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Intestinal calcium transport in the chicken Bhatti, Mohammad Suleman 1998

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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 THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR T H E DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of A n i m a l Science)  We accept this thesis as conforming to the required standard  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A April,  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)  ABSTRACT 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 i n this thesis was conducted i n 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 C a C k and H-mannitol were used to delineate the cellular 4 5  3  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  disappearance mechanisms.  not  influenced by  l,25-(OH)2D3  from in situ intestinal loops was  administration.  not affected  Further,  by vitamin  mannitol  D-dependent  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 mechanisms.  demonstrated  to be nonsaturable  suggesting  both occur by paracellular  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 i n the duodenal compared to the distal jejunal or distal ileal loops. The capacity of paracellular transport i n 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 i n in situ loops and i n intact rapidly growing young broiler cockerels occurs largely by a paracellular process, which can take place i n the absence of vitamin D .  ii  TABLE OF CONTENTS ABSTRACT  ii  TABLE OF CONTENTS  iii  LIST O F F I G U R E S  xi  LIST O F T A B L E S  xvi  ABBREVIATIONS  Xviii  ACKNOWLEDGMENTS  xx  DEDICATION  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. C a l c i u m Homeostasis  6  2.2. Vitamin D i n Calcium Metabolism  10  2.3. Calcium Transport Across the Intestinal Barrier  13  2.3.1. Physiological Anatomy of the Intestinal W a l l 2.3.2. Mechanisms Involved i n Intestinal Calcium Transport 2.3.2.1. The Cellular Pathway 2.3.2.1.1. Classical M o d e l of the Cellular Pathway  13 15 18 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  iii  2.3.2.1.2. The Vesicular M o d e l of the Cellular Pathway 2.3.2.2. The Paracellular Pathway  23 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 i n Intestinal Calcium Transport Research  33  2.4.1. Intestinal Preparations for Transport Studies  33  2.4.2. Techniques U s e d i n 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 F r o m 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. A n i m a l 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 3.17. Factors that M a y Influence In Situ Intestinal Loop Transport Transport  52 56  C H A P T E R 4. Regulation of Intestinal Calcium Transport i n Rapidly G r o w i n g Young Broiler Cockerels by Vitamin D-Dependent Mechanisms  64  4.1. Introduction  64  4.2. Methods  67  4.2.1. Effects of H i g h 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 i n Response to H i g h Calcium Intake  68  4.2.1.3. Ileal Calcium Transport in Response to H i g h 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 o f V i t a m i n D-Deficiency Rickets and 1,25-(OH)2D3 Treatment on Calcium Transport i n In Situ Duodenal and Ileal L o o p 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 -Treated Chicks 3  72  4.2.2.3.3. Ileal Calcium Transport in Rachitic and l,25-(OH)2D -Treated Chicks 3  73  4.2.2.3.4. Intestinal Calcium Transport i n Intact Rachitic Chicks 4.3. Results  74 75  4.3.1. Responses to H i g h Calcium Intake  75  4.3.1.1. Body Weight, and Plasma and Excreta Calcium Concentrations in Response to H i g h Calcium Intake  75  4.3.1.2. Duodenal Calcium Transport i n Response to H i g h 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 V i t a m i n D and l,25-(OH)2D3 Treatment 4.3.2.1. Rachitogenesis  80 80  vi  4.3.2.2. Body Weight, Tibia Fat-Free Dry Weight, Tibia A s h Weight, and Tibia Length i n Rachitic and Control Chicks  83  4.3.2.3. Plasma Total-Calcium Concentration and Hematocrit Values i n Rachitic and Control Chicks  85  4.3.2.4. Plasma Total-Calcium Concentration i n Response to l,25-(OH)2D  3  Treatment i n Rachitic and Control Chicks  87  4.3.2.5. Duodenal Calcium Transport in Rachitic and Control Chicks W i t h or Without l,25-(OH)2D  3  Treatment  89  4.3.2.6. Ileal Calcium Transport i n Rachitic and Control Chicks With or Without l,25-(OH)2D Treatment 3  91  4.3.2.7. Calcium Retention by Duodenal and Ileal loop Tissues in Rachitic and Control Chicks W i t h or Without l,25-(OH)2D Treatment 3  4.3.2.8. Calcium Transport in Intact Rachitic Chicks 4.4. Discussion  93 93 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 i n Intact Rachitic Chicks  101  4.4.5. Calcium Metabolism and Vitamin D  103  vii  4.4.6. Vitamin D-Dependent Intestinal Calcium Transport: A r e Juvenile Chickens Different from Neonatal Rats? 4.4.7. Mannitol as a Marker of Paracellular Calcium Transport 4.5. Conclusions  104 104 106  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 T w o Different Regimens of Increasing Calcium Intake 5.3 Results  Ill 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 F e d a H i g h Calcium Diet or Provided 40 m M C a C h  117  5.3.3. Characteristics of Intestinal Calcium Transport i n Intact Birds with T w o Different Regimens of Increasing Calcium Intake  120  5.4. Discussion  123  5.5. Conclusions  130  viii  C H A P T E R 6. Age-Related Changes and Regional Differences i n Paracellular Absorption, Regional Differences i n Paracellular Secretion, and Regional Differences i n 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 i n In Situ Duodenal, Jejunal, and Ileal L o o p Preparations i n Broiler Cockerels  134  6.2.2. Regional Differences i n 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 L