix Figure 1). Similarly, a moderate to high degree of correlation was found to exist between the amount of 3H-mannitol disappeared (pmol g"1 10 min"1) from the lumen of in situ intestinal loops and the plasma 3H-mannitol concentration (dpm mL" 1 10 5dpm injected"1 kg"1) at termination of the experiment (see Appendix Figure 2). The appearance of 4 5 C a in the vascular fluid has been previously used as an index of intestinal 4 5 C a transport (deBoland and Norman, 1990). 3.11. Collection of Plasma and Tibiae When required, a blood sample was collected via a wing vein puncture prior to the induction of anesthesia. When required, another blood sample was collected via intracardiac 49 puncture 30 sec before termination of the experiment. Blood collections were made with pre-heparinized syringes produced by rinsing the syringes with 3 mg/mL of lithium heparin and oven drying. Plasma was separated by centrifugation in a microcentrifuge and aliquoted for calcium and phosphorus analyses. When required, the right tibiae were removed, cleared of adherent soft tissue, and defated with each of 95% ethanol, and petroleum ether for a 24 h period in a Soxhelet apparatus. The tibiae were then dried to a constant weight at 50° C , and ashed at 600° C for 12 h. The ash was dissolved in concentrated HC1, by heating to ensure complete dissolution, and made to volume. 3.12. Balance Experiments The balance experiments were conducted to determine calcium retention in a group of intact birds. These experiments were conducted for a duration of 24 h. In certain cases these experiments were concluded 12 h prior to determinations made with in-situ loops in the same birds. The birds were weighed at the beginning and termination of the experiment. Feed consumption during the experimental period was recorded. Total excreta produced during the course of the experiment was collected on wax paper, dried, weighed, pulverized, thoroughly mixed and then an aliquot was ashed for determination of calcium and phosphorus concentrations as described for the tibiae. Water consumption was recorded when calcium was supplemented via the drinking water. 50 3.13. Analytical Procedures Total calcium in the ashed samples and plasma was determined by atomic absorption spectrophotometery using a Perkin Elmer Model 560 apparatus. For these measurements, the samples were dissolved in a lanthanum solution to achieve a final concentration of 200 ppm of lanthanum. Inorganic phosphorus in samples was determined by the colorimetric procedure detailed by Itaya and U i (1966). 3.14. Animal care The husbandry of the chicks and the surgical procedures were carried out according to the general guidelines issued by the Canadian Council on Animal Care (1993). 3.15. Statistical Analyses The data pertaining to parameters such as intestinal calcium and mannitol transport, calcium retention by the loop tissue, feed and calcium intake, body weight, plasma, tibia, and excreta calcium concentrations and tibia ash concentrations were analyzed by analysis of variance via the general linear model ( G L M ) procedures of the Statistical Analysis System (SAS, 1985). The difference between means was considered significant at P < 0 . 0 5 . When significance of the means was indicated, a mean separation procedure was carried out by Tukey's multiple range test (SAS, 1985). The correlations were performed by G L M procedures of S A S . 51 3.16. Effect of Short-Term Easting on Intestinal Calcium and Mannitol Disappearance from In Situ Duodenal Loop Preparations The gut atrophies in response to fasting. Emery et al . (1986) reported that intestinal mass loss proceeds at a rate that surpasses any other organ during short term fasting. They reported a 16 to 23% loss in small intestinal protein content after a 24 h fast which increased to 30 to 35% when the duration of fast was increased to 96 h. It is important to note, however, that gut atrophy in response to fasting is readily reversed after normal feed intake is resumed (Goodlad et a l . , 1987). A decrease in cell proliferation rate together with decrease in protein synthesis may well be expected to down-regulate intestinal absorption via the cellular mechanism and, may lead to speculation that absorption by the paracellular mechanism may also change. Whether such changes occur after a short term fasting is not known. The procedures in the present research involve fasting for a 12 h period to deplete the intestinal lumen of digesta. A l so , calcium concentration in the lumenal ingesta of various intestinal regions is high (Figure 3.4). A n empty lumen ensures maximum accessibility for the solutes in the test solution to the absorptive sites. Fasting is mainly required for emptying the distal intestine as compared with the proximal intestine, which normally contains relatively little ingests. To empty the intestinal lumen, other researchers have used pre-rinsing (Pansu et a l . , 1993), a fast of 24 h (Pansu et a l . , 1975) or of 48 h (Charpin et a l . , 1992), or removal of digesta by a manual pushing. A l l of these methods may have detrimental effects on intestinal permeability but these effects have not been evaluated. It was, therefore, necessary to investigate the effects of short-term fasting on intestinal calcium and mannitol transport. 52 E = E i 3 C 3 - .22. a s S E - 3 - 3 3 3 2 '«? c = § i r i -"2 * « S 2 s s s § 1 II II II II II Q 1 *1 M M CU S3 CU C/3 Ct-I O a © '53D CU 6 a co 00 ^ s " a <L) P3 ° .52 b 53 Intestinal calcium and mannitol transport was determined with in situ duodenal loops in 7-d-old broiler cockerels. The birds used in this study were either continuously fed or fasted for 12 h prior to in situ loop transport determinations (n=12). The intralumenal test solution contained 75 m M each of 4 5 C a C h (S .A . , 1.33 n C i pmol"1) and 3H-mannitol (S .A . , 1.33 n C i pmol"1). A t the termination of the in situ loop experiment, the intestinal tissue was ashed as described earlier and the amount of 4 5 C a associated with the tissue was determined. General procedures for fasting, in situ loop transport experiments, and analyses of samples have been previously described in this chapter. The percentage of calcium or mannitol that disappeared from the lumen of in situ duodenal loop preparations, and the percentage of calcium retained by the duodenal tissue during the experimental procedures was not significantly different between fed and fasted birds (Table 3.1). However, the plasma total-calcium concentration (P < 0.05) as well as the relative weight of the duodenal tissue (g/cm) (P < 0.01) were significantly decreased in response to a 12 h fast (Table 3.1). The fact that plasma calcium concentration was significantly decreased in response to fasting, supports the concept that the intestine is an organ of calcium homeostasis (Hurwitz, 1996; Bronner and Stein, 1995). Despite a decrease in plasma calcium concentration there was no change in calcium disappearance (% of dose) from the in situ duodenal loop preparations in response to fasting. It is probable, that the intestinal tissue of fasting birds undergoes metabolic adjustments sufficient enough to compensate for the amount of tissue loss (Table 54 00 ON V a. s V a. TJ TJ <u +-» CO <+-l I I 43 ON +1 oo oo ON o ON +1 oo oo (N d +1 m C O O o d +l oo m O d +l io •a PL, O ON +1 oo o ON +1 ON C4 d + l >/-) O o d +i oo oo o d +l ON r-(N a .2 CO > S-i <u , to o o a , - S o o CO « CO CO C '<U. <u & O -« 3 .5 a o co 1  I '-3 a CO O •a o i ^ U 4-* s OH & CO TJ TJ — S '§ -S cO D <u o TJ r-H p .2 0) co O TJ O a c o o C ^ <U CO CD 0) 1-1 CO S TJ 2 <~ O O ^ &5 U w 'S 1) co CO ' _> %—» _c0 6 o "cO o Is o •4—> e CO CO 55 3.1) in response to a short-term fasting. The intestinal calcium transport capacity was, therefore, considered not to be compromised as a result of 12 h fast. The paracellular transport capacity was also not compromised by 12 h fasting since mannitol transport was not affected. 3.17. Factors that May Influence In Situ Intestinal Loop Transport The intralumenal calcium concentration decreases during a 10 min incubation period (Figure 3.5). Since percent disappearance of calcium is inversely related to concentration, a long incubation period may result in an increase in the percent disappearance, which would be an experimental artifact related to time. A very rapid decrease in intralumenal calcium concentration occurred when hyper-osmolar solutions (i.e. 200 m M ) were used, especially during 1 h incubation period (Figure 3.6). This rapid decrease in calcium concentration was probably caused by movement of body fluids into the intestinal lumen. The movement of body fluids into the intestinal lumen wi l l impede net solute absorption (Madara, 1983; Reuss and Finn, 1977). Peeters et al . (1994) have also noted a significant decrease in the permeation of inert solutes across the intestine when provided in hyper-osmolar solutions. In contrast, however, are the findings of Pansu et al. (1975) that hyper-osmolar conditions inside the intestinal lumen, independent of the nature of the solute, increase calcium transport flux two to three fold. It appears that the 2 h incubation period used by Pansu et al . (1975) might have resulted in almost complete absorption of the solutes. In the preliminary experiments in the present research, the intestinal lumen was found to be partially collapsed 56 [ W U I ] U0IJBJJU33U03 UIlipiBD jeuaumnUJUJ 1 © "•8 C3 u a et O C/D CO 03 C O 57 3 c 1 o o s o S .ai, s ? 3 g w a 2 g l-i (3 o « S3 o t+- 33 -13 o to TO (-1 S3 * ^ « o O M T) t« d 2 z «.a o c to _|_| O . 2 s 2 ?? « cu &, C "3 & S O ° S a 4-> is "o DJD 1) — "C ~ o fa 3 .a U0IJBJJU9DU03 UinpiB3 IBU9UII1IBJ1UI 58 after incubation periods of 1 h, indicating that the absorptive surface was not completely bathed with the test solution. Whether or not mannitol is degraded by the chicken intestine or the body is unknown. Plasma mannitol concentration, however, significantly decreases from 1 to 10 min after an intravenous injection (Figure 3.7). This decrease may possibly be associated with a slow diffusion of mannitol into extracellular spaces, or a degradation within the body. Pappenheimer (1990) and Wick et al. (1954) have demonstrated that mannitol is degraded by the rat liver but not by the rat small intestine. Degradation of mannitol by the chick intestine or other organs has not been reported in the literature. The outcome of experiments when mannitol is used as a marker of intestinal paracellular transport could be influenced by mannitol degradation by intestinal tissue. A significant decrease (P < 0.05) in plasma calcium concentration as a result of 12 h fasting raises the question, whether the prandial state may affect plasma total-calcium concentrations in birds and, therefore, whether it can affect intestinal calcium transport responses. Since, to determine the prandial state is a subjective measurement, plasma total-calcium concentrations were determined over a 24 h period. There was no significant circadial variation in plasma total-calcium concentration in 18-d-old birds (Figure 3.8). These results were expected since the birds were subjected to a 24 hour light program with continuous access to feed and water. These results are supported by the findings of Hurwitz et al . (1994) who reported a lack of significant circadial variation in the plasma total-calcium concentrations of broiler cockerels raised under a 22 h light period with constant access to feed and water. 59 fN *t •a -a •a -a .la .!= .!= .!= CQ CQ PQ pa • • 4 » U P C a , a o o CO o a S3 © a ft t> 2 2 u u § ' a 1 - * co O a ?a o U > I_| ,«> /-N - 3 ^ § £ •a a T - i a « .2 U ^ e m a § ° : i a a oo a .2 1 — 1 cd » ci_, * ° l l > EL a M a a «n .a DC S " 'S M fa E ^ [JAJUI] UOI;BJ;U33UOD lojiuireui B U I S B I J 60 « o s H ca bp i-i bD O ca •=1 ^ 8 .5 .S3 v W bp &0 3 bo 3 33 CD A s <* a _o %—» ca 3 cu o 3 o o ca 3 O T3 cu CO cu 5 3 ^ P 9. ca o J-l o o •I-" 1-1 ca J ? s •? . 00 00 CU L-i H O ° g a« 3 b o 3 bJ3 O £ a, H 3 ^ U • 3 > x! | j f c N ' 5 io T3 > ?a g«- ^ —. ca £ ^ - H a cu > £ ^ 2 3 & § cu J> *-> • « CU fa OH CJ 3 cu 2 ca S 3 3 T5 3 - ° » 3 £ » . S C S « o 2 u " S 3 cu « 2 * 8 £ £ s* 2 § S U0IJBJJU93U03 UIIipre3-[Omo; BUISBld 61 Finally, it would be of interest to know about calcium secretion from blood into the intestinal lumen during the in situ loop experimental procedures. Although no direct measurements were made, it appears that the secretion was negligible since there was no significant change in specific radioactivity (S.A.) of a 100 m M 4 5 C a C k test solution between 2 to 20 min after the intralumenal injection (Figure 3.9). A n initial 5% decrease in S . A . occurred at 2 min after the injection, probably caused as a result of resident calcium in the lumen of the intestine. 62 (N O o o o 8 8 «/-) o CN CN o o o o o o i n O v~i a o u 0> (N «D - s cu cu E H 5 x> (T.Iouiri mdp) A":u>i:peoipej aiipadg 63 C H A P T E R 4 Regulation of Intestinal Calcium Transport in Rapidly Growing Young Broiler Cockerels by Vitamin D-Dependent Mechanisms 4.1. Introduction Vitamin D regulation of intestinal calcium transport is well established (Wasserman and Fullmer, 1995). However, the essential vitamin D-dependency of intestinal calcium transport has been questioned by some researchers. Kollenkirchen et al . (1991) demonstrated that normocalcemia could be maintained in vitamin D-deficient rats, from weaning to 19 weeks of age, when the diet contained 2% calcium, 1.25% phosphorus, and 20% lactose. They further demonstrated that rats on this dietary regimen maintained control concentrations of P T H throughout the course of the study. In addition, they demonstrated that tibia mineral content did not differ between rats on this dietary regimen and rats fed a vitamin D adequate control ration containing only 0.8% calcium and 0.5% phosphorus. These results indicate that, under suitable conditions, sufficient quantities of calcium can be absorbed from the gastrointestinal tract under vitamin D-deficient conditions. Vitamin D has also been suggested to stimulate intestinal calcium transport via the paracellular pathway. Evidence supporting this view, however, is limited. Studies by Dostal and Toverud (1984) using in situ loops tend to show that the paracellular component of calcium transport in the rat small intestine is enhanced by vitamin D . Karbach (1991, 1992) demonstrated that l,25-(OH)2D3 treatment stimulates bi-directional calcium transport in all 64 segments of the rat small intestine mounted on Ussing chambers. Karbach (1992) reported further that 1,25-(OH)2D3 treatment of rats increases mannitol transport across the duodenal, jejunal and ileal tissues. In contrast to this evidence are the findings of Pansu et al. (1983b), obtained with the saturation technique, that treatment of rats with l,25-(OH)2D3 does not increase the nonsaturable, paracellular, component of intestinal calcium transport. In normal layer-type young chicks, a very rapid, (within 2 to 10 min), stimulation of intestinal calcium transport following perfusion of the intestinal arteries with l,25-(OH)2D3, has been demonstrated and termed transcaltachia (Nemere and Norman, 1990). Because of the time course of l ,25-(OH) 2 D3 action in this case, the transcaltachia was considered to be a non-genomic action of l,25-(OH)2D3. There exists the possibility that a l,25-(OH)2D3 stimulation of paracellular calcium transport is involved in transcaltachia. The effects of vitamin D on paracellular intestinal calcium transport in the chick remain a subject requiring more study. Regional differences in the small intestine for the vitamin D-dependent component of calcium transport in the rat remain a subject of controversy. For example, using the saturation technique with in situ intestinal loops, Bronner, Pansu and their associates maintain the view that the vitamin D-dependent component of intestinal calcium transport decreases from the proximal to the distal sections of the rat small intestine (Pansu et a l . , 1983b; Bronner, 1992) such that ileal calcium transport is essentially devoid of a vitamin D-dependent component. On the other hand, researchers who have used the Ussing chamber technique to study intestinal calcium transport have concluded that 30 to 40% of intestinal calcium transport in all regions of the rat small intestine, cecum and colon is vitamin D-dependent (Karbach, 1992; Karbach 65 and Feldmeier, 1993; Nellans and Kimberg, 1978). The contribution of vitamin D-dependent calcium transport in different regions of the small intestine of rapidly growing broiler chicks is not known. Most of the existing information related to mechanisms of intestinal calcium transport was obtained using the laboratory rat. It is well known that the role of vitamin D in the etiology of rickets in rats differs from that in chickens. In contrast with chickens, rats do not become rachitic from the simple exclusion of vitamin D from the diet, but must also be given a diet with a grossly altered calcium to phosphorus ratio (Coates and Holdsworth, 1961). A l so , calcium metabolism in mature birds is different than in mature mammals. For example, these birds can develop a temporary reservoir of calcium in the form of medullary bone in their long bones. Calcium is rapidly mobilized from the medullary bone to the developing egg shell. Possibly some aspects of intestinal calcium transport in rapidly growing broiler chickens may differ from those in young growing rats, since chicken hatchlings are more mature than rat neonates. The objectives of the following experiments were: 1) to determine the contribution of the vitamin D-dependent component of calcium transport in the proximal and distal small intestine in rapidly growing young broiler cockerels, and 2) to determine whether the paracellular pathway is regulated by vitamin D in these birds. This is the first report to describe these aspects of intestinal calcium transport in rapidly growing young broiler cockerels. 66 4.2. Methods 4.2.1 Effects of High Calcium Intake on Calcium Transport in In Situ Duodenal and Ileal Loop Preparations 4.2.1.1. Diets A high calcium diet (HCD) (-1.65 fold N R C recommendation) was prepared by adding calcium-gluconate plus calcium-lactate (3:1) (Both salts obtained from B D H Inc. Toronto, Ontario, Canada) to a commercial broiler starter diet used as the control diet. The lactate and gluconate salts of calcium were used to supplement dietary calcium because of their high solubility relative to calcium carbonate salts used as the primary source of dietary calcium in commercial diets. One consideration involved in the preparation of the H C D was that dietary calcium concentration does not become excessively high since concentrations approaching 2% may interfere with nutrient utilization in broiler chickens (Shafey et a l . , 1990). The calcium and total-phosphorus concentrations ( % D M ) in the diets were analytically determined (Table 4.1). Table 4.1. Calcium and phosphorus concentrations in the experimental diets Diets Calcium Total phosphorus ( % D M ) ( % D M ) Control diet 1.09% 0.82% High calcium diet 1.65% 0.75% 67 4.2.1.2. Duodenal Calcium Transport in Response to High Calcium Intake Two experiments were conducted with the objective of determining the effects of high calcium intake on calcium transport in the in situ duodenal loop preparation. Birds were fasted for 12 h before the experiment, to minimize the amount of residual dietary calcium in the duodenal lumen of birds fed the H C D . The intralumenal test solution contained 75 m M each of 4 5 C a C h ( S . A . , 1.33 n C i pmol"1) and 3 H-Manni tol (S .A . , 1.33 n C i pmol' 1). A l l experiments were terminated 10 min after intralumenal injection of the test solution. The details of the in situ loop experimental procedures and analytical techniques are described in Chapter 3. The first experiment was conducted in 4-, 7-, 14-, and 21-d-old chicks fed the H C D or control diet from hatch (n = 10 to 12). Ten pens each containing 13 newly hatched chicks were used in this experiment. Five pens each were randomly allocated to the H C D regimen or the control diet regimen. The second experiment was a crossover experiment conducted to confirm the duodenal calcium transport response to H C D and to determine whether the duration of feeding the H C D affected in situ duodenal loop calcium transport (n = 9 or 10). Chicks were fed a control diet or H C D from hatch to 9 d of age, after which they were crossed over to the control diet or H C D for 24 h. Six pens each containing four chicks were used in this experiment. Three pens each were randomly allocated to the H C D regimen or the control diet regimen for this experiment. 68 4.2.1.3. Ileal Calcium Transport in Response to High Calcium Intake The effects of high calcium intake on calcium transport from the lumen of in situ ileal loop preparations were determined in 4-, 7-, and 14-d-old chicks (n = 7 to 12). These birds were fed the H C D for 48 h and then fasted for a 12 h period before the in situ loop experiments were performed. The intralumenal test solution contained 75 m M each of 4 5 C a C h (S .A . , 1.33 n C i pmol"1) and 3 H-Manni to l (S .A . , 1.33 n C i pmol" 1). A l l experiments were terminated 10 min after the intralumenal injection of the test solution. The details of the in situ loop experimental procedures and analytical techniques are described in Chapter 3. 4.2.2. Effects of Vitamin D on Intestinal Calcium Transport 4.2.2.1. Diets A diet primarily based on corn and soybean meal was prepared, both with or without the addition of vitamin D 3 . Since the commercial diet used in the other experiments described in this thesis was claimed by the manufacturer to contain 2200 I .U. /kg of vitamin D 3 , the same vitamin D concentration (2200 I .U . /kg wet weight) was used in the control diet in this experiment. The composition and calculated nutrient concentrations of the diets used in this experiment are provided in Table 4.2. 69 Table 4.2. Composition of the rachitogenic and normal diets' Ingredients Inclusion (g/100g) Ground yellow c o r n 2 Soybean meal (solvent extracted, dehulled, 48.5% protein) 2 Corn gluten meal (60% protein) 2 Dicalcium phosphate3 Calcium carbonate3 2 2 55 35 1.3 Iodized sodium chloride 3 0.24 Canola o i l 4 4 Vitamin and mineral premix 5 0.4 'Calculated Concentration of Nutrients in the Diet [based on N R C (1994) composition of feed ingredients] protein, 23.06%; metabolizable energy, 3129 kcal/kg; calcium, 1.005%; phosphorus (total), 0.741%; sodium, 0.149%; magnesium; 0.069%; manganese, 60 mg/kg; zinc, 40 mg/kg; copper, 8 mg/kg; iron, 200 mg/kg; selenium, 0.15 mg/kg; lysine, 1.265%; methionine, 0.5%; methionine plus cystine, 0.86%; vitamin A , 1500 I .U. /kg ; vitamin D 3 , (none in the rachitogenic diet, 2200 I . U . / kg in control diet); vitamin E , 10 I .U . /kg ; vitamin K , 0.50 mg/kg; riboflavin, 3.6 mg/kg; pantothenic acid, 10 mg/kg; niacin, 27 mg/kg; vitamin B12, 0.009 mg/kg; biotin, 0.15 mg/kg; folic acid, 0.55 mg/kg; thiamin, 1.8 mg/kg; pyridoxine, 3 mg/kg. 2 Otter Co-op, Langly B . C . 3 V a n Waters and Rogers L td . , Abbotsford, B . C . 4 Neptune Foods, Richmond, B . C . 5 United states biochemical corporation, Cleveland, O H , U . S . A . 70 4.2.2.2. Rachitogenesis This experiment was conducted to determine the time course of development of rickets in broiler cockerels fed a vitamin D-deficient diet (Table 4.2). Newly hatched broiler cockerels were randomly assigned to either rachitogenic or control diets. A t 7, 12, and 21 d of age, blood samples were collected by wing vein puncture immediately before the chicks were killed (n = 6 to 18). Plasma total-calcium concentrations were determined in these samples. Tibiae were collected and analyzed for ash and calcium concentration as described in Section 3.3. Based on plasma total-calcium concentrations, birds became rachitic by 7 d of age. Gradually, the birds became severely rachitic between 12 to 16 d of age. Since a major loss of general well being of chicks occurred after 16 d of age, the following experiments involving rachitic chicks were conducted in birds from 12 to 16 d of age. 4.2.2.3. Effects of Vitamin D-Deficiency Rickets and l,25(OH)2D3 Treatment on Calcium Transport in In Situ Duodenal and Ileal Loop Preparations 4.2.2.3.1. General Procedures Crystalline l,25-(OH)2D3 was a gift kindly provided by Hoffman LaRoche, Misissauga, Ontario, courtesy of D r . Youngblut, and M s . Linda Jane. This crystalline vitamin D compound was dissolved in a water: propylene glycol: ethanol (v:v:v) (5:4:1) mixture, as prescribed by the manufacturer. The solution was made in brown glass bottles under a nitrogen environment and immediately loaded into 1 m L syringes with air excluded which were subsequently vacuum packed in sealed plastic containers. The syringes were then stored at -20 °C until used. 71 When the in situ loop experiments were performed, hematocrit values were determined on the blood samples. Plasma was separated from blood samples by centrifugation for determination of calcium concentration. Tibiae were collected from chicks in all experiments and tibia length and ash concentrations were determined. The tibia calcium concentration was not determined since it was noted in the experiment on rachitogenesis, that tibia ash concentration is very closely related to tibia calcium concentration. The details related to collecting the blood samples and tibiae, and analytical techniques used in these experiments are described in Chapter 3. 4.2.2.3.2. Duodenal Calcium Transport in Rachitic and l,25-(OH)2D3-Treated Chicks Calcium transport in in situ duodenal loop preparations was determined in 12- and 13-d-old unfasted chicks fed the rachitogenic or control diets from hatch (n = 9 or 10). The intralumenal test solution contained 75 m M each of 4 5 C a C h (S .A . , 1.33 n C i pmol"1) and 3 H -Mannitol (S .A . , 1.33 n C i pmol' 1). The experiments were terminated 10 min after the intralumenal injection of the test solution. A t termination of the experiment, the intestinal tissue was ashed to recover 4 5 C a retained during the experimental procedures. The details of the in situ loop experimental procedures and analytical techniques are described in Chapter 3. In 13-d-old chicks only, an intramuscular injection of l,25-(OH)2D3 (10 pg/kg) was administered 6 h before the start of the in situ loop calcium transport experiment. Troy et al . (1994) have reviewed that an initial rapid stimulation of calcium transport reaches a peak after 6 h of a l,25-(OH)2D3 treatment. Two blood samples were collected in these birds from a 72 wing vein; the first immediately before the 1,25-(OH)2D3 injection and the second immediately before the induction of anesthesia to conduct the in situ loop calcium transport experiment. In 12-d-old chicks, the solvent medium was injected 6 h before the in situ loop experiment and only one blood sample was collected from a wing vein immediately before the induction of anesthesia for conducting the in situ loop calcium transport experiment. The 12-d-old birds, therefore, were used as noninjected controls for this experiment. Due to the length of time involved in experimental preparations and procedures, it was logistically difficult to conduct the whole experiment on the same day. Although, day-to-day variations in intestinal calcium and mannitol transport responses are possible, many other experiments presented in this thesis show that such a variation is usually not significant. 4.2.2.3.3. Ileal Calcium Transport in Rachitic and l,25-(OH)iDi-Treated Chicks Calcium transport in in situ ileal loop preparations was determined in 15-d- (n = 9) and 16-d-old chicks (n = 7 or 9) fed the rachitogenic or control diets from hatch. The intralumenal test solution contained 75 m M each of 4 5 C a C k ( S . A . , 1.33 n C i pmol"1) and 3 H -Mannitol ( S . A . , 1.33 n C i pmol"1). Experiments were terminated 10 min after the intralumenal injection of the test solution, and intestinal tissue was ashed to recover 4 5 C a retained during the experimental procedure. The details of the in situ loop experimental procedures and analytical techniques are described in Chapter 3. The chicks were fasted for 12 h prior to the in situ calcium transport experiment. The Sixteen-day-old chicks were given an intramuscular injection of l,25-(OH)2D3 (10 ug/kg) 6 h 73 before beginning the in situ loop calcium transport experiment. Two blood samples were collected from these birds using wing vein puncture; the first sample immediately before the l,25-(OH)2D3 injection and the second immediately before the induction of anesthesia used to conduct the in situ loop calcium transport experiment. In 15-d-old chicks the solvent medium was injected 6 h before the in situ loop experiment and only one blood sample via wing vein puncture was collected immediately before the induction of anesthesia. The 15-d-old birds, were, therefore, used as noninjected controls for this experiment, for reasons described in the preceding section. 4.2.2.3.4. Intestinal Calcium Transport in Intact Rachitic Chicks A 24 h balance experiment was conducted to determine the effects of the rachitic condition on absorption of dietary calcium. Ten pens each containing six chicks were used in this experiment. Five pens each were randomly allocated to rachitogenic or control diets from hatch. The experiment began at 11 d of age. Feed consumption and body weight gain were recorded. Total excreta from individual pens was collected on wax paper. The excreta was dried in aluminum pans at 60 °C to a constant weight, pulverized, thoroughly mixed, and aliquoted for ashing. Calcium concentration in the ash was determined. The details of the experimental procedures and analytical techniques are described in Chapter 3. 74 4.3. Results 4.3.1. Responses to High Calcium Intake 4.3.1.1. Body Weight, and Plasma and Excreta Calcium Concentrations in Response to High Calcium Intake Table 4.3 shows the effects of high calcium intake on body weight, and plasma and excreta calcium concentrations in 4-, 7-, 14-, and 21-d-old chicks. In 4-, 7-, and 14-d-old birds, the body weight was not different between chicks fed the H C D and those fed the control diet. The body weight of 21-d-old birds fed the H C D was significantly less (P < 0.05) than for birds fed the control diet. A t 4, and 7 d of age, the plasma total-calcium concentration of chicks fed the H C D was not significantly different from that of chicks fed the control diet. A t 14, and 21 d of age, however, the plasma total-calcium concentration was significantly (P<0.05) less in chicks fed the H C D compared to chicks fed the control diet. A t all ages under investigation, the calcium concentration ( % D M ) in the dry excreta of chicks fed H C D was 1.8 to 2.1 fold greater ( P < 0.001) compared with that of chicks fed the control diet. 4.3.1.2. Duodenal Calcium Transport in Response to High Calcium Intake Figure 4.1 shows the effects of high calcium intake on calcium and mannitol transport in the in situ duodenal loop preparations from 4-, 7-, 14-, and 21-d-old chicks fasted for 12 h. Calcium and mannitol transport is described as the percentage loss of dose from the lumen during 10 min. A t all ages under investigation, calcium transport from the lumen of the in situ duodenal loop preparations of chicks fed the H C D was significantly less compared with chicks fed the control diet (P<0.01) . The average percentage reduction in calcium transport in 75 .2 B o '53 Q U X CD I '-3 Q U CD 16 Q U CD I'-3 +-» c i5 o >n o O CN NO d d d d + l + l + l +1 ON CN 00 ON uo co 8 CN 8 CO o o CO o o NO o V a. o o V a. o CN o V a. NO o V a. d d d d + i + i + i +1 00 in NO NO o 1—1 r-H I T ) o CN o q d d d d + l + i + i +1 o CN ON >n r-~ m o >o co t s . CN <*> CN m o CN m o CO O Qs II &, <o q c> II a. o o V a. q o V a. d d d d +1 +1 + l +1 o co co in CO m NO CO CN CN CN ON +1 +1 + 1 r-~ o r—( oo r-- T—H CN 00 CN O vq ON r-H + l +1 +1 NO T—H O ON i—i CN +1 NO o d V ft. CO i — i +1 o ON CN 76 o I 73 u O • 4 a I—f_ * H : <N — — 2 ca H TO © * — 3 r-- cu a S S <D 03 03 2 (=1 CU CU CO CU 03 S3 CU ^ si 43 5 « co a CU •§ 2 a > (mm ox «! asop jo %) 93UBJB3ddesip lojmueiu pm? uinpre^ o Q eg 77 chicks fed the H C D compared with chicks fed the control diet was 17.4% at 4 d, 24.4% at 7 d, 26% at 14 d, and 17.1% at 21 d of age. A t all ages under investigation, mannitol transport from the lumen of the in situ duodenal loop preparations in chicks fed the H C D was not significantly different from that of the chicks fed the control diet. Except at 14 d of age, mannitol transport from the lumen of in situ duodenal loop preparations was significantly less than that of calcium transport (P<0.05) , when chicks were fed the control diet. In contrast, chicks fed the H C D , exhibited no difference between the calcium and mannitol transport. Figure 4.2 shows the effects of feeding H C D to 10-d-old chicks on calcium and mannitol transport from the lumen of in situ duodenal loop preparations, in the cross-over experiment. Calcium transport from the lumen of the in situ duodenal loop preparations in chicks fed the H C D was significantly (P < 0.05) less compared to that in chicks fed the control diet. There was a 23.6% decrease in calcium transport in chicks fed H C D compared to chicks fed control diet. Mannitol transport in chicks fed the H C D was not significantly different from that in chicks fed the control diet. In chicks fed the control diet or the H C D , there was no difference between mannitol transport and calcium transport. The results of the cross-over experiment were similar to those described in Section 4.3.1.2. in which the diets were not crossed-over. In the crossover experiment, the calcium concentration in the excreta of birds fed the H C D was significantly greater ( P < 0.001) compared with that of the chicks fed the control diet. The plasma calcium concentration in chicks fed the H C D was not significantly different from that of chicks fed the control diet, after the cross over (results not shown). 78 s s U 0> o 13 c cu 3 ^ 00 ro tti CO ^ 5 O P •a a 2 < B 00 (uira o i ui asop JO %) 33UBJBaddBsip JOJIUUBUI pire uinpir>3 g u o 79 4.3.1.3. Ileal Calcium Transport in Response to High Calcium Intake Figure 4.3 shows the effect of high calcium intake on calcium and mannitol transport from the lumen of in situ ileal loop preparations in 4-, 7-, and 14-d-old chicks. Calcium and mannitol transport are described as the percentage loss of dose from the lumen during the 10 min experimental period. A t all ages under investigation, calcium transport from the lumen of the in situ ileal loop preparations of chicks fed the H C D was not significantly different compared with calcium transport in ileal loops in chicks of the same age fed the control diet. A t all ages under investigation, mannitol transport from the lumen of in situ ileal preparations in chicks fed the H C D was also not significantly different compared with that in chicks of the same age fed the control diet. It is clear that, at all ages under investigation, no significant differences between calcium transport and mannitol transport from the ileal loop preparations were observed whether the chicks were fed the H C D or the control diet. 4.3.2. Responses to Dietary Vitamin D and l,25-(OH)2D3 Treatment 4.3.2.1. Rachitogenesis Table 4.4 shows the effects of the rachitogenic diet on body weight, and plasma and tibia calcium concentrations in 7-, 12-, and 21-d-old broiler cockerels. A t 7 d of age, body weights of chicks fed the rachitogenic and control diets were not different; however at 12, and 21 d of age, body weights were significantly (P < 0.01) lower in birds fed the rachitogenic diet. In chicks fed the control diet, there was an almost 100% increase in body weight from 12 to 21 d of age. In contrast, during the same time period, there was only a 41 % increase in body weight of chicks fed the rachitogenic diet. 80 2 4 2 o u 3 2 .= u ox I u + 1 u 2 no CD 1 © 1 M C8 a 2 O • i « ^ o (UIUI ox ui asop jo %) 33UBJB3ddBSip JOJIUUBUI pUB Ultipp33 81 1-1 cu +-* -i—< S B 2 "J3 ?^  73, ~o ° CD CT3 <U H .ca .5? '53 6-0 nic oo CN 00 CN CD bn _> d d d hito die +l +1 +l hito die in —. r- CN Rac i >—i ON Rac d 00 o in o o q o o diet 00 r-~1 P< oo CN p< o co P< diet d d d 75 +l +l +1 r~- ON ON G CN CN u CN CO CN i—i o '3 CD >n oo oo o O o d d d O CD .Si '-rt +l +1 +i co o Rac o Rac CN § o q CN o q o o V V V u in ft. 00 ft. ON ft. O o O d d d 75 t-l +1 +l +1 -t—' eg ,—i o O o ON CN u co CN CO o ;en: •st O O CN O <u +1 +1 +1 achit r— CN CN CN CN in CO «•> <N T l q o C> II o o V V CD a. ft. ON T—( ft. t-75 + 1 +1 +1 ontr o CO co in CN co O in U r-- CN CN 82 A t all ages under investigation, the plasma total-calcium concentration was significantly less (P< 0.001) in chicks fed the rachitogenic diet compared to chicks fed the control diet. In chicks fed the rachitogenic diet, the plasma total-calcium concentration at 12 d of age was significantly (P< 0.001) less than the plasma total-calcium concentration at 7 d of age. In 21-d-old birds fed the rachitogenic diet, however, the plasma total-calcium concentration was not significantly different from plasma total-calcium concentration at 12 d of age. Based on the differences in plasma total-calcium concentration between birds fed the rachitogenic diet and those fed the control diet, it was considered that rickets was established by 7 d of age. Tibia calcium concentrations (on a fat-free dry weight basis) in chicks fed the rachitogenic diet gradually and continuously decreased from 7 to 21 d of age (P< 0.001). A t all ages under investigation, the tibia calcium concentration was significantly less in birds fed the rachitogenic diet than in birds fed the control diet. Tibia calcium concentration at 7 d of age was not significantly different from tibia calcium concentration at 21 d of age in control birds. Based on these data, it was considered that birds fed the rachitogenic diet were severely rachitic between 12 and 21 d of age. 4.3.2.2. Body Weight, Tibia Fat-Free Dry Weight, Tibia Ash Weight, and Tibia Length in Rachitic and Control Chicks Table 4.5 shows the body weight, tibia fat-free dry weight, tibia ash weight, and tibia length of 12-, 13-, 15-, and 16-d-old rachitic and control chicks. A l l parameters had lower values in rachitic chicks compared with control chicks of the same age. A t all ages under 83 bb es G Tibi -t—» bfl wei •S ,29 a ccs Tibi > bfl wei dry U Of) el) w C+H .3 /-\ H 43 W> '53 ^ 'So Body ^ 3 r - H CO i 1 co T - H T - H T - H T - H •3 +1 +1 +1 +1 oi <N NO 00 oo 0.02 CN m 0.98 CO in 0.04 in 0.29 ontrol -(—» 2H ± 1.2 P< ± 1.3 II a. ± 1.2 P< ± 1.2 II a. ontrol •3 o CN CO u CN in CN in in NO in diet 0.017 0.019 0.018 0.020 diet +i +i +l +i 04 co o r-00 T - H in 00 d d 0.07 d o d 0.01 rol oo T-H o P<< o CN O P<i 00 T-H o p<\ oo T-H o P<| rol > .SH *3 d d d d + i +l +l +l Co 0.307 0.301 0.393 0.408 ON CO o o q o d d d d +l +l +1 +1 04 0.620 0.01 0.702 0.01 0.756 100 0.810 0.01 rol •<* o p< r-o P< o P< o p< rol diet d d d d Cont diet 0.847 ± 0.864 ± 1.097 ± 1.137 ± diet co T - H CN diet +1 +1 +1 +1 04 00 CN CN 0.01 T-H CN 0.01 CN CN TOO in CN TOO Control -t—» TH CN T-H +1 V a. T-H +1 V a, CN T-H +1 P< CN +1 V a. Control '•3 ON ON CN NO ON CN T-H CN CO T-H CO CN T - H CO T-H m NO 8 4 investigation, the body weight of chicks fed the rachitogenic diet was 18 to 24% less than for chicks fed the control diet (P<0.01). Similarly, the tibia fat-free dry weight was 18 to 31% less in chicks fed the rachitogenic diet compared with chicks fed the control diet (P< 0.01) for all ages studied. In contrast, the effect of rickets on tibia ash weight was more prominent compared with its effects on body weight and tibia fat-free dry weight. The tibia ash weight was in the range of 43 to 53% less in chicks fed the rachitogenic diet compared with tibia ash of chicks fed the control diet. On the other hand, the tibia length was much less affected by rickets than tibia fat-free dry weight or tibia ash weight. For example, the tibia length in chicks fed the rachitogenic diet was only up to 7% less compared with chicks fed the control diet. The differences between rachitic and control chicks for the tibia length were, however, not consistent. Tibia length was significantly less in 12-, and 15-d-old rachitic chicks compared with that of the control chicks (P<0.05). In 13-, and 16-d-old chicks, however, there were no significant differences in tibia length between rachitic and control chicks. 