o o p Preparations in Broiler Cockerels 6.4. Discussion  141 144  6.4.1. Age-Related Changes i n Paracellular Absorption in In Situ Duodenal, Jejunal, and Ileal Loop Preparations i n Broiler Cockerels 6.4.2. Regional Differences i n the Small Intestine of Broiler  ix  144  Cockerels for Paracellular Absorption  145  6.4.3. Regional Differences i n Paracellular Secretion from the Blood into the Lumen of In Situ Duodenal, Jejunal, and Ileal L o o p Preparations in Broiler Cockerels 6.5. Conclusions  146 148  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  REFERENCES  156  APPENDICES  171  x  LIST O F F I G U R E S  Figure 2.1 Schematic representation of the epithelial barrier of the intestinal absorptive surface  16  Figure 2.2. Schematic diagram showing some o f the possible pathways involved i n 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 C a C l 2 and H-mannitol radionuclides (Mean ± S E ) 4 5  3  from the lumen of duodenal, distal jejunal, and distal ileal loops in vitro (n=5 to 7) i n 13-d-old broiler cockerels, 2 m i n after an intralumenal injection of a solution containing 75 m M each of C a C l 2 and H-mannitol 45  3  ( S . A . , 1.33 n C i p m o l )  45  1  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 5 C a C l and 3H-mannitol ( S . A . , 1.33 n C i p m o l ) , i n 14-d-old 4  1  2  broiler cockerels (n=6)  47  Figure 3.3. Disapperance of calcium (Mean ± S E ) from the in situ duodenal loop preparations at 2, 4, 6, 8, and 10 m i n after injection of an intralumenal test solution containing 75 m M each of C a C l 2 and H-mannitol ( S . A . , 1.33 4 5  n C i pmol" ), i n 14-d-old broiler cockerels (n=6) 1  xi  3  48  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 % C a of D M )  53  Figure 3.5. Intralumenal calcium concentration (Mean + SE) at different times after a 100 m M C a C h solution was injected into the lumen of in situ 4 5  duodenal loop preparations i n 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; M e a n + S E ; n = 2 to 5) after an intralumenal injection into the in situ duodenal loop preparation i n 10-d-old broiler cockerels  58  Figure 3.7. Plasma mannitol concentration at different times after a 300 m M H 3  Mannitol solution ( S . A . , 0.33 n C i pmol" ) was injected @ 1.5 m L kg" 1  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 i n 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  CaCl  2  test solution (Mean  + SE) at 2, 4, 8, 12, 16, and 20 m i n after intralumenal injection into the in situ duodenal loop preparation i n 10-d-old broiler cockerels (n=5) 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  xii  63  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 i n 10-d-old broiler cockerels fasted for 12 h, i n 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 i n 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 ) D treatment (i.m. injection @10 pg /kg) on 2  3  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 , 2 5 - ( O H ) D 2  3  treatment (i.m. injection @ 10 pg/kg) on calcium and mannitol disappearance (Mean + SE) from in situ duodenal loop preparations i n 12-, and 13-d-old broiler cockerels  90  Figure 4.6. The effects of vitamin D-deficiency rickets, and l , 2 5 - ( O H ) D 2  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 Figure 4.7. Calcium retention by duodenal and ileal loop tissues (Mean + SE)  xiii  92  after in situ loop calcium and mannitol transport experiments i n rachitic and nonrachitic-control broiler cockerels with or without 1,25-(OH) D 2  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) i n 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 H-mannitol that disappears (Mean + SE) from the lumen of in situ 3  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- 10 m i n ' ) (left panel), and the 1  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 for a 24 h period on feed 2  xiv  (left panel) and water (right panel) intakes (Mean + SE) i n 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, i n 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) i n 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 i n 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 % C a of D M ) or high calcium diet ( H C D ) (1.65% C a of D M ) from hatch  76  Table 4.4. Body weight, and plasma total-calcium and tibia calcium concentrations (Mean ± SE) i n 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) i n 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) i n 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) i n 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  xvii  ABBREVIATIONS  l,25-(OH)2D  3  1,25-dihydroxyvitamin D 3  AP  alkaline phosphatase  ATP  adenosine triphosphate  cAMP  cyclic adenosine monophosphate  cGMP  cyclic guanosine monophosphate  CIF  calcium influx factor  Da  dalton(s)  DM  dry matter  HCD  high calcium diet  I.U.  international unit  i.m.  intramuscular  i.v.  intravenous  kcal  kilocalorie  Kev  kilo-electron-volt  M.W.  molecular weight  n  sample size  nCi  nanocurie(s)  NRC  National Research Council  P  probability  PEG  polyethylene glycol  PTH  parathyroid hormone  xviii  standard error (of mean) specific radioactivity statistical analysis system volume  xix  ACKNOWLEDGMENTS  Special thanks go to my research supervisor D r . Thompson for his very valuable instruction, guidance and support.  H e is a good man.  D r . Thompson accepted me as a  graduate student after D r . Hart's departure for California.  Thanks go to D r . 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.  A m o n g them, D r . M a r c h has been very concerned and encouraging, D r . Blair  has been very supportive and helpful, and D r . Kitts, besides being very stimulating and helpful, became involved to the extent o f 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 D r . Youngblut, and M s . Linda Jane of Roche Canada for  sending me a gift of 1,25-dihydroxyvitamin D 3 . D r . Norman from the University of California Riverside was very kind to send me antibody for calcium binding protein.  Thanks go to my  friend M i c h e l i n 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 E g g 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 o f A n i m a l Science for providing the research and teaching assistantships and for the Professor B . E . M a r c h Travel A w a r d . I thank D r . Leichter and D r . Samuels for being helpful i n my  comprehensive  examination, and D r s . 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 Canada,  xxi  Pakistan,  to my teachers  and the United  in  States.  CHAPTER 1 General Introduction  The importance  of calcium in human and animal nutrition is well  recognized.  Numerous disorders such as osteoporosis i n humans and skeletal abnormalities i n rapidly growing broiler chickens have been linked to calcium malnutrition.  Calcium nutrition has a  significant role i n public and animal health, and animal production.  The intestine is the sole port o f 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 o f improving calcium nutrition, information pertaining to regulation mechanisms of intestinal calcium transport is of great significance.  Intestinal calcium transport involves translocation o f 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. pathway,  calcium ions move via the cytosolic compartment  In the cellular  whereas i n 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.  l  The  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).  M o s t o f 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 i n all segments of the rat small intestine mounted on Ussing chambers has a significant cellular component.  W i t h 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 i n 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 o f 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. T o 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 i n 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 i n in situ ileal loops i n 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 i n 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. O n 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 o f research related to regulation mechanisms o f 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 o f the mucosal epithelial lining  of the intestine is high (Fromter and Diamond, 1972), large quantities o f water and solutes are expected to cross the mucosal barrier v i a 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 o f laboratory rodents via the paracellular pathway.  The  significance o f the paracellular pathway i n intestinal calcium transport needs to be studied. The present research is the first to explore the mechanism and regulation o f intestinal calcium transport i n rapidly growing young broiler cockerels.  1.1. Research Objectives 1.  T o develop an in situ intestinal loop experimental system to study the cellular and paracellular components of intestinal calcium transport i n rapidly growing young broiler chickens.  2.  T o determine the fractional contribution of the vitamin D-dependent and vitamin D-independent components of calcium transport i n the small intestine o f rapidly growing young broiler chickens.  3.  T o determine the significance of paracellular calcium transport in intact young broiler chickens.  4.  T o determine: a) the age-related and regional differences in paracellular absorption, and b) the regional differences in paracellular secretion i n the small intestine o f young broiler chickens.  5  CHAPTER 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 i n the signal transduction cascade, calcium ions are ubiquitously involved i n 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 i n the cytosol and calcium phosphates, being very insoluble, may precipitate i n the cytosol when the calcium ion concentration is high. The cytosolic concentration of calcium ions is typically 0.1 pM,  several orders of magnitude less than its concentration i n the extracellular space.  (Fullmer, 1992).  Persistently subnormal plasma calcium concentrations may lead to the  development o f pathological states such as tetany,  rickets, or osteomalacia.  In effect,  intracellular calcium concentration is one of the most tightly regulated parameters i n 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.  M o s t o f total body calcium is associated with the skeleton in the form of hydroxyapatite crystals, while the rest resides i n 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 i n 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 i n the free ionized form, and 40% is protein bound. Calcium i n the urine and cerebrospinal fluid is either i n the free ionized or complexed form but is not protein bound. The plasma calcium concentration i n normal animals rarely varies more than 10% from its mean 2.5 m M value.  Most o f the intracellular calcium is normally kept sequestered by cellular organelles such as the mitochondrion, endoplasmic reticulum, or G o l g i apparatus, and is released in response to stimuli destined to regulate cellular functions.  The resulting increase i n 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 o f 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 i n the urine.  Bone is subject to continuous remodeling and i n this  process replenishes or sequesters plasma calcium.  