4.3.2.3. Plasma Total-Calcium Concentration and Hematocrit Values In Rachitic and Control Chicks Table 4.6 shows plasma total-calcium concentrations and hematocrit values for rachitic and control chicks. At all ages under investigation, the plasma total-calcium concentration was significantly less in rachitic chicks compared with control chicks (P< 0.001). The plasma total-calcium concentration was in the range of 30 to 37 % less in rachitic chicks relative to that of control chicks. Hematocrit values were in the range of 3 to 12% greater in rachitic chicks relative to controls. Hematocrit values were significantly greater in 12-, 13-, and 15-d-old 85 J-l 1*1 a '3 CD W) _ O CD o cd o 3 -3 U o '3 CD O CD O ca o 3! ;3 O ^ U NO d + i q C O C O d + l d C O •-I C 5 V a. NO o d + i oo NO O d + i o ON CN V ft, CN r-d + l ON C O co oo d +1 •<* NO co O V a. >n o d + i o oo q d + l o CN V a. d +! CN >n co d + i V a. o d +1 >n NO o d + l o CN V a. i-O oo d +1 CN co d +1 ON NO o d + l r-in o d + i co oo CN NO CN V a. 86 chicks fed the rachitogenic diet compared to chicks fed the control diet (P<0.05) . In 16-d-old birds, however, there was no signifiacnt difference in hematocrit values between rachitic and control chicks. 4.3.2.4. Plasma Total-Calcium Concentration in Response to l,25-(OH)2Di Treatment in Rachitic and Control Chicks Plasma total-calcium concentration was determined before, and 6 h after an intramuscular injection of l ,25-(OH )2D3 (10 pg/kg) in 13- and 16-d-old chicks. The 13-d-old birds were unfasted throughout whereas the 16-d-old birds had been fasted 6 h at the time of the injection of l ,25-(OH )2D3 and continued to be fasted for another 6 h after injection. Figure 4.4 shows the effect of l ,25-(OH )2D3 treatment on plasma total-calcium concentration in 13-d and 16-d-old chicks. In response to l ,25-(OH )2D3 injection of 13-d-old unfasted chicks fed the control diet, the plasma calcium concentration significantly increased 6 % above the pre-injection value (P<0.05) . In rachitic chicks of the same age, the plasma calcium concentration significantly increased 24 % above the pre-injection value ( P < 0.001). In response to l ,25-(OH )2D3 injection in 16-d-old fasting chicks fed the rachitogenic diet, the plasma calcium concentration significantly increased 13 % above the pre-injection value (P<0.001); surprisingly however in control chicks, the plasma calcium concentration slightly but significantly decreased 4% below the pre-injection value. 87 in -S .4> O i CO CA IS C M O & •> H ca -T-> O GO O • in CN ca JH «• g S3 o S3 - <u • E g H c ca O ft-3 $ 2 - - o |> SP-£ || 2 s r ® ? § s *? i 8 s " III a § -a a o vo G -J3 ^ 3 3 -2 <D O <u a 2 S ° -9 § § £ | ^ ca ca ca T3 O X5 NO CO J3 G <*-> 2 O cG h i GO < £ P .CD , (11 TH > H 2 & ca X J (SB 6 -S O to . • - i ca <*- « '—^ «-> r-H f i , _a cu 7 N >H " — ' CO I 1 O ea £ SP •S G a * -^ TS -SP OX) <*> s > S -a a CD CO T) "2 * 2 T3 o <p o <4-H CU TS H O ca J H O n s c o o ,"3 C+H [j\[iu] uinp[BD-iB;o; euiss]j -a M — •-< ca o^ fa O T - H  CO ° f"3 • a ' s 2 I ca <C in ca a J H (U •si <4H CU I H CO <u ca 1 — 1 'co 2 o (90 v CU CL 00 O-«" TH c a ca -S c ™ c o " ft-  •H S a o S «s *s t> cu 88 4.3.2.5. Duodenal Calcium Transport in Rachitic and Control Chicks With or Without 1,25-(OH)2D3 Treatment Calcium transport or mannitol transport was defined as the percentage of calcium or mannitol that disappeared from the lumen of in situ intestinal loop preparations during the 10 min experimental period following intralumenal injection. Figure 4.5 shows calcium transport from the lumen of in situ duodenal loop preparations in 12-, and 13-d-old unfasted rachitic or control chicks, with or without 1,25-(OH)2D3 treatment. Duodenal calcium transport was significantly less in rachitic chicks compared to control chicks (P< 0.001). In 12-d-old unfasted rachitic chicks, the duodenal calcium transport was 38% less than in control chicks. A t 13 d of age, the rachitic as well as the control chicks were treated with l ,25-(OH )2D3. The difference between the rachitic and control chicks in terms of duodenal calcium transport existed even after a 1,25-(OH)2D3 treatment. In 13-d-old unfasted rachitic chicks, the duodenal calcium transport was 26% less than in the control chicks. When the data from 12-d-old and 13-d-old chicks was compared, it becomes apparent that nearly a 16% increase in the duodenal calcium transport in control chicks after a l ,25-(OH )2D3 treatment occurred, while the increase in calcium transport in rachitic chicks was nearly 39%. The justification of such as comparison may, however, be questioned since these data were collected on two consecutive days. There was no significant difference (P<0.05) between rachitic and control chicks for mannitol transport from the in situ duodenal loop preparation. In 12-d-old unfasted rachitic chicks, the duodenal mannitol transport was not significantly different from that that of the 89 e B o ? ID C3 h ^ ^ ^ ^ ^ ^ X) | I l l a a ca ca U cu s a cu S s s ca ca O ts ( v | — - H (UIUI oi ui asop jo %) 33UBJB3ddBSip [OJIUUEUI piIB I i m p i B 3 i m .5 g > 32 q, .3 C X o • cd cu co 90 control chicks. In 13-d-old unfasted rachitic chicks that were treated with l ,25-(OH )2D3 6 h before the in situ loop experiments, the duodenal mannitol transport was also not significantly different from that of the control chicks: 4.3.2.6. Ileal Calcium Transport in Rachitic and Control Chicks With or Without 1,25-(OHhDs Treatment Figure 4.6 shows calcium transport, expressed as the percentage of dose lost from in situ ileal loop preparations, from 15-, and 16-d-old 12 h fasted rachitic or control chick, with or without l ,25-(OH )2D3 treatment. In both rachitic and control chicks, ileal calcium transport was not significantly different, with or without l ,25-(OH )2D3 treatment. In 15-d-old 12-h-fasted rachitic chicks, the ileal calcium transport was not significantly different from that of the control chicks. Similarly, in 16-d-old 12-h-fasted rachitic chicks treated with l ,25-(OH )2D3 6 h before the in situ loop experiments, the ileal calcium transport was not significantly different from that of control chicks. In 15-d-old 12-h-fasted rachitic chicks, ileal mannitol transport was not significantly different from that in the control chicks. Similarly, in 16-d-old 12-h-fasted rachitic chicks treated with l,25-(OH)2D3 6 h before the in situ loop experiments, ileal mannitol transport was not significantly different from that in the control chicks. When the data from 15-d-old and 16-d-old chicks are compared, it becomes apparent that l,25-(OH)2D3 treatment did not influence ileal mannitol transport. 91 e ca ca U ca U (mm oi ui asop jo %) 33UBJB3ddBSip (OJIUUBIU piIB UinpjB3 co s © c cu s 13 cd ON 92 4.3.2.7. Calcium Retention by Duodenal and Ileal Loop Tissues in Rachitic and Control Chicks With or Without l,25-(OH)2D3 Treatment Tissue calcium retention was defined as percentage of calcium injected into the loop lumen that was retained by the tissue at 10 min after injection. Figure 4.7 shows calcium retention by duodenal and ileal loop tissues in 12-, 13-, 15-, and 16-d-old chicks. Calcium retention by duodenal tissue was not significantly different between rachitic and control chicks or between l ,25-(OH )2D3 -treated or -untreated chicks. Similarly, calcium retention by ileal tissue was not significantly different between rachitic and control chicks or between 1,25-(OH)2D3 -treated or -untreated chicks. It is surprising, however, that unlike other experiments in the present study, calcium retention by ileal tissue was significantly (P < 0.05) lower than calcium retention by duodenal tissue. The reason for this difference is not clear. 4.3.2.8. Calcium Transport in Intact Rachitic Chicks The effects of the rachitogenic diet on feed intake, body weight gain, and net absorption of dietary calcium (percent of intake) by intact chicks are shown by Figure 4.8. The feed intake (g bird"1 24 h"1) was 29% less in rachitic than in control chicks (P<0.001) . The body weight gain (g bird"1 24 h"1), during the experiment period was 40% less in rachitic than in the control chicks ( P < 0.001). Feed utilization was, therefore, adversely affected in rachitic chicks since the feed conversion ratio (gram body weight gain per gram feed intake) in rachitic chicks (Mean ± SE) (1.95 ± 0.08) was more than the control chicks (1.64 ± 0.85). 93 CU 3 CA CA a o o ca CU 4 ^ x x x x x x x x ^ H ^ ^ ^ ^ 1 1 1 1 1 <n o V© IT) CU 3 cn a o 73 e cu © 3 o 3 M o 3 1? +3 cu 2 >n (uiiu 01 ;B 3S0p JO %) U0IJU3J3J umpn?3 o o J S O -H 94 (95[Bjui jo %) uopdjosqe UiniOIBD }3JsI fi n. u © C/3 -Q es U "S OA •SP "5 O cu CO cu cu fa ( l - W Z T-PJm §) W P 3 ^ CO 3 B ^ •r; cu cu is cu > =• £ u £ C £ .22 ~ 2 cu x> B ^3 B B -3 Q 60 60 .3 S .3 Q a .a o CO V £ 5i CU 7! .B -B CU > - y cu .a o u X) CU „ CD ca S Ta B ^ CU " c£ ^ B rt § g> a -g CD 3 •c JS . cu >> ^  CD cu 2 $ 8 S CU 8 S o B cu '53 IS CD T3 cu |-1 - B O ~ O O cN S3 7 3 ^ "2 B rt cu >, 00 Jo o . B .3 a cu CD I-H . B O H 1 - 1 ca . B -i-> CD CU '•3 Q 2 B - ° *3 -»-> B ° ca & • B 3 > • <4-^  B o • CO •a *4-l CD J - H CD CO CD D , " 4-1 CU -B-W 00 +1 B ca CD rr .2 s ^ 3 O ftl) co fa •§ o o o iJ H B CD O H t-H CD cu 60 co ca M cw o o 3 T3 ° CN ^ i— i CU c3 1 3 >->—i cU •§ 3 co ca 3 ^ cu 2 p B r-; O o •a C S3 'B 1 7 3 60 g -lo .2 <u "3 ^ c3 ^ CO ^ 53 i n T3 « B 3 cu "3 1 - 1 3 « .a $ CD CD H g 60 '•a ^ CD ca 60 - ° O 3 2 ca JB 1-H O H 95 Calcium intake during the course of the experiment (g kg"1 body weight) was 1.56 ± 0.05 g kg ' 1 in chicks fed the rachitic diet, compared with 2.01 ± 0.05 g kg ' 1 in chicks fed control diet. Net calcium excretion (g kg"1 body weight) during the course of the experiment was (1.19 ± 0.05 g kg"1) in chicks fed the rachitic diet compared with 0.79 ± 0.05 g kg"1 in chicks fed control diet. A s a percentage of intake, the net absorption of dietary calcium in intact rachitic chicks was 2.5 fold less than that observed in intact control chicks. 96 4.4. Discussion 4.4.1. Relative Contribution of Vitamin D-Dependent Mechanisms to Duodenal Calcium Transport The present research has substantiated the existence of both cellular and paracellular pathways of intestinal calcium transport in the chick small intestine. The two pathways were clearly and consistently separable in the duodenum in response to: 1) a high calcium intake, 2) under rachitic conditions, and 3) after a l ,25-(OH )2D3 treatment of control and rachitic chicks. While there is little doubt that vitamin D plays a role in regulating intestinal calcium transport, the contribution of the vitamin D-dependent component of intestinal calcium transport has not yet been clearly delineated in the broiler chick. The fractional contribution of the vitamin D-dependent component of calcium transport calculated under rachitic conditions (38%), and after l ,25-(OH )2D3 treatment of rachitic chicks (39%) is different from that calculated with chicks fed a H C D (17 to 26%). Part of this difference may be explained by calculating 'overstimulation' of calcium transport, above that of the control, by l ,25-(OH )2D3 treatment. After treating 13-d-old control chicks with 1,25-(OH)2D3, there was a 16% increase (overstimulation) in calcium transport, over that of the 12-d-old control chicks not treated with l ,25-(OH )2D3; although, as pointed out earlier, this comparison may not be completely valid since data from two consecutive days are compared. The fractional contribution of the vitamin D-dependent component of calcium transport after l ,25-(OH )2D3 treatment of the rachitic chicks (39%), therefore, should be corrected for the 'overstimulation' (16%) factor. The new corrected value of the fractional contribution of the 97 vitamin D-dependent mechanism would be 23 percent, similar to the values determined under conditions of high calcium intake. 4.4.2. Ileal Calcium Transport Largely Occurs Independent of Vitamin D In the present research, calcium transport in the in situ ileal loop preparation appears to be primarily a paracellular process. This conclusion is based on the findings that there were no significant decreases in ileal calcium transport under three different experimental situations; 1) after a high calcium intake, 2) under rachitic conditions, and 3) in response to treatment of chicks with l , 2 5 - ( O H ) 2 D 3 . The acceptance of the concept that ileal calcium transport is primarily a paracellular process, is not universal. A complete absence of the cellular pathway in the ileum has been questioned in a number of experiments conducted with the Ussing chamber technique. Lee et al. (1981) gave large doses of l ,25-(OH )2D3 repeatedly to mature rats and were able to stimulate ileal calcium transport above that obtained with control rats. Nellans and Kimberg (1978) demonstrated the existence of a saturable component in rat ileal tissue mounted on Ussing chambers in response to increases in intralumenal calcium concentration. These researchers also suggested the existence of a cellular component of calcium transport in the ileum with evidence that the cellular component was abolished at a subphysiological temperature of 10 °C. Moreover, a low calcium intake was found to stimulate calcium transport in the ileum, thereby further supporting the presence of a cellular calcium transport component in the rat ileum (Nellans and Kimberg, 1978). In more recent studies with the Ussing chamber, Karbach (1992) demonstrated the existence of a saturable component of 98 calcium transport in the rat duodenum jejunum, and ileum. This researcher, reported that l ,25(OH )2D3 treatment of rats induced a significant increase in ileal calcium transport, thus implicating the existence of a cellular component of calcium transport in the rat ileum. The data obtained with the Ussing chamber technique also tend to disagree with the view that the contribution of vitamin D-dependent intestinal calcium transport decreases with increasing distance from the stomach (Pansu et a l . , 1983b; Boass and Toverud, 1996). For example, Karbach and Feldmeier (1993) reported that the saturable component in the rat cecum contributes nearly half of the total calcium transport. A l so , Nellans and Goldsmith (1981) reported that calcium transport in the rat cecum is down-regulated in response to an increased load of dietary calcium. The conclusion that ileal calcium transport is largely a vitamin D-independent mechanism obtains support from the findings of Behar and Kerstein (1976), that calcium transport from in situ ileal loops was not different between vitamin D-deficient and -replete rats. Pansu et. a l . , (1983b) demonstrated the lack of a saturable component of calcium transport in the in situ rat ileum, using the saturation technique. These researchers also reported that l,25-(OH)2D3 treatment did not stimulate ileal calcium transport. In contrast, however, Wasserman (1962) demonstrated that calcium transport from in situ ileal loops from leghorn chickens was significantly greater in vitamin D-sufficient chicks than in the rachitic chicks. More recently, Takito et al . (1992) were able to stimulate calcium transport in in situ duodenal, jejunal, and ileal loops of leghorn cockerels after a l,25-(OH)2D3 treatment of the vitamin D-deficient chicks. Similar data on broiler cockerels are not currently available. The present research extends these findings by making use of mannitol as marker of paracellular 99 transport and shows in three different ways that calcium transport from in situ ileal loop preparation, in broiler cockerels up to 14 d of age, is largely a vitamin D-independent mechanism. 4.4.3. Paracellular Transport is Not Regulated by Vitamin D Since mannitol transport in the in situ duodenal or ileal loops did not change after high calcium intake, under rachitic conditions, or for that matter in response to l ,25-(OH )2D3 treatment, it can be concluded that paracellular transport is likely not controlled by vitamin D . This conclusion is in agreement with that of Pansu et a l . , (1983b). Bronner (1992) and Bronner and Stein (1995) also maintain the view that paracellular calcium transport is not under a vitamin D regulatory control. The present research has, for the first time, used concomitant transport of calcium and mannitol in in situ duodenal and ileal loops in broiler cockerels to help delineate the cellular and paracellular components of calcium transport. The data are consistent with the theory that vitamin D does not significantly regulate paracellular transport. Karbach (1992) demonstrated that in response to l ,25-(OH )2D3 treatment, the absorption as well as the secretion of both calcium and mannitol were stimulated in the rat duodenal, jejunal, and ileal tissues mounted on Ussing chambers. Since, the permeability of the isolated intestinal tissue (Karbach, 1992) may be different from that of the in situ intestine, it is difficult to compare Karbach's data with the data obtained in the present study. 100 4.4.4. Calcium Absorption in Intact Rachitic Chicks In intact rachitic 12-d-old intact chicks, calcium absorption from the entire gastrointestinal tract was 40% that of the controls thus demonstrating that potentially 60% of the calcium absorbed in the control chicks was absorbed in a vitamin D-dependent manner. A discrepancy in the outcome, in terms of the fractional contribution of the vitamin D-dependent mechanisms in intestinal calcium transport, is apparent between the data obtained with intact chicks and the data obtained with in situ duodenal loops. A part of this discrepancy may be attributable to intralumenal calcium concentrations. Since there must be an upper limit to the ability of the intestine to absorb calcium via the cellular pathway, the fractional contribution of vitamin D-dependent calcium transport w i l l decrease with an increase in intralumenal calcium concentration. Expecting a five fold dilution of ingesta (Zornitzer and Bronner, 1971), and a 0.59% calcium concentration in the dried duodenal ingesta of 12-d-old unfasted intact chicks (Chapter 3; Figure 3.4), the calcium concentration in the duodenal lumen can be calculated to be approximately 25 m M . Since the calcium concentration of the intralumenal test solution was 75 m M , the fractional contribution of vitamin D-dependent calcium transport obtained with the in situ loop technique would be expected to be smaller than that obtained with intact birds. It is not clear whether the relatively low calcium absorption in intact rachitic chicks was associated with lower bioavailability caused by rickets. It is possible that vitamin D deficiency impairs dietary phosphorus absorption which in-turn may impair calcium absorption by decreasing its bioavailability (DeLuca and Schnoes, 1983). Feed utilization, as indicated by the feed conversion ratio, was markedly deteriorated in the intact rachitic chicks, compared 101 with the controls. However, using tibia ash (and calcium) concentrations in rachitic chicks as an indicator, it appears from the low bone ash and calcium results that impaired calcium absorption from the intestinal tract occurred. This is based on the fact that the amount of calcium absorbed by intact chicks was 70% less than by the control chicks whereas, the feed intake was reduced only 29%. In support of this conclusion are the findings of Weinstein et al . (1984), who demonstrated that tibia mineral concentration in rachitic rats could be normalized by intravascular injections of calcium. It also should be pointed out that calcium in the intralumenal test solutions was provided as the chloride salt whereas for intact chicks it was provided in the form of carbonate and phosphate salts. Since the solubility of calcium in the form of chloride salt is considerably greater than in the form of carbonate or phosphate salts, calcium transport via the paracellular pathway is more likely to be facilitated. The vitamin D-dependency of intestinal calcium transport in intact animals is worth evaluating. Kollenkirchen et al . (1991) reported that rats fed a vitamin D-deficient diet containing 20% lactose from weaning to 19 wk of age, maintained normocalcemia and achieved control tibia mineral concentrations. Many researchers have reported increased calcium utilization in rats fed the milk sugar lactose (Pansu et a l . , 1981) and phosphopeptides derived from, casein (Kitts et a l . , 1992). Mykkanen and Wasserman (1980) reported that inclusion of casein phosphopeptides in intralumenal test solutions resulted in a significant increase in calcium transport from in situ duodenal loops prepared from rachitic chicks, implicating that the stimulatory response was not dependent upon molecular changes induced 102 by vitamin D . It appears, therefore, that there is a relatively limited role of vitamin D in calcium absorption from the intestine, since when calcium concentration and bioavailability are not limiting, adequate amounts of calcium may be absorbed in the absence of vitamin D . 