After an increase by a bolus intravenous injection of calcium, the plasma calcium concentration returns to normal i n about 40 m i n i n growing birds (Hurwitz et a l . , 1983), and i n about 1 h i n rats (Bronner and Stein, 1992). normalizing the  experimentally perturbed  The shortness of the period involved in  plasma calcium concentration  has  led to  an  evaluation of the concept that surfaces of the mineral in bone act as a calcium buffer ( M c L e a n 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 o f calcium homeostasis by this process is pending.  Parathyroid hormone ( P T H ) , 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 i n 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 i n rat osteoblastlike 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 i o n  concentration, by inhibiting bone resorption (Raisz and Niemann, 1967).  The importance of  calcitonin i n 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  V i t a m i n D acts i n 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 ) . common plant steroid precursor, ergosterol.  Ergocalciferol is derived from a  Cholecalciferol is produced from its precursor 7-  dehydrocholesterol primarily i n 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. property (anonymous).  Precursors of vitamin D have no antirachitic  The precursors of vitamin D are widely distributed i n 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 D3.  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 D e L u c a , 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). undergoes microsomes  two hydroxylations in the body. of the  hydroxy vitamin D 3 .  T o become fully active, vitamin D 3  The first hydroxylation takes place i n the  liver (Ponchon and D e L u c a ,  1969) to convert vitamin D  3  to 25-  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 1,25dihydroxyvitamin D 3 ( l , 2 5 - ( O H ) 2 D 3 ) .  Although other metabolites of vitamin D have been  identified, Brommage and DeLuca (1985) provided strong evidence that the only active form was l , 2 5 - ( O H ) 2 D 3 .  This form o f vitamin D is also linked to the autosomal recessive disorder  vitamin D-dependency rickets type I, which is likely a defect i n the enzyme la-hydroxylase (Fraser e t a 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 (DeLuca and Schnoes, 1976; Fraser, 1980). The effects of high 3  calcium intake on l,25-(OH)2D biosynthesis appear to be secondary to P T H since P T H 3  concentration varies in a reciprocal manner to serum calcium and phosphorus concentrations (Fraser, 1980).  Hypocalcemia i n 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 D e L u c a , 1981).  11  The vitamin D metabolite, l , 2 5 - ( O H ) 2 D 3 , 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  localizes i n the nucleus of target tissues.  l,25-(OH) D3 2  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 , 2 5 - ( O H ) 2 D 3 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,25-(OH)2D  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 , 2 5 - ( O H ) 2 D 3 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 i n the nucleus of crypt and villus cells of the intestine after injecting radiolabelled l , 2 5 - ( O H ) D 2  3  in vivo (Stumpf et a l . , 1979).  stimulated i n response to l , 2 5 - ( O H ) 2 D 3 treatment. mediated by C a B P .  Intestinal calcium transport is  This effect is considered to be primarily  Wasserman and Taylor (1966), isolated a C a B P with a molecular weight  of approximately 28,000 D a (calbindin-28k) from the mucosa of the chicken intestine. A C a B P of 9,000 D a molecular weight (calbindin-9k) was discovered soon after i n 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 i n mammalian tissues, whereas calbindin-28k has four such domains and has been isolated from a  12  variety of avian and mammalian tissues, but not i n the mammalian intestine (Christakos et a l . , 1989). It must be pointed out that the expression of some o f the C a B P is vitamin D-dependent only i n 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 o f a number o f bone-specific proteins i n 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 i n 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 i n bone mineralization. that bone development  Underwood and D e L u c a (1984)  demonstrated  and mineralization are normal i n 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 o f mineral and matrix i n their bones than d i d those rats only treated with vitamin D , pointing out that bone mineralization was dependent on availability o f 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  o f the intestine  is organized into two  morphologically and functionally distinct cellular compartments: the crypts o f 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 borderbearing 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.  A m o n g the four general types o f 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 o f the actin cytoskeleton (Madara et al., 1987). A n extensive actin network supports the apical microvilli i n 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 i n 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 actinmyosin 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 v i a the cytosolic compartment of the enterocytes.  In the paracellular pathway, the  movement o f calcium ions takes place via the extracellular spaces bounded by the tight junction between adjacent enterocytes.  15  u  X5 H  co .3  £ '3, a  s  •5 &3  S3  u  .a E  au  03  CJ  4->  CO t< e  CD  J3 co IS 5 © O 03 CO 03 ^ s ° H o3  u  53  2  U  X3 4-> 03  o o  '£  03  CI,"  °co 2^ « 3  •2 -5 B •2, 3 £ -2 .2? o  X> Xi 13 " "3 c^  4->  £3  < ^ > 3.  o c _o *4-t 03 •*-»  c  1/3  tu l-l Ui  .2 >  < D j3 O N ^1 -3 O N 03 4-"  s O  G O  U  d  I ts  CO - H  __  M  »  +->+->  03  c ^ o o  .6.2 Jo  16  ^  CO  2 § 6  o3  ob  <u o o  u c  •-  «  ^  ^ £  g  i  17  2.3.2.1. The Cellular Pathway The general scheme of events for the cellular pathway o f intestinal calcium transport involves three sequential steps: 1) calcium entry into the cytosol v i a the apical membrane, 2) transmural calcium movement from the apical membrane to the basolateral membrane o f the enterocyte,  and 3) calcium extrusion at the basolateral membrane into the lateral space  occupied by the lamina propria. circulation.  F r o m the lateral space, calcium diffuses into the portal  Details o f the mechanisms involved i n these steps has been recently reviewed  (Fullmer, 1992; Stein, 1992; Wasserman et a l . , 1992a).  T w o 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 i n 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 o f calcium entry into the enterocyte through the  brush border membrane is lacking. Based on the difference i n the intralumenal free calcium concentration (-10 m M ) and the intracellular free calcium concentration (10" -10 m M ) , it is 7  6  generally believed that calcium entry is a process o f simple diffusion proceeding down an  18  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 ( A P ) activity associated with the brush border has been implicated to be involved i n 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 i n 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 i n the fluid state of the brush border membrane is likely to enhance calcium permeation.  Matsumoto  et  al.  (1981)  demonstrated  an  increased  biosynthesis  of  phosphatidylcholine i n isolated enterocytes in response to l , 2 5 - ( O H ) 2 D 3 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 o f choline and ethanolamine into the brush border lipid 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  lipid 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 i n 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 i n 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 i n 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 transport is accomplished by a cascade of events.  Ca  + +  In this cascade, the C a - b i n d i n g 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 o f transmural diffusional transport (Bredderman and Wasserman, 1974; Glenney and Glenney,  1985; W i l s o n 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 o f  calcium from the basolateral side o f enterocytes into the extracellular space i n the lamina propria takes place against a steep electrochemical gradient.  In addition to a positive  electropotential difference o f 58 m V , an approximately 50,000 fold greater concentration of free  calcium exists i n the extracellular fluid  compartment  compared to the cytosolic  compartment (Wasserman and Fullmer, 1995). T w o 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.  properties o f calcium ATPase.  Penniston and Enyedi (1994) have recently reviewed the  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 i n the synthesis o f calcium ATPase (Wasserman et a l . , 1992b) and an increase i n the gene expression o f calcium ATPase (Cai et a l . , 1993) have been reported i n the duodenum of layer-type chicks i n response to l,25-(OH)2D3 treatment. The N a / C a +  + +  exchanger, on the  other hand is reported to be independent o f vitamin D and accounts for nearly 20% of calcium  22  extrusion from the rat duodenum (Ghijsen et a l . , 1983). The capacity o f 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), G o l g i apparatus (Freedman et a l . , 1977), and more recently microsomes (Rubinoff and Nellans, 1985) are involved i n intracellular calcium sequestration i n many tissues.  The role o f these organelles during enterocyte transmural  calcium transport is not clear.  Davis and Jones (1981) and Nemere and Szego (1981)  suggested the involvement o f lysosomes i n vitamin D mediated calcium transport responses i n 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 i n 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 o f 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 liquid 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. F r o m 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 i n the tight junction complex is an area of emerging information.  A  change i n the actin cytoskeleton could induce a functional alteration i n the tight junction by varying the tension applied to the junctional complex.  A n elevation i n 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). A l s o , an increase i n 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 i n 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 i n 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: J ^s=  A  m  where A is the net saturable C a  + +  calcium flux as a function of [Caf ], +  flux,  + P[Ca; ] +  P is the slope (or rate) of the net nonsaturable  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. mathematical expression of the saturable component, A , therefore is:  ~ K, + [Ca; ] +  26  The  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 [ C a , ] i s the intralumenal free calcium concentration. ++  transport is plotted as function of [Ca* ], +  If the rate of net C a  + +  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 v i a the enterocytes, the saturable component is hypothesized to represent transport via the cellular pathway.  B y 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 o f this process.  A regression line  drawn through the data points obtained at high intralumenal calcium concentrations w i l l create an intercept representing the maximum rate of calcium transport via the cellular pathway, and a slope representing the rate o f 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 i n 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 i n 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  co  cu O CO it w" CV,  3  2  to «  IS c  cu  CN  aJ  CJ —  . cu  CO  a a co  CU  C3  oo  n  TJ S  cu  E  .3  "J3 i cS  M ?  73  cu 03 03  C+H cd X !  