4.4.5. Calcium Metabolism and Vitamin D The findings that plasma and tibia calcium concentrations were significantly reduced in rachitic chicks by 7 d of age are consistent with observations made in rats (Brautbar et a l . , 1981). Vitamin D has an indirect effect on bone mineralization, as suggested by observations that low-calcium diets can impair mineralization (Morrissey and Wasserman, 1971). In rachitic chicks, the decrease in tibia fat-free dry weight was proportional to the decrease in body weight (18 to 31 % of control), but the decrease in tibia ash content was relatively large (43 to 53%). It may be concluded that the growth of the tibial organic matrix is not affected by vitamin D . Interestingly, Tsonis (1991) has reported that l ,25-(OH )2D3 stimulates transcription of collagen type II in chick limb bud mesenchymal cells. A more interesting finding was that in rachitic chicks, the tibia length was decreased only to a limited degree, compared with the decrease in tibia fat-free dry weight. The longitudinal growth of the organic matrix of bone in the present study does not seem to have been seriously decreased by vitamin D deficiency. A moderate increase in hematocrit values in the rachitic chicks may reflect decreased water consumption because of impairment of locomotion. It is not clear whether such a degree of dehydration can reduce calcium absorption in intact chicks by decreasing its solubility in the intestinal lumen. 103 4.4.6. Vitamin D-Dependent Intestinal Calcium Transport: Are Juvenile Chickens Different from Neonatal Rats? Since calcium transport from in situ duodenal loops in 4-d-old broiler cockerels was significantly decreased in response to high calcium intake, the vitamin D-dependent component of calcium transport in this species appears to exist at least by day 4 of age. Pansu et al. (1983a) and Dostal and Toverud (1984) reported that the vitamin D-dependent component in laboratory rats is not apparent until weaning. A possible reason for these differences between the two species could be related to dietary differences. Newly hatched broilers are fed diets relatively similar to diets fed to mature chicks. In contrast, newly born rat pups are nursed by their dams. The intestine of newly hatched chicks very likely needs to be more mature compared with the intestine of newly born rats on anatomical and physiological bases, i f it is to be able to absorb sufficient amounts of dietary calcium. 4.4.7. Mannitol as a Marker of Paracellular Calcium Transport The use of an inert marker molecule of paracellular transport, such as mannitol, to obtain a valid estimate of paracellular calcium transport may be questioned. Since at present, there exists no technique to provide direct evidence for the pathways calcium ions take when crossing from the intestinal lumen to the lateral space, indirect evidence is used to delineate these components. The marker molecule technique used in the present research appears to provide a valid estimate of the relative contributions of the cellular, putatively vitamin D -dependent, and the paracellular, putatively vitamin D-independent components of intestinal calcium transport when 75 m M intralumenal calcium and mannitol is used. 104 O n a molar basis, mannitol transport was significantly lower than calcium transport in chicks fed the control diet. After providing a high dietary calcium intake, which would down regulate the cellular component of calcium transport by down regulating l ,25-(OH )2D3 biosynthesis and consequently calcium binding protein biosynthesis (Boass and Toverud, 1996), it is interesting to note that calcium transport was not significantly different from that of mannitol transport; an effect consistently noted in birds at four different ages and also during the cross-over experiment. Mannitol transport, therefore, may be considered to provide a valid estimate of paracellular calcium transport in the experimental model used in these studies. 105 4.5. Conclusions It is concluded that in rapidly growing young broiler cockerels, calcium transport from in situ duodenal loop preparations has a vitamin D-dependent component, whereas this component is absent in the distal ileal loop preparations. Paracellular mannitol transport from the in situ duodenal and distal ileal loop preparations is not acutely regulated by vitamin D . A t 75 m M intralumenal C a C h , the fractional contribution of vitamin D-dependent calcium transport in in situ duodenal loops ranges from 15 to 25% of the total. The vitamin D -dependent mechanisms of calcium transport are present at 4 d of age in rapidly growing young broiler cockerels. 106 C H A P T E R 5 Paracellular Absorption of Dietary Calcium in Rapidly Growing Young Broiler Cockerels 5.1. Introduction The data reported in Chapter 4 indicates that calcium transport in the in situ intestinal loop preparations is largely a vitamin D-independent process when intralumenal calcium concentration is high. Pansu et al . (1981) demonstrated that vitamin D-independent calcium transport from the in situ intestinal preparation in laboratory rats takes place in a nonsaturable manner. It is generally believed that nonsaturable calcium transport occurs via the paracellular pathway (Dostal and Toverud, 1984; Bronner, 1992). Paracellular calcium absorption increases as a linear function of intralumenal calcium concentration in in situ intestinal loops (Zornitzer and Bronner, 1971). Since, the paracellular pathway is considered to offer considerably less resistance to the transport of water and hydrophilic solutes (Bronner, 1987) compared to the cellular pathway (Boass and Toverud, 1996), the linear relationship between paracellular absorption and concentration is explained. Whether the linearity of the relationship that exists between absorption and concentration in in situ loop experimental model also exists in the intact animal model, has yet to be determined. The fact that the absorptive epithelium of the intestine is a highly permeable tissue (Ballard et a l . , 1995), suggests that paracellular calcium transport could play an important role under normal dietary conditions in intact chicks. 107 To date, only limited research has been undertaken to address the significance of paracellular pathway to intestinal calcium transport in intact animals. Pansu et al . (1993) fed laboratory rats diets ranging in calcium concentration from 1.5 to 2.94%. They provided dietary calcium with varying degrees of solubility ranging from very low, such as calcium carbonate, to very high, such as calcium gluconate. They demonstrated that net calcium absorption increased when calcium was provided as the more soluble gluconate salt compared to when it was provided as the relatively less soluble carbonate salt (Pansu et a l . , 1993). Since the concentration of calcium binding protein (calbindin-9k) in the duodenal mucosa decreases with an increase in net calcium absorption, they concluded that the fractional contribution of the saturable (cellular) component of calcium transport was diminished in rats fed calcium in the form of calcium gluconate. Thus, the fractional contribution of calcium transport via the paracellular pathway appeared to increase with increased solubility of dietary calcium. In a more recent report, Duflos et al . (1995), determined the intestinal sojourn time, and the amount of soluble calcium in the small intestine of rats fed diets containing calcium concentrations higher than normal. The authors concluded that the nonsaturable (paracellular) calcium transport was in part co-determined by calcium solubility and intestinal sojourn time. Long ago, the question pertaining to the relationship between calcium solubility and absorption was addressed in a more direct manner by Marcus and Lengemann (1962). They measured calcium absorption in rats by either providing C a C h as liquid dose via stomach tube, or by providing the same dose mixed in diet by normal feeding. They reported calcium absorption value of 45% with the liquid dose and 19% when the same liquid C a C h dose was provided mixed in the diet, indicating that calcium solubility enhances absorption. However, a 108 proper interpretation of their data in terms of a relationship between solubility and absorption is difficult since the dose mixed in the diet supplied 38 mg of calcium whereas the liquid dose supplied only 6.6 mg. The experiments reported in this chapter were conducted with the objective of determining whether calcium solubility is related to paracellular calcium transport in broiler chickens. In addition the question, whether intestinal calcium transport in intact chickens is largely a paracellular process under certain conditions as suggested by the findings obtained with the in situ loop technique in Chapter 4. In the first experiment, the characteristics of intestinal calcium and mannitol transport were determined with in situ duodenal loops. In the second experiment, the characteristics of intestinal calcium transport in intact birds were determined under conditions of moderately high calcium intake, accomplished by either adding calcium gluconate + calcium lactate salts to normal diets, or by providing a 40 m M C a C h solution as drinking water to birds fed a normal diet. It was estimated for these experiments that provision of 40 m M C a C h solution as drinking water to birds fed a normal diet could establish an intralumenal calcium concentration similar to that obtained in the in situ intestinal loop preparations. A l s o , the physiological relevance of the data obtained with the in situ intestinal preparation were be established since the form, solubility, and probable intralumenal concentration of calcium were similar between the in situ loop model and the intact bird model. 109 5.2. Methods 5.2.1. Characteristics of Intestinal Mannitol and Calcium Transport from In Situ Duodenal Loops Preparations Intestinal mannitol transport was determined in in situ duodenal loop preparations in 0-, 4-, and 21-d-old birds fasted for a 12 h period before the experiment. Intralumenal test solutions contained 50 (2 n C i pmol 1 ) , 100 (1 n C i pmol"1), 150 (0.75 n C i pmol"1), 200 (0.5 n C i pmol" 1), 250 (0.41 n C i pmol" 1), or 300 (0.33 n C i pmol"1) m M 3 H-mannitol. The solutions were made iso-osmolar with N a C l , where necessary. Four or five birds were used in each treatment. A t the termination of the 10 min experiments, the duodenal loops were removed from the body and flushed with 'cold (nonradioactive)' test solution to ensure maximum recovery of unabsorbed mannitol from the lumen. The amount of mannitol that disappeared from the lumen was calculated by subtracting the total amount recovered from the lumen from the amount injected. Intestinal calcium transport was determined in 21-d-old birds fasted for a 12 h period before the experiment. Intralumenal test solutions contained 2.5 m M (40 n C i pmol"1) (n=14), or 100 m M (1 n C i pmol"1) 4 5 C a C h (n=24). The 2.5 m M C a C h solution was made iso-osmolar with N a C l . A t the termination of each 10 min experiment, the duodenal loops were removed, ashed at 600°C for 8 h, and the amounts of 4 5 C a in the lumen+tissue were individually determined. The amount of calcium absorbed during the incubation period was calculated by subtracting the total amount recovered in the lumen and in the tissue from the amount injected. The details of experimental procedures and analyses are provided in Chapter 3. 110 5.2.2. Characteristics of Intestinal Calcium Transport in Intact Birds with Two Different Regimens of Increasing Calcium Intake Calcium retention in intact birds was determined during 24 h balance experiments. The control birds were fed a normal commercial broiler starter diet. A high calcium intake was accomplished by either providing a high calcium diet (HCD) or drinking water with high calcium concentration. The H C D was prepared by adding (3:1) calcium-gluconate plus calcium-lactate salts ( B D H Inc. Toronto, Ontario, Canada) into the commercial broiler starter (control) diet. Alternately, a 40 m M C a C h solution was provided during the experimental period. A preliminary trial indicated that water intake significantly decreases when the calcium concentration in drinking water is increased to more than 40 m M C a C h . The calcium concentration in the H C D was adjusted after a preliminary trial so that at any given age, the calcium intake of birds fed the H C D was approximately equal to the calcium intake of the birds that drank the CaCh. solution. Calcium and phosphorus concentrations of the diets were determined to be as follows: high calcium diet, calcium (% dry matter) (1.65%), phosphorus (0.75%); control diet, calcium (1.09%), phosphorus, (0.82%). The 24 h balance experiments were concluded in birds at 4, 7, and 14 d of age. Total excreta was collected on wax paper, dried, pulverized, thoroughly mixed, and an aliquot taken for ashing. The ash was analyzed for calcium content. The drying, ashing and analytical procedures are described in Section 3.3. In experiments conducted with the H C D , the diet was provided from hatch. The experiment with 4-d-old birds was conducted with 10 pens containing 13 birds each; five pens i l l were allocated each of the H C D and the control diets. The experiment with 7-d-old birds was conducted with 10 pens containing eight or nine birds each; five pens were allocated to each of the H C D and the control diets. The experiment with 14-d-old birds was conducted with six pens containing three or four birds each; three pens were allocated to each of the H C D and the control diets. In experiments where calcium intake was increased via the drinking water, a 40 m M C a C h solution was provided throughout the experimental period. These birds were fed the control diet from hatch throughout the experiment. The experiment with 4-d-old birds was conducted with eight pens each containing 12 or 13 birds; four pens allocated to 40 m M C a C h solution and to distilled water. The experiment with 7-d-old birds was conducted with 10 pens each containing eight or nine birds; five pens allocated to 40 m M C a C h solution and five pens to distilled water. The experiment with 14-d-old birds was conducted with four pens each containing 6 birds; two pens allocated to 40 m M C a C h solution and two pens to distilled water. 112 5 . 3 . Results 5.3.1. Characteristics of Intestinal Mannitol and Calcium Transport from In Situ Duodenal Loop Preparations Figure 5.1 shows the amount of mannitol disappeared (pmol g"1 10 min"1) from in situ duodenal loop preparations in 0-, 4-, and 21-d-old chicks as a function of intralumenal mannitol concentration. The amount of mannitol disappeared increased as a direct function of the concentration, in each age group. Regression analyses described the relationship between intralumenal mannitol concentration and the rate of transport as y = -1.29 +0.197x at 0 d, y = -2.7 + 0.253x at 4 d, and y = -0.66 + 0.371x at 21 d of age. The y-axis intercept was not significantly different from zero at 0 d (P < 0.51), 4 d (P < 0.69), and at 21 d of age (P < 0.90). These data clearly indicate that mannitol transport from the in situ duodenal loop preparation is a nonsaturable process. A n essential characteristic of the nonsaturable transport process is highlighted by the fact that the percentage of intralumenal mannitol that disappeared from the lumen of in situ duodenal loop preparation did not change with change in concentration (Figure 5.2). Figure 5.3 shows calcium transport in in situ duodenal loop preparations in 21-d-old chicks containing 2.5 m M or 100 m M intralumenal calcium. The results indicate that intestinal calcium transport in in situ duodenal loop preparations is a saturable phenomenon, in contrast to intestinal mannitol transport. The amount of calcium disappeared (pmol g"1 10 min"1) obtained with 2.5 m M intralumenal calcium was 22 fold greater than the amount obtained with 100 m M intralumenal calcium, despite a 40 fold difference in the concentration. In other 113 T3 cu Ui cs cu a 03 7 2 S ® o S o ° i c 3 O 140 120 H 100 80 60 40 20 0 y = -1.29 + 0.19x 0 d 0 50 100 150 200 250 300 350 4 d i — i — r 0 50 100 150 200 250 300 350 21 d i i r 0 50 100 150 200 250 300 350 Intralumenal Mannitol [mM] Figure 5.1. Mannitol disappearance (Mean + S.E) from in situ duodenal loop preparations in 0-, 4-, and 21-d-old broiler cockerels (n=4 or 5), as a function of intralumenal mannitol concentration. 114 TS r H rN i r >o V T3 ^ ^ ^ ^ ^ ^ ^ T = E = cu -= co crj o H ^ \ \ \ \ \ \ \ \ \ \ W 8 eg u cu "t3 (UIUI 01 UI 3S0p JO %) 3DUBJB3ddBSip lojiuuepv 115 ( m u i oi i n asop j o %) p a j c a d d e s i p u i n p i e o j i r a o j a j ( T .UIUI oi x - § loiurl) pa jBaddes ip u i n p i e o j o j u n o u i y 03 13 cs E 13 u .a a 03 £ ^ £ •o +, o o3 1=1 CD CD l-< CD OH O O O •t—» o 03 CD CD s C eS _ ca c3 a « « 03 C u » CD OH ™ O OH -ca to ^ . 2 2 7 D a -a g * l 8 r 8 i-H CD O fci r. CD i 3 ii a c <N Z P ^ CD O •8 1 V "53 .2? w T SH CD of) g .-73 —1 S ° o a. ^ 3 - M - ° ^ O • § <^ s s .s o CD C3 3 O a 03 CD 03 co « O ca T3 OH , CD £ O OH 1 = 3 Tci . o c U - 5 .CD 3 cd T3 § r-" <4-H 3 h O 13 CD 53 ft £ o3 CD 2 .a a "o £ „ co CD u co s CD S 03 +-» - O > CD 4-H W X! O 3 § •a < -a 2 • >^  § ^ § o * a a ^-2? CD cs o ?S CD w ,3 116 words, the amount of calcium absorbed from in situ duodenal loop preparations did not increase as a linear function of concentration. Unlike mannitol, the percentage of calcium that disappeared from the in situ duodenal loops (% of dose in 10 min) containing 2.5 m M calcium was two fold greater ( P < 0.001) than the percentage that disappeared from the loop containing 100 m M calcium. Since this result occurred despite a 40 fold difference in concentration, it suggests the saturable nature of calcium transport in the in situ duodenal loop preparations. 5.3.2. Feed, Water, and Calcium Intakes in Intact Birds Fed the High Calcium Diet or Drank 40 mM CaCh. Figure 5.4 shows the effects of H C D on feed intake in 4-, 7-, and 14-d-old birds. A t 4, and 14 d of age, there was no significant difference in feed intake (g bird"1 24h - 1) between birds fed the H C D and those fed the control diet. A t 7 d of age, however, feed intake in birds fed the H C D was significantly (P < 0.05) less compared with the intake in controls birds. Figure 5.5 shows the effects of drinking 40 m M C a C h on feed and water intakes. In 4-and 7-d-old birds, there were no significant differences in feed intake (g bird"1 24h"') between birds consuming 40 m M C a C h in drinking water and the controls which were given distilled water. However, in 14-d-old birds, the feed intake by birds which drank 40 m M C a C h was significantly (P < 0.05) less compared to the birds which drank distilled water. In 4- and 14-d-old birds, there were no significant differences in water intake (mL bird"1 24h"') between birds which drank the 40 m M C a C h and birds which drank distilled water. In 7-d-old birds, water intake by birds which drank the calcium solution was significantly (P < 0.05) greater than the intake by birds which drank distilled water. 117 CD < Q a t : m CO CO CU '•3 CU P-i ca .Xr cu cu i2 -C cu VH 1) ° 3 " 8 cu CD CU OS h1 Os CD CD CO ca 118 cd x) -o CN cd OA 13 3 5 cd 5 x3 u o r -1) on + 1 ^ CO cd >* d B O O S ? ^5 q CN in 119 Table 5.1 shows the amounts of calcium consumed and calcium absorbed (g kg"1 24 h"1) by 4-,7-, and 14-d-old birds in which calcium intake was increased by either feeding the H C D or by provided 40 m M C a C k in drinking water In birds fed the H C D , calcium intake significantly (P<0.01) exceeded controls by 48 to 60%. While birds which drank 40 m M C a C h , consumed 31 to 53% more calcium than control birds (P<0.01) . The amount of calcium absorbed by birds either fed the H C D or provided 40 m M C a C h as drinking water, was significantly greater than for their respective controls (P<0.01) . A s may be expected, therefore, the amount of calcium absorbed increased with an increase in calcium intake. 5.3.3. Characteristics of Intestinal Calcium Transport in Intact Birds with Two Different Regimens of Increasing Calcium Intake Figure 5.6 shows the percentage of calcium retained (percent of intake in 24 h) by birds fed the H C D and by birds which drank 40 m M C a C h , at 4, 7, and 14 d of age. A t all ages under investigation, the percentage of dietary calcium retained by birds fed the H C D was 16 to 22% less compared to birds fed the control diet (P<0.01) . In contrast, the percentage of calcium retained by birds which drank distilled water was not significantly different from birds which drank 40 m M C a C h . These data indicate that calcium absorption by intact birds was a saturable process when calcium intake was increased by dietary supplementation with calcium gluconate and lactate salts, since percent calcium retention differed with intake. In contrast, the absorption appeared to be a nonsaturable process when the intake was increased by chloride salt dissolved in drinking water, since percent calcium retention did not differ with intake. 120 o « 43 CD CN CU u is .2 ^ .3 'oo 5 i "c3 bO CD ca T 3 o CO > 3 „ TH £ L 43 ft 3^ _ cu <A § 3 3 8 ca 3 T3 CU T3 '> O u ca U ftS e s 3 u l a U o ca T 3 cu '> o CU -4—» ca o CO -2 <2 ft 73 CU coj » ca c o O "Ho .3 |o "ca o 43 00 a o •= c o 1) oo a o U e 3 "ca o 43 oo c o U _3 73 "ca o 43 00 s 3 O CJ co o d + l r-m CN o O +1 CN o CN O CD +1 00 CN o d +i r -os V OH V a. o d + l co § O d + l co co CN V ft. 8 V o d +1 CO 00 CN o d + l m o d +l as CN T - H V o a, d +i in o CO O d +1 co co o d + l o\ >n CN V o d +i sO CN O d +1 SO OS CO 1—I d + l o CN O d + i co ON sO O d +1 r-CN CN sO o d + l CN V ft. o o d V ft. 8 v a, co o co T - H • in o d d d +1 +1 +l CN CN Os CO o r-CO 0.00 CO 0.00 CN 0.00 r-o p< o p< CN O P< d d d + l +1 +l o CN CN 00 CN CN 12 1 cs '•5 2 *> T3 B a> E i> a a. s E _3 'o a "3 u « to 3 Q. <u os S o u J3 Tab in CS OX re •° ^ ^ ^ ^ ^ re H 1 1 1 1 1 1 iioiju9j3.i mnpn?3 2 o co *-* -i< 3 SB " "S 2 ^-N 3 1 "2 I -t; O , H 3 cd f! M & T3 a, C CD — c ' cd » OH -3 T t 43 H Cd © -5 3 -g xi co :jg cd W co > a oo co ex ^ 3 +1 *3 .£3 3 « CO QJ .3 CO IS ^ cs jo cd ^ w CD OH "o 33 u c S CO 3 o l-i '3 co co •3 w £ B S 5 » C ' 3 S 2 S ° I H - 2 J3 ° ' S O ft £H .3 CD <£j c^  aj O, co 3 O | < _g 8 £ .S „ 3 to K « o » r CO 33 CO CO H 2 ^ J3 ft U ° « 2 • ^  U J) S « o x; ^  u O w « 13 3 3 ° ft 5 33 2 ^ ft Of) O ft CN ctl JD .13 13 CO * o co 4-H to 3 O 122 5.4. Discussion The present results are consistent with those presented by Krugliak et al . (1994) showing that intestinal mannitol transport in the in situ intestinal preparations of laboratory rats occurs by a first order process. Three observations from the present study are notable in this respect: 1) the amount of mannitol that disappears from the in situ duodenal loop preparations increased as a direct function of intralumenal concentration, 2) the y-axis intercept created by the absorption curve is not significantly different from zero, and 3) the percent disappearance of mannitol is independent of the intralumenal concentration. The fact that the intercept was not different from zero indicates that mannitol transport takes place solely via the paracellular pathway, since the involvement of the cellular pathway would be expected to create a positive value for the y-axis intercept. Mannitol is generally considered to be a molecule restricted to the extracellular space. Wick et al . (1954), and more recently, Pappenheimer (1990) have demonstrated that significant amounts of mannitol can be degraded by the rat liver but not by the intestinal tissue. Whether or not the chick intestinal tissue can degrade mannitol remains to be studied. The paracellular pathway appears to be restricted only by the size of the tight junction gaps (Czaky, 1987). K i m (1996) has demonstrated that transport of polyethylene glycol ( P E G , a marker of paracellular transport) oligomers in the distal part of the rat small intestine in situ is inversely proportional to the square of the molecular weight of the oligomers. Since mannitol is a relatively small size molecule, its use as a marker of paracellular transport has become a common practice in studies of intestinal calcium transport, determined with Ussing Chambers (Karbach, 1992; H u et a l . , 1993). 123 In contrast to mannitol transport, calcium transport in the in situ duodenal loop was shown to be a saturable process, since the amount disappeared was not a direct function of the intralumenal calcium concentration and the percentage absorbed varies significantly greatly with concentration. Since the intestine is permeable to mannitol, and to other markers of paracellular transport which are much higher in molecular weight than mannitol such as inulin (5500 Da , Pappenheimer and Reiss, 1987) and PEG-2000 (2000 Da ; Donovan et a l . , 1990), it is unlikely that calcium w i l l be excluded from the paracellular pathway. The existence of cellular and paracellular pathways for intestinal calcium transport has been widely recognized as reviewed by Wasserman and Fullmer (1995). The most striking finding of the present study was the difference in intestinal calcium transport characteristics between birds in which a high calcium intake was accomplished by feeding the H C D , and the birds in which it was accomplished by providing 40 m M ' C a C k as drinking water. In intact birds fed the H C D , the percent calcium retention (% of intake kg"1 24 h"1) decreased in the range of approximately 17 to 22% of controls, while calcium intake (g kg"1 24 h"1) increased 48 to 60 percent of controls. Thus a net calcium retention increased in response to consuming the H C D . Also , calcium retention in these intact birds appears to be a saturable process since percent calcium retention in birds fed the H C D would not be different from controls, i f the retention was a nonsaturable process. On the other hand, in birds which drank the 40 m M C a C h , the percentage of calcium retained was not different from that of the control birds which drank distilled water. This result occurred despite the fact that calcium intake was approximately 32 to 53% greater in these birds than in control birds. Al so , the net calcium balance achieved in birds which drank the calcium solution was greater compared to 124 that in birds fed the H C D . This occurred because the amount of calcium retained by birds which drank the calcium solution was 1.2 to 1.4 fold greater than by the birds fed the H C D . Conflicting results have emerged in terms of characteristics of intestinal calcium transport in intact birds obtained by the two modes of increasing calcium intake. Calcium transport appears to be a saturable process when calcium intake is increased by adding calcium gluconate with calcium lactate salts to the diet. In contrast, calcium transport appears to be to a nonsaturable process when calcium intake is increased by providing a calcium chloride solution as drinking water. This apparent conflict may be explained by the difference in solubility of the calcium salts used in these experiments. Pansu et al . (1993) reported that the net calcium absorption, when calcium was present in the diet as a gluconate salt, was nearly two fold greater compared to when calcium was present as the carbonate salt in the diets of intact rats fed similar amounts of calcium (~3% dietary calcium). It may be pointed out that at p H 7.4, the solubility of calcium carbonate is nearly 4 mmol /L (Washburn, 1928), whereas the solubility of calcium gluconate is nearly 118 mmol /L (Dean, 1979). In comparison, C a C h is many fold more soluble than gluconate or lactate salts. To more clearly address the relationship between calcium solubility and absorption, C a C h was provided in aqueous solution instead of mixing it in the feed in the present experiment. This experimental protocol mimics in situ loop experimental conditions, since calcium in intralumenal test solution is routinely provided as the chloride salt. It was concluded in Chapter 4 that when intralumenal calcium concentration was high and solubility of calcium was not limiting, the fractional contribution of the saturable component of calcium 125 transport in the in situ duodenal loops of broiler cockerels was small. Moreover, the distal part of the small intestine in chicks was nearly devoid of the saturable component, a finding confirmed in other animal species by other researchers (Behar and Kerstein, 1976; Pansu et a l . , 1983b; Yuan and Kitts, 1992; Boass and Toverud, 1996). In the present study, when intact birds were provided calcium in the form of a highly soluble salt in a solubilized form, the fractional contribution of the nonsaturable calcium transport was so large that the relative saturable component of calcium transport becomes barely detectable. The probable reason for this apparent lack of a saturable component is that the chloride salt likely remains in solution even in the distal small intestine of chickens where a relatively high p H of 7.96 (Coates and Holdsworth, 1961) favors calcium precipitation under normal conditions (Allen, 1982). The bioavailability of calcium may, therefore, be increased, facilitating paracellular absorption (Duflos et a l . , 1995). It is clear that the fractional contribution of the paracellular component of calcium transport in intact birds is high under conditions of high calcium intake when solubility is not a limiting factor. The physiological relevance of data obtained with in situ or in vitro intestinal preparations has not been established. In the words of Pansu et al . (1993), " i f a rat intestine is capable, when studied as a ligated loop in situ, of readily absorbing calcium concentrations of 100-150 mmol /L at the appreciable rate of 16%/h (Bronner, 1987; Bronner et a l . , 1986), why does this not seem to occur in whole animal studies?". One is prompted to consider that the permeability of in situ intestinal loop preparations may be different from the permeability of the intestine in intact birds, although such a difference remains to be illustrated. It is widely reported that the contribution of the saturable component of calcium transport is down 126 regulated with increased calcium absorption (Pansu et a l . , 1981; Boass and Toverud, 1996). It is possible that the down regulation of the saturable component was relatively complete in birds which drank the calcium solution, since they retained more calcium (g kg"1 24 h"1) compared to birds fed the H C D . Under these conditions calcium transport w i l l occur predominantly via the paracellular pathway, thus showing that the outcome of experiments conducted with in situ intestinal loop preparations is similar to the outcome obtained in intact birds under similar experimental conditions,. In other words, the data obtained with in situ intestinal preparation is physiologically relevant. In birds which drank the calcium solution, most of the calcium absorbed would have come from the chloride rather than the carbonate form, a major form of calcium in commercial diets. In this context, Pansu et al . (1993) demonstrated that when rat diets are constituted with two or more calcium salts of widely differing solubilities, calcium w i l l be absorbed from each salt in direct proportion to its solubility. Since calcium in most commercial diets is primarily provided as the carbonate salt, the question arises as to how this poorly soluble form of calcium is absorbed. The low p H of the stomach ensures solubilization of calcium carbonate (Bronner et a l . , 1991; Pak et a l . , 1989) which facilitates calcium absorption in the proximal small intestine. Since the intestinal p H rises in the caudal direction (Duflos, et a l . , 1995), some calcium may re-precipitate in the distal small intestine (Schedl et a l . , 1968) and therefore become poorly absorbed. When both C a C k and CaCCh are present, the chloride, because of its relatively higher solubility, w i l l tend to stay in solution whereas the carbonate wi l l precipitate (Moore and Verine, 1985). Thus, these relative solubility characteristics explain the reason why calcium absorption was found to be increased as a direct function of intake 127 when the chloride form was fed. Relatively poor solubility of the gluconate and lactate salts compared to the chloride salt appears to be a reason for the relative decrease in the amount of calcium absorbed in birds fed the high calcium diet compared to birds which drank the calcium solution. Solubility appears to be the primary cause of the differences in the outcome of in situ loop experiments and intact animal experiments. Balance experiments conducted with rats show that calcium absorption plateaus with an increase in calcium intake (Cohn et a l . , 1968; Pansu et a l . , 1993) when the solubility of calcium is low. O n the other hand, the rate of calcium transport from in situ intestinal loops increases as a direct function of intralumenal calcium concentration (Pansu et al . 1981, 1993). Since calcium was provided as the chloride salt in in situ loop experiments, solubility was not a limiting factor. A l so , the intestinal preparations used in in situ experiments are usually emptied by procedures such as fasting (Wasserman, 1962; Charpin et a l . , 1992) or rinsing (Pansu et a l . , 1993), which reduces the chances of calcium precipitation by ingesta contents such as phytate. It may be concluded that the results obtained with the in situ intestinal loop preparations are reproducible in intact animals provided calcium solubility and bioavailability remain similar. Calcium solubility is an important determinant of bioavailability. Mykkanen and Wasserman (1980) demonstrated that purified casein phosphopeptides (CPP) may stimulate intestinal calcium transport in both normal and rachitic chicks. Yuan and Kitts (1994) showed that calcium absorption and bone utilization in spontaneously hypersensitive rats was decreased in rats fed heat-damaged casein, in which the C P P production becomes limited. Gerber and 128 Jost (1986) demonstrated that C P P inhibit the formation of insoluble calcium phosphate complexes in vitro indicating that the effect of C P P on stimulating intestinal calcium absorption may be caused by an increased solubilization of dietary calcium. Provision of dietary calcium in the form of oyster shell in the diets of layer-type chickens is one example of improving egg shell quality and egg production, thus indicating the importance of increased bioavailability of calcium to its digestibility. Keshavarz and Scott (1993) reported that the in vitro solubility of pulverized oyster shell was 77.8 percent compared to 46.6 percent of the pulverized lime stone. It may be noted from a nutritionist's view that nearly 50 % of dietary calcium is absorbed in the chicken. The dietary inclusion of calcium may be reduced i f the bioavailability of dietary calcium is not limiting. 129 5.5 Conclusions The present experiments demonstrate that intestinal mannitol transport is a nonsaturable and, therefore, likely a paracellular process. On the other hand, intestinal calcium transport has both saturable and nonsaturable components. When concentration and solubility of dietary calcium are not limiting factors, the intestinal calcium transport in intact birds occurs largely by a nonsaturable process, likely via the paracellular pathway. These data obtained with intact birds demonstrate the physiological relevance of the data obtained with in situ loop preparations, since majority of calcium transport in both experimental systems occurs via a nonsaturable paracellular pathway, when solubility is not a limiting factor. The results emphasize the importance of calcium solubility that facilitates paracellular calcium absorption. 130 C H A P T E R 6 Age-Related Changes and Regional Differences in Paracellular Absorption, and Regional Differences in Paracellular Secretion in the Small Intestine of Rapidly Growing Young Broiler Cockerels 6.1. Introduction The mucosa of the gastrointestinal tract can be considered a protective boundary between the external and internal milieus, allowing uptake of water, nutrients, and electrolytes but excluding many other compounds ( K i m , 1996). Absorption of nutrients and other molecules across the mucosal barrier of the intestine takes place via cellular or paracellular pathways. Absorption via the paracellular pathway is limited primarily to gaps between tight junctions on adjacent enterocytes (Madara, 1989). The greater permeability of certain epithelia is inversely related to the density of tight junction strands (Claude, 1978). The mucosal absorptive layer of the intestine is highly permeable to ions and water and is, therefore, considered to be a leaky epithelial tissue as reviewed by Ballard et al . (1995). The significance of nutrient absorption via the paracellular pathway has only been investigated in the recent past. These investigations indicate that a significant fraction of nutrients such as glucose and certain amino acids may be absorbed via the paracellular pathway in laboratory rodents. For example, Pappenheimer (1990) reported that 50 to 65% of creatinine ingested by rats is recovered from urine compared with 75 to 85% recovered in urine after intraperitoneal or subcutaneous injection. These results demonstrate a high 131 paracellular permeability of the intestine to this molecule which is restricted to the extracellular space. Pappenheimer and Volpp (1992) have suggested that the paracellular pathway of nutrient absorption from the intestine is under a regulatory control, similar to the cellular pathway. While confirmation of these findings from other laboratories is still awaited, they stimulated interest of the author to explore the role of the paracellular pathway in nutrient absorption from the intestine of the broiler chick. Possible differences in the paracellular permeability of various regions of the intestine may have clinical importance in diseases affecting different regions of the intestine. Abnormal permeability of various regions of the intestine may also be important in the pathogenesis and pathophysiology of various diseases. For example, intestinal permeability invariably increases in Crohn's disease in humans (Jenkins et a l . , 1986). It is not known whether poultry diseases such as coccidiosis and enteritis which may localize at certain intestinal regions alters intestinal permeability. The experiments reported in Chapter 4 highlight the significance of paracellular pathway in calcium absorption from the gut of intact broiler chickens. Since these birds are selected for a high growth rate, it is possible that the paracellular permeability of their intestine is also high to match their needs for an efficient nutrient extraction from the diet. It was, therefore, of interest to know whether the paracellular permeability in different regions of the small intestine of rapidly growing broiler chickens varies with age. To the author's knowledge, there are no published reports in the literature available on this subject. 132 The experiments described in this chapter were conducted with the objectives: 1) to determine the age-related changes in paracellular absorption from the lumen of in situ duodenal, jejunal, and ileal loop preparations, 2) to determine regional differences in the small intestine for paracellular absorption from in situ loops, and 3) to determine i f there are differences between the duodenum, jejunum, and ileum for paracellular secretion from the blood into the lumen of in situ intestinal preparations. 133 6.2. Methods 6.2.1. Age-Related Changes and Regional Differences in Paracellular Absorption in In Situ Duodenal, Jejunal and Ileal Loop Preparations in Broiler Cockerels Paracellular absorption of mannitol from in situ loop preparations of the duodenum (n = 10 to 30), distal jejunum (n = 10 to 13), and distal ileum (n = 13 to 23) was determined in broiler cockerels at 0, 2, 4, 7, and 14 d of age. The birds were fasted for 12 h before each experiment. The intralumenal test solution contained 300 m M 3H-mannitol (S .A . , 0.33 n C i pmol"1) which is iso-osmolar. In some of the experiments, simultaneous determinations were made with loops prepared from both the duodenum and distal jejunum, or from both the distal jejunum and distal ileum. Where two loops from the same chick were used, the test solution was injected into the duodenal loop first and then 30 sec after into the jejunal or ileal loop. Ten min postinjection, the loops were removed, in the same order in which they were injected. The contents of the loops were removed by flushing with excess quantities of 300 m M mannitol to ensure maximum recovery of the labeled mannitol. The emptied loops were longitudinally opened, blotted dry and their weight and length were recorded to determine whether the efficiency or capacity of paracellular absorption changes with increase in the growth of the intestinal tissue. The details of in situ loop preparation and analytical procedures are described in Chapter 3. 