o „ 3 .9 CO cu 03  ^  a,  W  3  O  >>  2 g  T5 .2 e DC  co H  o "3 S  2 &  o T3 2  a3 • £  .2  ccS co 2 S  °  o  M^ 3  co  CO CU  -S  cu  T3 CU  H  S 3  2  £  co co 3 O co \j3 cu cu 3 o3  c  73  < 8 •f co j j o d s u B j ; u m p r e D jo  3;B^  29  is  03  "cu WD cu  a  cu  c  a  03  -3  l,25-(OH)2D3 receptor occupancy is correlated with the expression of calbindin i n 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 i n 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 o f intestinal calcium transport via the cellular pathway.  The role o f vitamin D in regulating intestinal calcium transport, however, may not be limited to mechanisms involving biosynthesis of calcium binding proteins. (1978) reported that following  Spencer et al.  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 i n 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)2D perfusion o f the intestinal vasculature.  3  The effects have been demonstrated i n vitamin D -  normal but not i n vitamin D-deficient chickens. M o r e recently, deBoland and Norman (1990) reported activation of protein kinase C and cAMP-dependent protein kinase in enterocytes i n  30  response to l,25-(OH)2D3 perfusion of the intestinal vasculature.  These researchers proposed  that activation o f these proteins results i n a transient increase in the intracellular calcium concentration which i n turn stimulates calcium extrusion from the basolateral cell membrane. 1,25-(OH)2D3-induced rapid activation of second messenger  systems such as the  cGMP  (Barsony and M a r x , 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 i n the literature.  Wasserman and Fullmer  (1995) questioned the physiological relevance of the transcaltachia response.  They noted an  initial pulse i n 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 Y u a n (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 i n the rat ileum. Sanyal et al. (1994), similarly, reported that premicellar taurocholate enhanced calcium uptake i n all regions of the rat small intestine. Since the physicochemical environment i n 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 ( A u and Raisz, 1967).  M i l l e r et al. (1988)  reported that dietary lactose improved endochondral bone growth and bone mineralization i n 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.  32  Favus and Backman  (1984) showed that lactose increases net calcium absorption i n the rat ileum i n the absence of transepithelial electrochemical or osmotic gradients. They proposed that since lactose induced hyperpolarization of the brush border, a cellular pathway was involved i n 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 D e L u c a (1980), however, reported earlier that intestinal calcium  transport increases during pregnancy and lactation i n vitamin D-deficient mothers suggesting that the effects o f 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 i n 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 i n 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). W i t h mucosal surface out, the loop is incubated i n 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 o f the solute i n 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 o f the in situ loop since the circulation of the everted gut sacs is not intact. A serious shortcoming o f the everted gut sac is, that to complete the transport process i n either direction, unlike the physiological state, the solute is required to move across all four histological layers of the intestine.  A l s o , the viability of the intestinal tissue that has been  removed from the animal is compromised which may also result i n a change i n 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 i n 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 i n 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 i n 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 Therefore,  capacity of cellular mechanisms of calcium transport is expected to be finite. these mechanisms w i 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 w i l l achieve a constant value whereas the rate via the paracellular pathway w i l l increase i n 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).  T o obtain a reliable value of the slope, intralumenal test solutions containing calcium i n 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 i n 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 s o , 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. calculations is misleading.  In this case, the significance of concentration in slope  The saturation technique may be used in in situ or i n in vitro  intestinal preparations, as well as i n 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 o f 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 i m , 1996). This technique is more suitable for tissues with high permeability so that any spontaneous change i n 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  CHAPTER 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 i n laboratory rats (Pansu et a l . , 1981, 1983a, 1993). These procedures involve the use of intralumenal calcium concentrations i n 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 i n 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 o f 12 battery brooder pens located in an environmentally controlled animal  38  housing facility in the Department of A n i m a l Science at The University of British Columbia. In each pen, 63 linear c m of feeder space and 66 linear c m 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, 2 3 % ; calcium, 1%; total phosphorus, 0.8%; vitamin D 3 , 2200 I . U . / k g 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 i n the in situ loop experiments. The objective of fasting was to minimize the amount of ingesta i n 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 m i n 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 o f 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 m i n in the case of the duodenum, and within 3 m i n 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 i n 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 i n 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  (Amersham Canada L t d . , Oakville Ontario) and/or  3  H-mannitol  CaCh  (Dupont Canada Inc.,  Mississauga Ontario) were added to the test solutions as required.  