134 6.2.2. Regional Differences in Paracellular Secretion from Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels Mannitol secretion from blood into the lumen of in situ duodenal, jejunal, and ileal loop preparations was determined in 18-d-old broiler cockerels. The birds were fasted 12 h before each experiment. Mannitol secretion was concomitantly determined in in situ duodenal (n = 20) and distal jejunal (n = 11) loop preparations, or in in situ duodenal and distal ileal (n = 9) loop preparations. The loops were prepared, filled by injection with a predetermined volume of 0.85% N a C l solution, and the abdominal wall was clamped by hemostats(s). A 300 m M 3H-mannitol solution of high specific radioactivity (3.3 n C i pmol"1) was injected into the wing vein (1.5 m L kg"1). Intravenous injection was completed within 3 to 4 sec which marked the start of the experiment. A blood sample was collected at 9-10 min via intracardiac puncture. The loops were removed from the body at approximately 10 min and their contents collected by flushing with an excess volume of 300 m M mannitol solution to maximize 3H-mannitol recovery. Plasma was separated from the blood and the plasma 3H-mannitol content was determined by liquid scintillation counting procedures. Calculations of the plasma 3H-mannitol concentration and the amount of mannitol secreted into the intestinal lumen was based on the specific radioactivity of the intravenously injected 3H-mannitol solution. The emptied loops were longitudinally opened, blotted dry with wiping papers and their weight and length was recorded. The details of the in situ loop preparation and analytical procedures are described in Chapter 3. 135 6.3. Results 6.3.1. Age-Related Changes and Regional Differences in Paracellular Absorption in In Situ Duodenal, Jejunal and Ileal Loop Preparations in Broiler Cockerels The relative weights (g/cm) of the duodenal, distal jejunal, and distal ileal loop tissues from 0 to 14 d of age are provided in Figure 6.1. The data for jejunal weights at 0, and 2 d of age was not collected. The relative weight (g/cm) of the duodenal, jejunal, and ileal tissues increased with age. A t any given age under investigation, the relative weight of the duodenum was significantly greater than that of the distal jejunum or distal ileum. A t any given age, the relative weight of the distal jejunal loop tissue was similar to the relative weight of the ileal tissue. Absorption of mannitol from the lumen of the in situ duodenal, distal jejunal, and distal ileal loop preparations is described by two parameters, namely the efficiency of paracellular absorption, and the capacity of paracellular absorption. In this chapter, the efficiency is defined as the percent dose lost during the experimental period and is independent of the amount or length of the intestinal tissue involved. The capacity of paracellular absorption is defined as the amount of mannitol absorbed in 10 min (pmol cm"1 10 min"1) which takes into account the length of the intestinal loop. The efficiency of mannitol absorption in one intestinal region can be validly compared with another region since, at any given age under investigation, the relative intralumenal volume (mL/cm) did not significantly differ between the duodenum, distal jejunum, and distal ileum (see Appendix Figure 3). 136 DX) < •a S» O co o 4 « CN G O JH ,*-> o ^ 3 CO T3 CN CL) O -s ~ ° V W G oo w +1 | G cu ca rG cu G CU J—i •3 43 T3 ca G .SP CO CU T3 ca PQ .•33 43 S 3 s—' co g 53 •a -a ca c ca 00 CO G -a o S o « T-H •B II 2 ^ T. 9 ^ y <U CH •*-H W / CO ,ca co <4-H CU cu ca C > CU - . o o o GCU T-l JTH 3 G a» -si, V - T— 3 ca WD to -G '-3 o 1-4D T3 2 ^ •1—» G ca T3 i -0" h t j H G DO (UID/S) 137 The efficiency of mannitol absorption in duodenal, distal jejunal, and distal ileal loop preparations in broiler chicks at 0, 2, 4, 7, and 14 d of age is shown by Figure 6.2. A t all ages under investigation, the efficiency of mannitol absorption was generally 1.5 to 2.5 fold greater in the duodenal than in the distal jejunal or distal ileal loops (P<0.01) . In other words, paracellular permeability of the in situ duodenal loop preparation is much greater than the paracellular permeability of the jejunal or ileal loops. A t 0 d, and 2 d of age, the efficiency of mannitol absorption in distal jejunal loops was significantly greater than the efficiency in distal ileal loops (P<0.01) . A t 4, 7, and 14 d of age, the efficiency of mannitol absorption was not different between the distal jejunal and distal ileal loops. In distal jejunal loops, the efficiency of mannitol absorption did not significantly change from 0 to 4 d of age but it became significantly less at 7 and 14 d of age. In distal ileal loops, the efficiency of mannitol absorption did not significantly (P < 0.05) differ from 0 to 14 d of age. Figure 6.3 shows the amount of mannitol absorbed (pmol cm"1 10 min"1) from the in situ duodenal, distal jejunal, and distal ileal loop preparations in chicks at 0, 2, 4, 7, and 14 d of age. In all regions of the small intestine examined, the amount of mannitol absorbed gradually increased from 0 to 14 d of age. A t any given age, the amount of mannitol absorbed from the lumen of in situ duodenal loop preparations was generally 1.5 to 2.5 fold greater (P<0.01) than the amount absorbed from either the distal jejunal or distal ileal preparations. A t all ages under investigation, the amount of mannitol absorbed by the distal jejunal loops was not significantly different from the amount absorbed by the distal ileal loops. 138 B a cu OS *-< CO 5 s 3 a a •3? CC3 - M VI 5 (mm oi ui asop jo ssoj %) uopdaosqe io}ium?pv[ OX) •S cu .3 3 O 3 7 3 cd cd a 3 cu o co 3 O CO ft cd o CO CN 3 +-> cu X ) a o C/3 CO 6 -H ft II a " CU 3 73 2i cd .3 _cu H +1 .23 2 3 cd cu ft 7 3 - S 3 ^ cd T3 - cu •—s -•—1 c^ 55 O co -t—< O x> cd o 3 cu ' 0 Cj-I cu H f N S O cu SH 3 CUD cu . . CU II -3 S 8 *a ft 3 3 "cd 2 co O -3 ^< ^ O ^ CO o S +-> cd U 3 CO CO ^ <= s 2 cu 'S 2 3 "cS cd > v a Hp l CU 3-1 ft 1 § ^ 0 3 co Jd a 'a ' cd 3 a .3° 'co cu s cd a .a O M 3 ft 5 cu O cu 2 a co 3 cu cu - f t ft — cu g IS cu -3 3 +2 cd £ cd 3 cu T3 O 3 7 3 C CU CN cd 3 00 X ! . 3 -co ? 73 139 CU DA +1 II C CN t*> ft • J3 S a f e ccj (T.U1UI 01 x-UI3 IOUIUI) uopdaosqB io;iuuep\[ 140 6.3.2. Regional Differences in Paracellular Secretion from Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels When the amount of mannitol secreted (pmol cm"1 10 min"1) was plotted against plasma mannitol concentration, a direct relationship existed between the concentration and the amount secreted in all three regions of the small intestine (Figure 6.4). The coefficient of correlation between these two parameters was 0.72 in the duodenum, 0.59 in the distal jejunum, and 0.58 in the distal ileum, and all correlations were significant (P<0.05) . This type of relationship was expected since mannitol is restricted to the intercellular space and is therefore likely to cross the epithelial barrier via the paracellular pathway. Since the plasma 3H-mannitol concentration varied between individual birds, it was decided to normalize the amount of mannitol secreted for plasma mannitol concentration. The amount of mannitol secreted from plasma into the in situ intestinal preparations is, therefore, expressed as pmol mM" 1 cm"1 10 min"1 in Figure 6.5. This figure shows that the amount of mannitol secreted into the duodenal loop preparations was not significantly different from the amount secreted into the distal ileal loop preparations. However, the amount of mannitol secreted into the distal jejunal loop preparations was significantly less (P < 0.01) than the amounts secreted into either the duodenal or the distal ileal loop preparations. 141 T3 cu -<-> cu J i ^ U in ».'s 3 a 1 i C M — O O a | o i i i r 0.0 0.3 0.6 0.9 1.2 1.5 1. i i i r 0.0 0.3 0.6 0.9 1.2 1.5 1. Duodenum Distal Jejunum Distal Ileum Plasma Mannitol [mM] Figure 6.4. Mannitol secretion into the lumen of the in situ duodenal (n=20), distal jejunal ( n = l l ) , and distal ileal (n=9) loop preparations, as a function of plasma mannitol concentration in 18-d-old broiler cockerels fasted for 12 h. For these determinations, a 300 m M 3 H-mannitol solution (3.3 n C i p m o l 1 ) @ 1.5 m L k g 1 was injected into the wing vein. Determinations in duodenal loops were made concomitant with determinations in either jejunal or ileal loops. 142 en a o • mm DX) cy u 13 a * - M CU s ca 2 -^1 ca ca a 3 T3 (j-Uiui oi r-Ui3 ^lo iurl j_jouiu) P3J3J33S l o n u u B i u jo j u n o u i y 45 S W) co 143 6.4. Discussion 6.4.1. Age-Related Changes in Paracellular Absorption in In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels The efficiency of mannitol absorption in the in situ duodenal loop preparations did not apparently change until the broiler cockerels reached at least 1 wk of age. The efficiency of mannitol absorption slightly decreased in the distal jejunal loops during this period, whereas the efficiency remained constant in the distal ileal loops during 0 to 14 of age. Pansu et al . (1983a) used the saturation technique with rat in situ duodenal preparations, to demonstrate that the slope of the calcium absorption curve (representing paracellular transport) decreases directly with increase in age from birth to 40 d of age, after which it plateaus. The finding reported herein with broiler cockerels indicates a slight or no decrease in paracellular permeability of the in situ duodenal loops during the first week posthatch period. It appears that the chick small intestine is relatively mature in terms of paracellular permeability. The suggestion that the small intestine of very young broiler cockerels is functionally mature in terms of paracellular transport is consistent with observations reported in Chapter 4 that the cellular component of calcium transport is present in the duodenum of broiler cockerels by 4 d of age. Yamauchi and Isshiki (1992) reported that the epithelial cells lining the intestine of broiler chickens are almost ultrastructurally mature at hatching. A detailed examination of ultrastructural changes in the tight junctions during the early posthatch period 144 would be needed to further verify the conclusions pertaining to age-related changes in the efficiency of paracellular absorption. Unfortunately, this information is currently not available. The amount of mannitol absorbed in all three regions of the small intestine increased with age, showing that the capacity of paracellular absorption increases with age. This outcome was expected since the size of the intestine and consequently its absorptive surface increases with age. The age-related increase in the capacity of mannitol absorption is more likely associated with an increase in the need for increased nutrient extraction by the absorptive surface, concomitant with growth. Although, the efficiency of paracellular transport either does not vary or is slightly decreased with increase in age, the capacity of paracellular transport continues to increase with age to meet the demands of the growing bird. 6.4.2. Regional Differences in the Small Intestine of Broiler Cockerels for Paracellular Absorption The present data show that at any given age from 0 to 14 d of age, the efficiency of mannitol absorption from in situ duodenal loops is significantly greater than the efficiency of mannitol absorption from distal jejunal or from distal ileal loops. The capacity of mannitol absorption follows the same pattern. This implies that the efficiency of paracellular mannitol transport, similar to the efficiency of cellular calcium transport (Chapter 4), decreases from the proximal to its distal portion. 145 The present data show that, in 4- to 14-d-old chicks, the efficiency of mannitol absorption in the distal jejunum was not significantly different from the efficiency of mannitol absorption in the distal ileum. A similar observation was made in the rat by Krugliak et al. (1994). These researchers demonstrated with in vivo perfused intestinal segments that the net absorption of mannitol was not different between the jejunum and ileum. Wi th isolated intestinal tissues mounted on Ussing chambers, however, Karbach (1992) demonstrated that mannitol absorption (from the mucosal to the serosal side) was significantly greater in the rat jejunum than in the duodenum or ileum. Differences in technique appears to be the likely factor accounting for this discrepancy between the two observations obtained with the rat. The physiological relevance of permeability studies on isolated intestinal tissue in the absence of an intact circulation is questionable. 6.4.3. Regional Differences in Paracellular Secretion from the Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal Loop Preparations in Broiler Cockerels Since the amount of mannitol secreted from the blood into the lumen of in situ duodenal, distal jejunal, and distal ileal preparations was a direct function of plasma mannitol concentration, mannitol secretion, similar to absorption, appears to be a nonsaturable paracellular process. This result was expected since mannitol is restricted to the extracellular space. Karbach (1992) demonstrated that mannitol secretion from the serosal to the mucosal side of rat intestinal tissue mounted on Ussing chambers is a paracellular process. 146 It is somewhat surprising that for the amounts of mannitol secreted (pmol mM"' cm"1 10 min"1) there was no significant difference between the in situ duodenal and distal ileal loop preparations, whereas for the amount of mannitol absorbed (pmol cm"1 10 min"1), it was significantly greater in the duodenal than in the distal ileal loop preparations. Data obtained by Karbach (1991) also show difference in mannitol absorption and secretion in the rat duodenal and jejunal tissues mounted on Ussing chambers. The mechanisms accounting for the differences in paracellular absorption versus paracellular secretion, need to be further studied. Krugliak et al . (1994) showed that mannitol absorption in in vivo perfused rat intestine was directly related to water absorption. They, however, also showed that significant mannitol absorption occurred even at zero net water absorption. These findings suggest that both the solvent drag effect and passive diffusion mechanisms play a role in regulating mannitol absorption across the small intestine The finding in the present research that mannitol secretion is a direct function of blood mannitol concentration indicates the existence of a passive diffusion mechanism for the intestinal secretion process. 147 6.5. Conclusions The efficiency as well as the capacity of paracellular absorption from the chicken in situ duodenal preparations is generally in the range of 1.5 to 2.5 fold greater than that from distal jejunal or distal ileal preparations, during the first 2 wk weeks of life. The efficiency of paracellular absorption in these intestinal regions slightly decreases with age whereas the capacity of absorption increases with age in all regions of the chicken small intestine. It appears, therefore, that the intestine of newly hatched broiler cockerels is relatively mature in terms of paracellular absorption. Further, mannitol secretion from blood into the lumen of in situ intestinal preparations is a concentration-dependent process indicating the involvement of a passive diffusion mechanism in intestinal secretion process. 148 C H A P T E R 7 General Discussion and Conclusions 7.1. General Discussion A remarkable increase in the rate of growth has been achieved in commercial broiler chickens during the past two decades. However, the incidence of disorders that may be associated with malfunction of calcium metabolism, such as the lower mineral density and higher porosity of tibial cortices, has increased concomitantly with the increase in growth rate (Letterrier and Nys , 1992). Leg abnormalities such as tibial dyschondroplasia and bone weakness are indeed a cause of considerable economic loss to the broiler industry. Comparison of older and newer reports in the literature suggest that accelerated growth is associated with an increase in the sensitivity of growth rate to dietary calcium intake. The dietary calcium requirement of commercial broiler chickens is well known. However, no work has been reported on the mechanisms that regulate intestinal calcium transport in this highly selected strain of chickens. A large body of research has addressed vitamin D regulation of calcium absorption from the intestine. Vitamin D fortification of diets is normal and a typical commercial broiler ration, including the one used in the present research, may contain vitamin D at levels greater than ten fold that recommended by N R C . Duflos et al . (1995) have stated that "whereas much has been written about the vitamin D-dependent process...., much less is known about the nonsaturable process". It should be noted that the nonsaturable process is generally considered 1 4 9 to be vitamin D-independent, taking place via the paracellular pathway. The first part of the present investigation, therefore, dealt with determining the fractional contribution of the vitamin D-dependent and vitamin D-independent mechanisms of calcium transport in in situ intestinal loop preparations. The results indicate the predominance of the vitamin D -independent, paracellular, component of calcium transport under the in situ loop experimental conditions, in which the intralumenal test solution contained 75 m M C a C h . The physiological relevance of the data obtained with the in situ loop experimental model may, however, be questioned. One usual difference between the in situ loop experimental model and the intact animal model is the form in which calcium is presented to the absorptive site in the intestine. Calcium in a normal diet is mainly provided in the form of calcium carbonate. In contrast, intralumenal test solutions used in in situ loops usually contain calcium in the form of the chloride salt. To mimic the in situ loop situation in vivo, therefore, calcium was provided to intact birds in the form of 40 m M C a C h as drinking water for 24 h. With this protocol, calcium absorption by intact birds, measured as calcium retention, occurred by a nonsaturable mechanism at all the three ages studied. These data obtained with the intact animal model demonstrate the physiological relevance of the data obtained with the in situ loop experimental model, and suggest that calcium transport in intact chicks may take place predominantly by paracellular mechanisms provided concentration and solubility are not limiting factors. One may question the practical significance of increasing calcium retention in intact animals by providing calcium as C a C h . In fact, there is a convincing evidence that high 150 chloride levels in the rations increase the incidence of tibial dyschondroplasia in broiler chickens. The results obtained in the present investigation, however, only highlight the significance of solubility and bioavailability factors in improving calcium nutrition. One way of improving calcium nutrition is by increasing intake. El l iot and Edwards (1992) and Edwards and Veltmann (1983) reported that increased dietary calcium levels decrease the incidence and severity of tibial dyschondroplasia and rickets is rapidly growing broiler chickens. Increasing dietary calcium concentration above the normal levels may, however, have other consequences. In the present investigations, a significant decrease in the body weight of chicks occurred at 21 d of age, when the newly hatched chicks were fed a diet containing 1.65% calcium on a dry matter basis (Table 4.3). Similarly, Shaffey et al. (1990) also reported a suppression of growth rate of rapidly growing young broiler chickens by providing dietary calcium levels ranging > 1 to 2%. Since approximately 50 % of the dietary calcium is absorbed under normal conditions, one may consider improving calcium nutrition of birds by decreasing its dietary inclusion but increasing its solubility. Provision of calcium to commercial poultry via the drinking water would not be an acceptable practice. However, special circumstances may permit such a practice. Solubility and bioavailability factors to improve calcium nutrition of humans and animals continue to be in the spot light of clinical nutritionists. Based on the mechanisms of intestinal calcium transport in broiler chicks, investigated in the present research, evidence is provided that solubility of dietary calcium is an important factor for its efficient absorption from the gastrointestinal tract of these birds. Some of the topics investigated in the present research have been a subject of continuing controversy. A n example is calcium transport in the ileum. Whereas some 151 researchers maintain that calcium transport in the rat ileum occurs solely via the paracellular pathway, others maintain that it occurs via a combination of the cellular and paracellular pathways. A limited number of studies have been conducted with layer-type chicks which indicate that ileal calcium transport can be stimulated by vitamin D , suggesting that a cellular pathway is involved. In the present study, using intact tissue located in situ with a patent circulatory system, evidence is provided that calcium transport in the in situ ileal preparation in rapidly growing young broiler cockerels occurs solely via the paracellular pathway. Whether or not vitamin D regulates paracellular pathway is also a subject of controversy. Some of the early reports which suggested the existence of such a regulation has not been revisited. The present investigation show that mannitol transport in the in situ duodenal or distal ileal loop preparations in young broiler cockerels is not affected under conditions of high calcium intake, under rachitic condition, and after l ,25-(OH )2D3 treatment of normal and rachitic chicks. This evidence suggest that vitamin D is not involved in regulation of the paracellular pathway in the in situ duodenal and ileal preparation in young broiler cockerels. 7.2. Conclusions In summary, the following conclusions can be drawn from this thesis. Chapter 3 1. A t an intralumenal calcium concentration of 75 m M C a C h , calcium disappearance in the in situ duodenal loops increases directly with time during a 2 to 12 min incubation period. 1 5 2 2. A 12 h fast does not affect calcium or mannitol disappearance in in situ duodenal preparations in 7-d-old broiler cockerels. 3. There is no significant circadian variation in plasma total-calcium concentration in 18-d-old broiler cockerels raised on a 24 h lighting program and provided continuous access to feed and water. Chapter 4. 1. Calcium transport in the in situ duodenal preparations in broiler chicks have both vitamin D-dependent and vitamin D-independent components. A t an intralumenal concentration of 75 m M C a C h , 75 to 85% of the transport in this intestinal region occurs independent of vitamin D . 2. Calcium transport in the in situ ileal preparations in broiler chicks occurs solely in a vitamin D-independent manner. 3. The paracellular pathway is not regulated by vitamin D 4. Vitamin D-dependent mechanisms of calcium transport are present in broiler cockerels by 4 d of age. 5. The proximal small intestine of young broiler chicks is a site of high efficiency calcium absorption compared with the distal small intestine, as measured with the in situ loop technique. 1 5 3 Chapter 5. 1. Mannitol transport in the in situ duodenal loop preparations in broiler chickens is a nonsaturable, and therefore, a paracellular process. In contrast, calcium transport is a saturable process. 2. When solubility of dietary calcium is not limiting, calcium absorption in intact birds occurs in a nonsaturable manner, and therefore, via the paracellular pathway. Since this conclusion is similar to the one drawn from the in situ loop data, it demonstrates the physiological relevance of the in situ loop experimental system. Chapter 6 1. The efficiency of paracellular absorption in the in situ duodenal loop preparations of 0-to 14-d-old broiler cockerels is 1.5 to 2.5 fold greater than the efficiency in either the distal jejunal or distal ileal preparations. 2. The efficiency of paracellular absorption in the in situ duodenal and jejunal preparations in 0- to 14-d-old broiler cockerels tends to decrease with age whereas the efficiency does not change in ileal preparations. 3. Mannitol secretion from blood into the lumens of in situ duodenal, jejunal, and ileal preparations is a paracellular process, similar to mannitol absorption. 154 7.3. Further Research Based on the findings of the present research, three areas of future research are identified to improve calcium nutrition of the broiler chickens. 1. Research should be conducted to explore how calcium nutrition in broiler chickens can be improved by increasing calcium bioavailability in chicken diets. 2 . Ultrastructural studies on the gap junction passage of the intestine should be conducted to provide a further insight to permeability characteristics of the chicken small intestine, since this insight is necessary to understand regulation of the paracellular pathway. 3. Since net calcium retention is determined by difference in the amounts of calcium absorbed and secreted, research should be conducted to study the mechanisms that regulate calcium secretion from blood into the intestinal lumen. Such an inquiry wi l l help us better understand the significance of intestine as an organ of calcium homeostasis. 155 R E F E R E N C E S Abou-Samra, A . B . , P . K . Goldsmith, L . Y . X i e , H . Juuppner, A . M . Spiegel, and G . V . Segre. 1994. Down-regulation of parathyroid (PTH)/PTHrP-related peptide receptor immunoreactivity and P T H binding in opossum kidney cells by P T H and dexamethasone. Endocrinology. 135: 2588-2594. Abou-Samra, A . B . , H . Juppner, T. Force, M . W . Freeman, X . F . Kong , E . Schipani, P. Urena, J . Richards, J . V . Bonventre, H . M . Kronenberg, and G . V . Segre. 1992. Expression cloning of a common receptor for parathyroid hormone and parathyroid hormone-related peptide from rat osteoblast-like cells; a single receptor stimulates intracellular accumulation of both c A M P and inositol phosphates and increases intracellular free calcium. Proc. Natl . Acad. Sci . U S A 89: 2732-2737. Al len , L . H . 1982. Calcium bioavailability and absorption: A review. A m . J . C l i n . Nutr. 35: 783-808. A u , W . Y . W . , and L . G . Raisz. 1967. Restoration of parathyroid responsiveness in vitamin D -deficient rats by parenteral calcium or dietary lactose. J . C l i n . Invest. 46: 1572-1577. Bakker, R . , and J . A . Groot. 1984. cAMP-mediated effects of oubain and theophylline on paracellular ion selectivity. A m . J . Physiol. 246: G213-G217. Ballard, S.T. , J . H . Hunter, and A . E . Taylor. 1995. Regulation of tight-junction permeability during nutrient absorption across the intestinal epithelium. Annu. Rev. Nutr. 15: 35-55. Bar, A . , S. Hurwitz, and I. Cohen. 1972. Relationship between duodenal calcium binding protein, parathyroid activity and various parameters of mineral metabolism in the rachitic and vitamin D-treated chicks. Comp. Biochem. Physiol. A . 43: 519-526. Barsony, J . , and S.J. Marx . 1991. Rapid accumulation of cyclic G M P near activated vitamin D receptors. Proc. Natl . Acad. Sci . ( U . S . A . ) . 88: 1436-1440. Behar, J . , and M . D . Kerstein. 1976. Intestinal Ca absorption: differences in transport between duodenum and ileum. A m . J . Physiol. 230: 1255-1260. Bikle, D . D . , S. Munson, and M . L . Mancianti. 1991. Limited tissue distribution of the intestinal brush border myosin I protein. Gastroenterology. 100: 395-402. Boass, A . , and S . U . Toverud. 1996. Duodenal active calcium transport in female rats increases with serum calcitriol concentrations, but reaches a plateau far below maximal calcitriol levels. J . Bone M i n . Res. 11: 1640-1645. 1 5 6 Boyle, I. T . , R . W . Gray, H . F . Deluca. 1971. Regulation by calcium of in vivo synthesis of 1,25-dihydroxycholecalciferol and 21, 25-dihydroxycholecalciferol. Proc. Natl . Acad. Sci . Sci . U S A 68: 2131-2134. Brautbar, N . , B . S . Levine, M . M . Wall ing, and J . W . Coburn. 1981. Intestinal absorption of calcium: role of dietary phosphate and vitamin D . A m . J . Physiol. 241: G49-G53. Bredderman, P . J . , and R . H . Wasserman. 1974. Chemical composition, affinity of calcium, and some related properties of the vitamin D dependent calcium- binding protein. Biochemistry. 13: 1687-1694. Brommage, R . , and H . F . DeLuca. 1985. Evidence that 1,25-dihydroxyvitamin D 3 is the physiologically active metabolite of vitamin D 3 . Endocr. Rev. 6: 491-511. Bronner, F . 1987. Intestinal calcium absorption: mechanisms and applications. J. Nutr. 117: 1347-1352. Bronner, F . 1992. Current concepts of calcium absorption: an overview. J. Nutr. 122: 641-643. Bronner, F . , D . Pansu, and W . D . Stein. 1986. A n analysis of intestinal Ca transport across the rat intestine. A m . J . Physiol. 250: G561-G569. Bronner, F . , and W . D . Stein. 1992. Modulation of bone calcium-binding sites regulates plasma calcium: an hypothesis. Calcified Tissue Int. 50: 483-489. Bronner, F . , and W . D . Stein. 1995. Calcium homeostasis-an old problem revisited. J. Nutr. 125: 1987S-1995S. Brooks, M . H . , N . H . Be l l , I. Love, P . H . Stern, E . Orfei, S.F. Queener, A . J . Hamstra, and H . F . DeLuca. 1978. Vitamin D-dependent rickets type II. Resistance of target organs to 1,25-dihydroxyvitamin D 3 . N . Engl . J . Med . 298: 996-999. Brumbaugh, P . F . , and M . R . Haussler. 1974. la,25-dihydroxycholecalciferol receptors in intestine. I. Association with la,25-dihydroxycholecalciferol with intestinal mucosa chromatin. J . B i o l . Chem. 249: 1251-1257. Butler, W . T . 1989. The nature and significance of osteopontin. Connect. Tissue Rev. 23: 123-136. Ca i , Q . , J.S. Chandler, R . H . Wasserman, R. Kumar, and J .T. Penniston. 1993. Vitamin D , and adaptation to dietary calcium and phosphate deficiencies, increase intestinal plasma membrane calcium pump gene expression. Proc. Natl . Acad. Sci . U . S . A . 90: 1345-1349. 157 Canadian Council on Animal Care. 1993. Guide to the Care and Use of Experimental Animals. E . D . Alfert, B . M . Cross and A . A n n M c W i l l i a m (Editors), 2nd ed. 2 vols, C C A C , Ottawa, O N . Care, A . D . , and R . F . L . Bates. 1972. Control of secretion of parathyroid hormone and calcitonin in mammals and birds. Gen. Comp. Endocrinol. 3: 448-458. Chandra, S., C . S . Fullmer, C A . Smith, R . H . Wasserman, a n d G . H . Morrison. 1990. Ion microscopic imaging of calcium transport in the intestinal tissue of vitamin D-deficient and vitamin D-replete chicks: a ^ C a stable isotope study. Proc. Natl . Acad. Sci . U . S . A . 87: 5715-5719. Charpin, G . , A . R . C h i k h l s s a , H . Guignard, G . Jourdan, C . Dumas, D . Pansu, a n d M . Descroix-Vage. 1992. Effect of sorbin on duodenal absorption of water and electrolytes in the rat. Gastroenterology. 103: 1568-1573. Christakos', S., C . Gabrielides, and W . B . Rhoten. 1989. Vitamin D-dependent calcium binding : chemistry, distribution, functional considerations, and molecular biology. Endocrine Rev. 10:3-26. Claude, P. 1978. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J . Membrane B i o l . 39: 219-232. Coats, M . E . , and E . S . Holdsworth. 1961. Vitamin D 3 and absorption of calcium in the chick. Brit. J. Nutr. 15: 131-147. Conn, S . H . , T. Teree, and E . Gusmano. 1968. Effect of varying calcium intake on the parameters of calcium metabolism in the rat. J. Nutr. 94: 261-267. Corradino, R . A . 1974. Embryonic chick intestine in organ culture: interaction of adenylate cyclase system and vitamin D3-mediated calcium absorption mechanism. Endocrinology. 94: 1607-1614. Czaky, T . Z . 1987. Intestinal permeation: an overview. Hand-book Exp. Pharmacol. 70: 50-89. Davis, W . L . , and R . G . Jones. 1981. Calcium lysosomes in rachitic and vitamin D 3 replete chick duodenal absorptive cells. Tissue and Ce l l . 13: 381-391. Dean, J . A . ed. 1979. Lange's handbook of chemistry, 12th ed. McGraw H i l l , New York , N . Y . deBoland, A . R . , and A . W . Norman. 1990. Evidence for involvement of protein kinase C and cyclic adenosine 3' 5'-monophosphate-dependent protein kinase in the 1,25-dihydroxyvitamin D3-mediated rapid stimulation of intestinal Ca transport (transcaltachia). Endocrinology. 127: 39-45. 158 DeLuca H . F . 1988. The vitamin D story: A collaborative effort of basic science and clinical medicine. F A S E B J . 2: 224-236. DeLuca, H . F . , and H . K . Schnoes. 1976. Metabolism and mechanism of vitamin D . Ann . Rev. Biochem. 45: 631-666 DeLuca, H . F . , and H . K . Schnoes. 1983. Vitamin D : Recent Advances. Ann . Rev. Biochem. 52: 411-439. Dickson, I .R., D . R . Eyre, and E . Kodicek. 1979. Influence of calcium and vitamin D on bone collagen. Effects of lysine hydroxylation and crosslink formation. Biochem Biophys. Acta. 588: 169-172. Donovan, M . D . , G . L . Flynn,and G . L . Amidon. 1990. Absorption of polyethylene glycol 600 through 2000: the molecular weight dependence of gastrointestinal and nasal absorption. Pharmaceut. Res. 7: 863-868. Dostal, L . A . , and S . U . Toverud. 1984. Effect of vitamin D 3 on duodenal calcium absorption in vivo during early development. A m . J . Physiol. 246: G528-G534. Drenckhahn, D . , and R. Dermietzel. 1988. Organization of actin filament cytoskeleton in the intestinal brush border: a qualitative immunoelectron microscope study. J . Cel l B io l . 107: 1037-1048. Duffey, M . E . , B . Hainau, S. Ho , and C . J . Bentzel. 1981. Regulation of epithelial tight junction permeability by cyclic A M P . Nature. 294: 451-453. Duflos, C , C . Bellation, D . Pansu, and F . Bronner. 1995. Calcium solubility, intestinal sojourn time and paracellular permeability co-determine passive calcium absorption in rats. J . Nutr. 125: 2348-2355. Edwards j r . , H . M . , and J .R. Veltmann. 1983. The role of calcium and phosphorus in the etiology of tibial dyschondroplasia in young chicks. J . Nutr. 113: 1568-1575. Ell iot , M . A . , and H . M . Edwards, Jr. 1992. Studies to determine whether an interaction exists among boron, calcium, and cholecalciferol on the skeletal development of broiler chickens. Poult. Sci . 71: 677-690. Emery, P . W . , L . Cotellessa, M . Holness, C . Egans, and M . J . Rennie. 1986. Different pattern of protein turnover in skeletal muscle and gastrointestinal smooth muscle and the production of N-methylhistidine during fasting in the rat. J . Biosci . Rep. 6: 143-153. Esvelt, R . P . , H . K . Schnoes, and H . F . DeLuca. 1978. Vitamin D 3 from rat skins irradiated in vitro with ultraviolet light. A r c h . Biochem. Biophys. 188: 282-286. 159 Favus, M . J . and E . Angeid-Backman. 1984. Effects of lactose on calcium absorption and secretion by rat ileum. A m . J. Physiol. 246: G281-G285. Feher, J .J . 1983. Facilitated calcium diffusion by intestinal calcium binding protein. A m . J . Physiol. 244: 303-307. Feher, J . J . , C . S . Fullmer, and G . K . Fritzsch. 1989. Comparison of the enhanced steady-state diffusion of calcium by calbindin-D9k and calmodulin: Possible importance in intestinal absorption. Ce l l Calcium. 10: 189-203 Feher, J .J . , and R . H . Wasserman. 1978. Evidence for a membrane-bound fraction of chick intestinal calcium-binding protein. Biochem. Biophys. Acta 540: 134-143. Fraser, D . R . 1980. Regulation of the metabolism of vitamin D . Physiological Review. 60: 551-613. Fraser, D . R . , and E . Kodicek. 1970. Unique biosynthesis by kidney of biologically active vitamin D metabolite. Nature. 288: 764-766. Fraser, D . R . , S .W. Kooh , H . P . K ind , M . F . Hol ick, Y . Tanaka, and H . F . DeLuca. 1973. Pathogenesis of hereditary vitamin D dependent rickets: A n inborn error of vitamin D metabolism involving defective conversion of 25-dihydroxyvitamin D to l a , 2 5 -dihydroxyvitamin D . N . Engl . J. M e d . 289: 817-822. Freedman, R . A . , M . M . Weiser, and K . J . Isselbacher. 1977. Calcium translocation by Golgi and lateral-basal membrane vesicles from rat intestine: Decrease in vitamin D-deficient rats. Proc. Natl . Acad. Sci . U . S . A . 74: 3612-3616. Fromter, E . , and J . Diamond. 1972. Route of passive ion permeation in epithelia. Nature New B i o l . 235: 9-13. Fullmer, C . S . 1992. Intestinal calcium absorption: Calcium entry. J . Nutr. 122:644-650. Gerber, H . W . , and R. Jost. 1986. Casein phosphopeptides: their effect in calcification of in vitro cultured embryonic rat bone. Calc. Tiss. Intern. 38: 350-357. Ghijsen, W . E . J . M . , M . D . De Jong, and C . H . Van Os. 1983. Kinetic properties of N a + / C a 2 + exchange in basolateral plasma membranes of rat small intestine. Biochim. Biophys. Acta. 730: 85-94. Glenney, J .R. , Jr., and P. Glenney. 1985. Comparison of Ca + + -regulated events in the intestinal brush border. J . Ce l l B i o l . 100: 754-763. Goodlad, R . A . , W . Lenton, M . A . Ghatei, T . E . Adrian, S.R. Bloom, a n d N . A . 1987. Effects of an elemental diet, inert bulk and different types of dietary fiber on the response of 160 the intestinal epithelium to re-feeding in the rat and relationship to plasma gastrin, enteroglucagon, and P Y Y concentrations. Gut: 28: 171-180. Halloran, B . P . , and H . F . DeLuca. 1980. Calcium transport during early development: role of vitamin D . A m . J . Physiol. 239: G473-G479. Holdsworth, E . S . 1970. The effects of vitamin D on the enzyme activities in the mucosal cells of the chick small intestine. J . Membrane. B io l . 3: 43-53. Howe, C . L . , T . C . S . I l l , Keller, M . S . Mooseker, and R . H . Wasserman. 1982. Analysis of cytoskeletal proteins and Ca 2 +-dependent regulation of structure in intestinal brush borders from rachitic chicks. Proc. Natl . Acad. Sci . U . S . A . 79: 1134-1138. H u , M . , L . H . Kayne, P . A . Willsey, A . B . Koteva, N . Jamgotchian, and D . B . N . Lee. 1993. Bile salts and ileal calcium transport in rats: a neglected factor in intestinal calcium absorption. A m . J. Physiol. 264: G319-G324. Hunzikar, W . , M . R . Walters, J . E . Bishop, and A . W . Norman. 1982. Effect of vitamin D status on the equilibrium between occupied and unoccupied 1,25-dihydroxyvitamn D intestinal receptors in the chicken. J . C l i n . Invest. 69: 826-834. Hurwitz, S., and P. Griminger. 1961. The response of plasma alkaline phosphatase, parathyroids and blood and bone minerals to calcium intake in the fowl. J . Nutr. 73: 177-185. Hurwitz, S. 1996. Homeostatic control of plasma calcium concentration. C l i n . Rev. Biochem. M o l . B i o l . 31: 41-100. Hurwitz, S., S. Fishman, A . Bar, M . Pines, G . Risenfeld, and H . Talpaz. 1983. Simulation of calcium homeostasis: modeling and parameter estimation. A m . J . Physiol. 245: R664-R672. Hurwitz, S., B . M i l l e r , and A . W . Norman. 1994. Oscillatory behavior of control-systems of calcium homeostasis in chickens. J . Ce l l . Biochem. 56: 236-244. Hurwitz, S, I. Plavnik, A . Shapiro, E . Wax, H . Talpaz, and A . Bar. 1995. Calcium metabolism and requirements of chickens are affected by growth. J . Nutr. 125: 2679-2686. Itaya, K . , and M . U i . 1966. A new micromethod for the colorimetric determination of inorganic phosphate. C l i n . Chim. Acta. 14: 361-366. Jenkins, R. T . , R. L . Goodacre, P. J . Rooney, J . Bienestock, T. Sivakumaran, and W . H . C . Walker. 1986. Studies of intestinal permeability in inflammatory diseases using polyethylene glycol 400. C l i n . Biochem. 19: 2298-2302. 161 Kallfelz, F . A . , A . N . Taylor, and R . H . Wasserman. 1967. Vitamin D-induced calcium binding factor in rat intestinal mucosa. Proc. Soc. Exp. B i o l . Med . 125:54-58. Kalu , D . N . , A . Hadji-georgopoulus, and G . V . Foster. 1975. Evidence for physiological importance of calcitonin in the regulation of plasma calcium in rats. J . C l i n . Invest. 55: 722-727. Karbach, U . 1991. Segmental heterogeneity of cellular and paracellular calcium transport across the rat duodenum and jejunum. Gastroenterology. 100: 47-58. Karbach, U . 1992. Paracellular calcium transport across the small intestine. J. Nutr. 122: 672-677. Karbach, U . , and H . Feldmeier, 1993. The cecum is the site with the highest calcium absorption in rat intestine. D ig . Dis . Sci . 38: 1815-1824. Kaune, R . , S. Munson, and D . D . Bikle . 1994. Regulation of calmodulin binding to the A T P extractable 110 kDa protein (myosin I) from chicken duodenal brush border by 1,25-(OH) 2 D3. Biochim. Biophys. Acta. 1190: 329-336. Kawashima, H . , S. Torikai , and K . Kurokawa. 1981. Calcitonin selectively stimulates 25-hydroxyvitamin D3-Ihydroxylase in the proximal straight tubules of rat kidney. Nature. 291:327-329 Keshavarz, K . , and M . L . Scott. 1993. The effect of two calcium sources with different solubility in presence and absence of oyster shell on shell quality. Poult. Sci . 72: 354 (Supplement). K i m , M . 1996. Absorption of polyethylene glycol oligomers (330-1122 Da) is greater in the jejunum than in the ileum of rats. J . Nutr. 126: 2172-2178. Kinoshita, Y . M . Fukase, A . Miyauchi , M . Taknaka, M . Nakada, and T. Fijita. 