The solutions with  equimolar concentrations o f calcium and mannitol (75 m M o f 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 volume of the test solution was experimentally predetermined.  and 12:00 hours.  The  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 o f the duodenum was found i n 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 ' c o l d ' 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.  F o r 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 . ) i n 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 i n the test sample, where window, and  4 5  3  H radioactivity was measured within the 0-8 K e v  C a radioactivity was measured within the 8-256 K e v 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.  42  3.8.  Parameters Used for Calcium and Mannitol Transport from In Situ Intestinal Loop  Preparations T w o 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 C a C h or H-mannitol tracer(s) 4 5  3  disappeared (dpm injected - dpm recovered) during the in situ loop procedures.  The amount o f calcium and/or mannitol that disappeared from the intestinal lumen in situ was calculated as follows: pmol" .  pmol disappeared = (dpm injected - dpm recovered) -H dpm  This parameter also takes into account the amount (weight) or length of the intestinal  1  tissue and duration o f the in situ loop transport procedure and, therefore, expressed as pmol g"  1  10 min" , or pmol c m 1  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 i n the calculation o f the intestinal transport.  To determine recovery of radioactive tracers, 13-d-old birds were killed 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  ( S . A . , 1.33 n C i pmol" ) and H-mannitol ( S . A . , 1.33 n C i pmol" ). 1  3  1  4 5  CaCh  T w o minutes after the  injection, the lumenal contents were collected by flushing with excess volume of the " c o l d " test solution.  The amount of radioactivity recovered from the lumens was determined as  described in the preceding text.  There were no significant differences i n the percent recovery of radioactivity between 3  H-mannitol and C a C k i n the duodenum (n=7), distal jejunum (n=5), or distal ileum (n=5). 4 5  N o r 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  CO  1 •a •«?  •a = §  C/2 0>  o  OS  O  et-i  O  en  ID  G O  '53D  1)  ( U I U I j je asop jo %) i(j3A033J He P  U B  B  45  3sf  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 C a C k ( S . A . , 1.33 n C i pmol" ) and H-mannitol ( S . A . , 1.33 n C i pmol" ). The in situ loop 4 5  1  3  1  experiments were terminated at 2, 4, 6, 8, 10, or 12 m i n after intralumenal injection (n=6).  Time related changes i n cumulative percent disappearance (% of dose in given time) of calcium and mannitol from the lumen o f the in situ duodenal preparation in 14-d-old birds are presented i n 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 m i n 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 o f 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 o f 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* i 60  a  ©  c  c5 3 Vi  03  O  CD  E (3UIIJ U 3 A l § J B 3S0p J O % ) 3DUBJB3ddBSip J O J I U U B U I p U B U i n p | B 3  47  .a  o  u  a  6  g  a  JO  v  u  °  0>  l-l  CD  to  •£2 U il  OS  a u u  CO  s  H  CT3  (9uip u9AiS ye asop jo %)  60 <u fcl  48  '  __  OH  from zero. A 10 m i n 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. A l s o , the results o f the in vitro recovery study (Section 3.9) indicated that the amounts o f 4 5  C a C b and H-mannitol associated with the intestinal tissue do not differ. 3  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 i n this thesis. It is worth noting, however, that a moderate to high degree of correlation was found to exist between the amount o f C a disappeared (pmol g" 10 m i n ' ) from the lumen o f in situ 4 5  intestinal loops, and the plasma  1  4 5  C a concentration (dpm m L  1  10 dpm injected" 5  1  kg" ) at 1  termination o f the experiment (see Appendix Figure 1). Similarly, a moderate to high degree of correlation was found to exist between the amount of H-mannitol disappeared (pmol g" 10 3  1  min" ) from the lumen of in situ intestinal loops and the plasma H-mannitol concentration (dpm 1  3  mL" 10 dpm injected" kg" ) at termination of the experiment (see Appendix Figure 2). 1  5  1  1  appearance of C a i n the vascular fluid has been previously used as an index o f intestinal 4 5  The 4 5  Ca  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 o f 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 m g / m L of lithium heparin and oven drying. Plasma was separated by centrifugation i n 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 i n 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 H C 1 , by heating to ensure  complete dissolution, and made to volume.  3.12. Balance Experiments The balance experiments were conducted to determine calcium retention i n a group o f intact birds. These experiments were conducted for a duration o f 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.  consumption during the experimental period was recorded.  