1986. Establishment of a parathyroid hormone-responsive phosphate transport system in vitro using cultured renal cells. Endocrinology. 119: 1954-1963. Kitts, D . D . , and Y . V . Yuan. 1992. Caseinophosphopeptides and calcium bioavailability. Trends in Food Science and Technology. 3:31-35. Kitts, D . D . , Y . V . Yuan, T. Nagasawa, and Y . Moriyama. 1992. Effect of casein, casein phosphopeptides and calcium intake on ileal 4 5 C a disappearance and temporal systolic blood pressure in spontaneously hypertensive rats. Brit . J . Nut. 68: 765-781. Kollenkirchen, U . W . E . , M . R . Walters, and J . Fox. 1991. Plasma Ca influences vitamin D metabolite levels as rats develop vitamin D deficiency. A m . J . Physiol . 260: E447-E452. 162 Krugliak, P . , D . Hollander, C . C . Schlaepfer, H . Nguyen, and T . Y . M a . 1994. Mechanisms and sites of mannitol permeability of small and large intestine in the rat D i g . Dis . Sci . 39: 796-801. Kyeyune-Nyombi, G . , K . - H . W . , Lau , D . J . Baylink, and D . D . Stong. 1989. Stimulation of alkaline phosphatase activity and its messenger R N A level in a human osteosarcoma cell line by 1,25-dihydroxyvitamin D 3 . A r c h . Biochem. Biophys. 275: 363-370. Lee, D . B . N . , M . W . Wall ing, B . S . Levine, V . Gafter, V . Sil is , A . Hodsman, and J .W. Coburn. 1981. Intestinal and metabolic effect of 1,25-dihydroxyvitamin D 3 in the normal adult rat. A m . J. Physiol. 240: G690-G696. Lee, Y . S . , T. Noguchi, and H . Naito. 1983. Intestinal absorption of calcium in rats given diets containing casein or amino acid mixture: the role of casein phosphopeptides. Brit. J . Nutr. 49: 67-76. Lee, Y . S . , T . J . Reimers, R . G . Cowan, C . S . Fullmer, and R . H . Wasserman. 1988. Calcium-dependent translocation of calbindin-D28k from intestine to blood. Arch . Biochem. Biophys. 261: 27-34. Letterier, C , and Y . Nys. 1992. Composition, cortical structure and mechanical properties of chicken tibiotarsi: effect of growth rate. Brit . Poult. Sci . 33: 925-939. Lian , J . B . , and C M . Gundberg. 1988. Osteocalcin: Biochemical considerations and clinical applications. C l i n . Orthop. Relat. Res. 226: 267-291. Lieberherr, M . , B . Grosse, and A . Bourdeau. 1991. Non gene mediated effects of calcitriol: A possible involvement of membrane receptor. In "Vitamin D : Gene regulation, structure function analysis and clinical applications" ( A . W . Norman, R. Bouil lon, and M . Thomaset, Eds.), pp, 368-375. de Gruyter, Berl in. Ludders, W . , J . Rode, G . S . Mitchel l , and E . V . Nordheim. 1989. Effects of ketamine, xylazine, and a combination of ketamine and xylazine in Pekin ducks. A m . J. Vet. Res. 50: 245-249. Madara, J . L . 1983. Increase in guinea pig small intestinal transepithelial resistance induced by osmotic loads are accompanied by rapid alterations in absorptive-cell tight-junction structure. J . Ce l l B i o l . 97: 125-136. Madara, J . L . 1987. Intestinal absorptive cell tight junctions are linked to cytoskeleton. A m . J. Physiol. 253: C171-C175. Madara, J . L . 1989. Loosening tight junctions: lessons from the intestine. J . C l i n . Invest. 83: 1089-94. 163 Madara, J . L . , R. Moore, and S. Carlson. 1987. Alteration of intestinal tight junction structure and permeability by cytoskeletal contraction. A m . J . Physiol. 253: 854-861. Madara, J . L . , and J .R. Pappenheimer. 1987. Structural basis for physiological regulation of paracellular pathway in intestinal epithelia. J . Membrane. B i o l . 100: 149-164. Mainoya, J .R. 1975. Effects of bovine growth hormone, human placental lactogen, and ovine prolactin on intestinal fluid and ion transport in the rat. Endocrinology. 96: 1165-1170. Marcus, C . S . , and F . W . Lengemann. 1962. Absorption of C a 4 5 and Sr 8 5 from solid and liquid food at various levels of the alimentary tract of the rat. J. Nutr. 77: 155-160. Marx , S.J. , C . J . Woodward, G . D . Aurbach, H . Glassman, and H . J . Keutman. 1973. Renal receptors for calcitonin: binding and degradation of the hormone. J . B i o l . Chem. 248: 4797-4802. Matsudaira, P . T . , and D . R . Burgess. 1982. Organization of the cross-filaments in intestinal microvi l l i . J . Ce l l B i o l . 92: 657-664. Matsumoto, T . , O. Fontain, and H . Rasmussen. 1981. Effect of 1,25-dihydroxyvitamin D3on phospholipid metabolism in chick duodenal mucosal cell . J . B i o l . Chem. 256: 3354-3360. McLean , F . C . , and M . R . Urist. 1968. Bone fundamentals of the physiology of skeletal tissue, 3rd ed., pp. 142-144, U . Chicago Press, Chicago, I L . Mi l l e r , S .C . , M . A . Mi l l e r , and T . H . Omura. 1988. Dietary lactose improves endochondral growth and bone development and mineralization in rats fed a vitamin D-deficient diet. J. Nutr. 118: 72-77. Moore, E . W . , and H . J . Verine. 1985. Pathogenesis of pancreatic and biliary CaC03 lithiasis: the solubility product ( K ' s p ) of calcite determined with the C a + + electrode. J. Lab. C l i n . M e d . 106: 611-618. Mooseker, M . S . , J.S. Wolenski, T . R . Coleman, S . M . Hayden, R . D . Cheney, E . Espreafico, M . B . Heintzelman, and M . D . Peterson. 1991. Structural and functional dissection of a membrane-bound mechanoenzyme: brush border myosin I. Curr .Top. Memb. 33: 31-55. Moreno, J . H . , 1974. Blockage of cation permeability across the tight junctions of gall bladder and other leaky epithelia. Nature: 251: 150-151. Morrissey, R . L . , and R . H . Wasserman. 1971. Calcium absorption and calcium-binding protein in chicks on differing calcium and phosphorus intakes. A m . J. Physiol. 220: 1509-1515. 164 Munck, B . G . , and S . N . Rasmussen. 1977. Paracellular permeability of extracellular space markers across rat jejunum in-vitro. Indications of a transepithelial circuit. J . Physiol. (Lond.) 271: 473-488. Munson, P . L . , and P . F . Hirsch. 1992. Importance of calcitonin in physiology, clinical pharmacology, and medicine. Bone Miner . 16: 162-165. Mykkanen, H . M . , and R . H . Wasserman. 1980. Enhanced absorption of calcium by casein phosphopeptides in rachitic and normal chicks. J . Nutr. 110: 2141-2148. Nasr, L . B . , J . -D . Monet, and P. A . Lucas. 1988. Rapid (10-minute) stimulation of rat duodenal alkaline phosphatase activity by 1,25-dihydroxyvitamin D 3 . Endocrinology. 123: 1778-1782. Nathanson, M . H . A . Gautum, O . C . N g , R. Bruk, and J . L . Boyer. 1992. Hormonal regulation of paracellular permeability in isolated hepatocyte couplets. A m . J . Physiol. 262: G1079-G1086. Nellans, H . N . 1990. Intestinal calcium absorption. Miner . Electrolyte Metab. 16: 101-108. Nellans, H . N . , and R . S . Goldsmith. 1981. Transepithelial calcium transport by rat cecum: high-efficiency absorptive site. A m . J . Physiol. 240: G424-G431. Nellans, H . N . , and D . V . Kimberg. 1978. Cellular and paracellular calcium transport in rat ileum: effects of dietary calcium. A m . J . Physiol. 235: E726-E737. Nellans, H . N . , and D . V . Kimberg. 1979. Anomalous calcium secretion in rat ileum: role of paracellular pathway. A m . J . Physiol. 264: E473-E481. Nemere, I. 1992. Vesicular calcium transport in chick intestine. J . Nutr. 112: 657-661. Nemere, I., V . Leathers, and A . W . Norman. 1986. 1,25-dihydroxyvitamin D 3 - mediated calcium transport. J. B i o l . Chem. 261: 16106-16114. Nemere, I., and A . W . Norman. 1989. 1,25-dihydroxyvitamin D3-mediated vesicular calcium transport in intestine: dose-response studies. M o l . Ce l l . Endocrin. 67: 47-53. Nemere, I., and A . W . Norman. 1990. Transcaltachia. vesicular calcium transport, and microtubule-associated calbindin-D28k: emerging views of 1,25-dihydroxyvitamin D 3 -mediated intestinal calcium absorption. Miner . Electrochem. Metab. 16: 109-114. Nemere, I., and C M . Szego. 1981. Early actions of parathyroid hormone and 1,25-dihydroxycholecalciferol on isolated epithelial cells from rat intestine: II Analysis of additivity, contribution of calcium, and modulatory influence of indomethacin. Endocrinology. 109: 2180-2187. 1 6 5 Nemere, I., Y . Yoshimoto, and A . W . Norman. 1984. Studies on the mode of action of calciferol. L I V . Ca transport in perfused duodena from normal chicks: Enhancement within 14 minutes of exposure to 1,25-dihydroxyvitamin D 3 . Endocrinology. 115: 1476-1483. Nicholson, G . C . , J . M . Moseley, P . M . Sexton, F . A . Mendelson, and T . J . Mart in. 1986. Abundant calcitonin receptors in isolated rat osteoclasts. J . C l i n . Invest. 78: 355-360. Norman, A . W . , 1990. Intestinal Ca transport: a vitamin D-hormone-mediated adaptive response. A m . J . C l i n . Nutr. 51: 290-300. Norman, A . W . , and H . F . DeLuca. 1963. The preparation of H3 and D3 and their localization in the rat. Biochemistry 2:1160. Norman, A . W . , A . K . Mircheff, T . H . Adams, and A . Spielvogel. 1970. Studies on the mechanism of action of calciferol; III. Vitamin D-mediated increase of intestinal brush border alkaline phosphatase activity. Biochim. Biophys. Acta. 215: 348-359. Okano, K . , S. W u , X . Huang, K . Pirola, H . Juppner, A . - B . Abou-Samra, G . V . Segre, J. Iwasaki, A . J . Fagin, and T . L . Clemens. 1994. Parathyroid hormone ( P T H ) / P T H -related protein (PTHrP) receptor and its messenger ribonucleic acid in rat aortic vascular smooth muscle and U M R osteoblast-like cells: cell specific regulation by angiotensin-II and P T H r P . Endocrnology. 135: 1093-1099. Pak, C . Y . C . , J. Poindexter, and B . Finlayson. 1989. A model system for assessing physicochemical factors affecting calcium absorbability from the intestinal tract. J . Bone Miner . Res. 4: 119-127. Palant, C . E . , M . E . Duffey, B . K . Mookergee, S. Ho , and C . J . Bentzel. 1983. C a 2 + regulation of tight-junction permeability and structure in Necturus gallbladder. A m . J. Physiol. 245: C203-C212. Pansu, D . , C . Bellaton, C . Roche, and F . Bronner. 1989. Theophylline inhibits transcellular Ca transport in intestine and Ca binding by CaBP. A m . J . Physiol. 257: G935-G943. Pansu, D . , C . Bellation, and F . Bronner. 1981. Effect of Ca intake on saturable and nonsaturable components of duodenal Ca transport. A m . J . Physiol. 240: G32- G37. Pansu, D . , C . Bellation, and F . Bronner. 1983a. Developmental changes in the mechanisms of duodenal calcium transport in the rat. A m . J. Physiol. 244: G20-G26. Pansu, D . , C . Bellation, C . Roche, and F . Bronner. 1983b. Duodenal and ileal C a + + transport in the rat and effects of vitamin D . A m . J . Physiol. 244: G695-G700. Pansu, D . , M . C . Chapuy, M . Mi l l an , and C . Bellation. 1975. Transepithelial calcium transport by xylose and glucose in the rat jejunal ligated loop. In: Calcified tissues 166 1975. Proceedings of the X l t h European Symposium on Calcified Tissues. (S.P. Nielsen and E . Hjorting-Hansen eds.) pp 45-52. Fadl's Copenhagen. Pansu, D . , C . Duflos, C . Bellation, and F . Bronner. 1993. Solubility and intestinal transit time limit calcium absorption in rats. J. Nutr. 123: 1396-1404. Pappenheimer, J .R. 1990. Paracellular intestinal absorption of glucose, creatinine, and mannitol in normal animals: relation to body size. A m . J . Physiol. 259: G290-G299 Pappenheimer, J .R. and K . Z . Reiss. 1987. Contribution of solvent drag through intercellular tight junctions to absorption of nutrients by the small intestine of the rat. J . Membrane B i o l . 100: 123-136. Pappenheimer, J .R. , and K . Volpp . 1992. Transmucosal impedance of small intestine: correlation with transport of sugars and amino acids. A m . J. Physiol. 263: C480-C493. Parfitt, A . M . 1994. Parathyroid growth, normal and abnormal. In: The Parathyroids, pp 373-405. (Bilezikian, J .P . , M . A . Levine, and R. Marcus. Eds.) New York: Raven Press. Peeters, M . , M . Hiele, Y . Ghoos, V . Huysmans, K . Geboes, G . Vantrappen, and P. Rutgeerts. 1994. Test conditions greatly influence permeation of water soluble molecules through the intestinal mucosa: need for standardization. Gut. 35: 1404-1408. Penniston, J .T . , and A . Enyedi. 1994. Plasma membrane C a 2 + pump: recent development. Ce l l . Physiol. Biochem. 4: 148-159. Ponchon, C , and H . F . DeLuca. 1969. The role of liver in the metabolism of vitamin D . J. C l i n . Inves. 48: 1273-1279. Raisz, L . G . and I. Niemann. 1967. early effects of parathyroid hormone and thyrocalcitonin on bone in organ culture. Nature. 214: 486-487. Rasmussen, H . , O. Fontaine, E . E . M a x , and D . B . P . Goodman. 1979. The effect of 1-ot-hydroxyvitamin D 3 administration on calcium transport in chick intestine brush border membrane vesicles. J . B i o l . Chem. 254: 2933-2999. Reuss, L . , and A . L . Finn. 1977. Effects of lumenal hyperosmolality on electrical pathways of Necturus gallbladder. A m . J . Physiol. 232: C99-C108. Robertson, W . G . 1976. Measurements of ionized calcium in body fluids. Ann . C l i n . Biochem. 13: 540-548. Rubinoff, M . J . , and H . N . Nellans. 1985. Active calcium sequestration by intestinal microsomes. J. B i o l . Chem. 260: 7824-7828. 167 Sampson, H . W . , J . L . Matthews, J . H . Mart in , and A . S . Kunin. 1970. A n electron microscopic localization of calcium in the small intestine of normal, rachitic, and vitamin D-treated rats. Calc. Tiss. Res. 5: 305-316. Sanyal, A . J . , J.I. Hirsch, and E . W . Moore. 1994. Premicellar taurocholate enhances calcium uptake from all regions of rat small intestine. Gastroenterology. 106: 866-874. S A S . 1985. S A S User's Guide Statistics SAS Institute Inc., Cary, N C . Schedl, H . P . , G . W . Osbaldiston, a n d l . H . M i l l s . 1968. Absorption, secretion, and precipitation of calcium in the small intestine of the dog. A m . J. Physiol. 214: 814-819. Shafey, T . M . , M . W . McDonald , and R . A . E . Pirn. 1990. The effect of dietary calcium upon growth rate, food utilization and plasma constituents in lines of chickens selected for aspects of growth or body composition. Brit . Poult. Sci . 31: 577-586. Shimura, F . , and R . H . Wasserman. 1984. Membrane associated vitamin D-induced calcium binding protein (CaBP): quantification by a radioimmunoassay and evidence for a specific C a B P in purified intestinal brush borders. Endocrinology. 115: 1964. Spenser, R. M . Charman, P . N . Wilson, a n d D . E . M . Lawson. 1978. Stimulation of intestinal calcium-binding protein m R N A synthesis in the nucleus of vitamin D-deficient chicks by 1,25-dihydroxycholecalciferol. Biochem. J. 170: 93-101. Spansos, E . , D . H . Brown, J . C . Stevenson, and I. Maclntyre. 1981. Stimulation of 1,25-dihydroxycholecalciferol production by prolactin and related peptides in intact renal cell preparations in vitro. Biochim. Biophys. Acta. 672: 7-15. Staehelin, L . A . 1973. Further observations on the fine structure of freeze-cleaved tight junctions. J . Ce l l Sci . 13: 763-786. Stein, W . D . 1992. Facilitated diffusion of calcium across the rat intestinal epithelial cell. J . Nutr. 122: 651-656. Stumpf, W . E . , M . Sar, F . A . Reid, Y . Tanaka, and H . F . DeLuca. 1979. Target cells for 1,25-dihydroxyvitamin D 3 in intestinal tract, stomach, kidneys, skin, pituitary gland, and parathyroid hormone. Science. 206: 1188-1190. Tanaka, Y . , and H . F . DeLuca. 1981. Measurement of mammalian 25-hydroxyvitamin D 3 24R-la-hydroxylase. Proc. Natl . Acad. Sci . U S A 78: 196-199 Takito, J . , S. T. Shinki, H . Tanaka, and T. Suda. 1992. Mechanism of regulation of calcium-pumping activity in chick intestine. A m . J . Physiol. 262: G797-G805. Tauber, S .D. 1967. The ultimobranchial origin of thyrocalcitonin. Proc. Natl . Acad. Sci . ( U . S . A . ) 58: 1684-1687. 168 Troy, K . S . , H . M . Darwish, and H . F . DeLuca. 1994. Molecular biology of vitamin D action. Vi t . and Horm. 49: 281-326. Tsonis, P. 1991. 1,25-Dihydroxyvitamin D 3 stimulates chondrogenesis of the chick limb bud mesenchymal cells. Dev. B i o l . 143: 130-134. Underwood, J.I . , and H . F . DeLuca. 1984. Vitamin D is not directly necessary for bone growth and mineralization. A m . J . Physiol. 246: E493-E498. Ussing, H . H . , and K . Zerahn. 1951. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta. Physiol. Scand. 23:110-127. Wada, S., T . J . Mart in, and D . M . Findlay. 1995. Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells. Endocrinology. 136: 2611-2621. Washburn, E . W . 1928. International critical tables of numerical data, physics, chemistry and technology. V o l . 3. pp, 377. McGraw H i l l , New York , N . Y . Wasserman, R . H . 1962. Studies on vitamin D 3 and the intestinal absorption of calcium and other ions in the rachitic chick. J . Nutr. 77: 69-80 Wasserman, R . H . , M . E . Brindak, S . A . Meyer, and C . S . Fullmer. 1982. Evidence for multiple effects of vitamin D 3 on calcium absorption: Response of rachitic chicks with or without partial vitamin D 3 repletion, to 1,25-dihydroxyvitamin D 3 . Proc. Natl . Acad. Sci . U S A 79: 7939-7943. Wasserman, R . H . , J.S. Chandler, S . A . Meyer, C A . Smith, M . E . Brindak, C . S . Fullmer, J .T. Penniston, and R. Kumar. 1992a. Intestinal calcium transport and calcium extrusion processes at the basolateral membrane. J. Nutr. 122: 662-671. Wasserman, R . H . , and C . S . Fullmer. 1995. Vitamin D and intestinal calcium transport: facts, speculations, and hypotheses: J. Nutr. 125: 1971S-1979S. Wasserman, R . H . , C A . Smith, M . E . Brindak, N . D . Talamoni, C . S . Fullmer, J .T. Penniston, and R. Kumar. 1992b. Vitamin D and mineral deficiencies increase the plasma membrane calcium pump of chicken intestine. Gastroenterology. 102: 886-894. Wasserman, R . H . , and A . N . Taylor. 1966. Vitamin D3-induced Ca-binding protein in chick intestinal mucosa. Science. 152: 791-793. Wasserman, R . H . , and A . N . Taylor. 1969. Some aspects of the intestinal absorption of calcium, with special reference to vitamin D . In: Mineral Metabolism. A n Advanced Treatise, edited by C . L . Comar and F Bronner. New York : Academic. 169 Wasserman, R . H . , A . N . Taylor, and F . A . Kallfelz. 1966. Vitamin D and transfer of plasma calcium to intestinal lumen in chicks and rats. A m . J . Physiol. 211: 419-423. Weinstein, R . S . , J . L . Underwood, M . S . Hutson, a n d H . F . DeLuca. 1984. Bone histomorphometery in vitamin D-deficient rats infused with calcium and phosphorus. A m . J. Physiol. 246: E499-E505. Webling, D . D . A . , and E . S . Holdsworth. 1966. Bile salts and calcium absorption. Biochem. J . 100: 652-660. Wick, A . N . , T . N . Mori ta , and L . Joseph. 1954. The oxidation of mannitol. Proc. Soc. Exp. B i o l . M e d . 85: 188-190. Wilson, P . , and D . E . M . Lawson. 1980. Calcium binding activity by chick intestinal brush border membrane vesicles. Pflugers A r c h . 389: 69-74. Yamauchi, K . , S. Iida, and Y . Isshiki. 1992. Post-hatching developmental changes in the ultrastructure of the duodenal absorptive epithelial cells in 1, 10 and 60-d-old chickens, with special reference to mitochondria. Brit . Poult. Sci . 33: 475- 488. Yuan, Y . V . , and D . D . Kitts. 1992. Effect of dietary calcium intake and protein source on calcium utilization and bone biomechanics in the spontaneously hypertensive rat. J. Nutr. Biochem. 3: 452-460. Yuan, Y . V . , and D . D . Kitts. 1994. Calcium absorption and bone utilization in spontaneously hypertensive rats fed on native and heat-damaged casein and soya-bean protein. Brit. J . Nutr. 71: 583-603. Zornitzer A . E . , and F . Bronner. 1971. In situ studies of calcium absorption in rats. A m . J. Physiol. 220:1261-1266. 170 APPENDICES 171 + - > s > a 1 o 1—1 a 3, T3 CU u a cu D-a « CA a s 'C 73 U O 7 3 % » 5 73 3 co c o ' S3 (TJ85j l .p3p3fui u i d p sox i - iui u idp) jCjIAipBOIpBJ B 3 s j 7 BUISBIJ 172 (TJ§5| j.papafui uidp soi T-iui uidp) ^MAipBOipBJ IO;iUUBUI-H£ BUISBU a B © "o S 3. T3 cu u CS cu a a a c« '•B c 1=1 O .— . -H •-H CU ft cu 5 cd »ri CU 12 & 2 cd W .S " T 3 cd 0 S 1 .3 3 ^ .3 3 e cd O cu '- ' x! a 1=1 d 8 .2 £ td X ) & OH ft 3 CM S3 & .2 o 1 ft cu OH X CU OH ft O 8 -2 '-ft — P 2 cd .3 g C CO S-9 =3 cu cu cd "73 T 3 ft , ft Cftl CU g O si H ^ cu o cd rt 11 . T3 (N O cu £ 3 a M -12 1X1 - ~ II s- a O. o . >^  ft M g « cu 3 .3 =*• § ft4 y-! ft o U cu o c o cu ft co *3 •S <=> ft & < % Hi ° £ ft -ft o w "3 co - I x? >~> 1 cu ^ " 7 3 ^ S % 7 3 'a .s C C cd rt o it ft m t(l ft cd II 3 £ 3 £ cd ' £ . s e s 173 4 v v WW//////. %/////////, G__ZI CU (3D R 1 2 2 8 S 8 o o o o o d (iro/qui) 3Uin(0A IBU3Uin|LMJUI 3AIJB[3^ > o ^ .-a '-3 £ T 3 cu cC cd 174 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0088740/manifest

Comment

Related Items