Feed  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 M o d e l 560 apparatus. F o r these measurements, the samples were dissolved i n 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 A n i m a l 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 ( S A S , 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 i n 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 i n 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 s o , calcium concentration i n 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. T o 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 i  = E  C 3 .22. a s  6 a  3  -  S  -3 3 3 2 '«? c = E  3  i  §  r  i  -  "2 * « S 2 s ss § 1  co  II II II II II  Q  1  *1 M  M  00  CU  ^  s "  S3 CU  <L) P3  C/3 Ct-I  O  a © '53D CU  53  ° .52 b  a  Intestinal calcium and mannitol transport was determined with in situ duodenal loops in 7-d-old broiler cockerels. The birds used i n 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 C a C h ( S . A . , 1.33 n C i pmol" ) and H-mannitol ( S . A . , 1.33 n C i 4 5  pmol" ). 1  1  3  A t the termination of the in situ loop experiment, the intestinal tissue was ashed as  described earlier and the amount of C a associated with the tissue was determined. 4 5  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 i n 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 i n calcium disappearance (% o f dose) from the in situ duodenal loop preparations i n 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  TJ  o  ON  +1  <u  +-» CO  I  +1  oo oo  oo  ON  oo  V a.  V a.  CO O  m  o d  d  ON  TJ  s  ON  +1  O  d  +l oo  m  +l io  (N  <+-l  I  43  o ON  d  +1  +1  +l  oo  •a  O  o d  o d  +l  +i  oo oo  ON  PL,  ON  r-  (N  C4  o a, -o S o  a  .2  CO « CO  CO  >  CO  C  a  '<U. <uo & O « 3 .5 co 13  <u S-i  , to  o  >/-)  O ON  I '-3 a  i  U  ^  4-  CO  O  •a o  s  a o o  'S  6 o  & TJ r-H  C ^  1)  "cO  co  Is o  * OH CO  TJ —  '§  cO  c  <u o  p TJ  .2 0)  co O S TJ -S O D  <U CO CD 0) 1-1 CO  S  TJ  _>  2 <~  _c0  O  O  ^ &5  U  55  CO '  w  %—»  o  4 •— >  e  CO CO  3.1) in response to a short-term fasting. therefore,  The intestinal calcium transport capacity was,  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 m i n 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 i n calcium concentration was  probably caused by movement of body fluids into the intestinal lumen.  The movement of body fluids into the intestinal lumen w i l l impede net solute absorption (Madara, 1983; Reuss and Finn, 1977).  Peeters et al. (1994) have also noted a significant  decrease i n 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 i n almost complete absorption of the solutes.  In the preliminary  experiments i n the present research, the intestinal lumen was found to be partially collapsed  56  1 ©  "•8  C3  O C/D  u  a  et  03 C O  [WUI]  U0IJBJJU33U03 UIlipiBD jeuaumnUJUJ CO  57  s o S s  .ai, ?  3  g  a 3  w  2  g  l-i  (3  o  «  S3 o 33 o  t+-  -13  to  (-1  TO  *  S3  ^  «  o  O M T)  t« d 2 z «.a o  to  .2  c  _|_|  O  s 2  ?? «  cu  &,  C "3 & S O °  c  S  1  a  o  4->  DJD  ~  o  fa  3  — "C  o  U0IJBJJU9DU03 UinpiB3 IBU9UII1IBJ1UI  58  is  "o  1)  .a  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 m i n after an intravenous injection (Figure 3.7). diffusion  of  mannitol  into  This decrease may possibly be associated with a slow  extracellular  spaces,  or  a  degradation  within  the  body.  Pappenheimer (1990) and W i c k 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 i n 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) i n 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, responses.  whether it can affect intestinal calcium transport  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 i n 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 .la . ! = . ! = . ! = CQ CQ PQ p a •a  •  •  4  »  o  a  S3 © a C O  U P  ft t> 2 2 u u § 'a -* 1  C a ,  a  o o  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 cd » ci_, 1—1  * ° l l EL  > a M a a  «n DC  .a  S "M 'S  fa E ^  [JAJUI] UOI;BJ;U33UOD  lojiuireui  BUISBIJ  60  ca  •=1 ^  ca  8 .5  bp i-i  bD  .S3 v  O  W bp &0 3  ° g  a«b  bo 3 33 3 C D A o  s <*  3  bJ3 O  £ a, a H 3 _o ca % — » 3O 3^ Ux!• ca >  « o s  H  T3 |  3  jfcN  5 io cu cCOu T'3 > ?a o 3 g « ^ o —. ca £a o ^ - H cu cu > 5 3  ^  £ ^ 2  J-l  3 & §  P ca 9. o o •I-"  ca  CJ c3u c Ju > *-> 2 S o ca  1-1  3 3  J  3 -°  T5 »  ? 3 £».S  s •? C S « .  00  00  CU L-i H  • «  fa  U0IJBJJU93U03 UIIipre3-[Omo; BUISBld  61  O CU  OH  o  2  "S3 cu  u «  2 *8 £ £ s* 2 § S  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 i n specific radioactivity ( S . A . ) of a 100 m M to 20 min after the intralumenal injection (Figure 3.9).  4 5  C a C k test solution between 2  A n initial 5% decrease i n S . A .  occurred at 2 min after the injection, probably caused as a result of resident calcium i n the lumen of the intestine.  62  (N  O  a o u  0>  (N  «D  -  s  cu cu o o o  8  «/-) CN  8 o  CN  o o  in  o o  O  o o v~i  ( .Iouiri mdp) A":u>i:peoipej aiipadg T  63  E H  5 x>  CHAPTER 4 Regulation of Intestinal Calcium Transport in Rapidly Growing Young Broiler Cockerels by Vitamin D-Dependent Mechanisms  4.1. Introduction Vitamin D regulation o f 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 i n 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 o f 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 i n 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) D3 action in this case, the transcaltachia was considered to be a non2  genomic action o f 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 i n 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. F o r 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. O n 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 K i m b e r g , 1978).  The contribution of vitamin D-dependent  calcium transport i n different regions of the small intestine of rapidly growing broiler chicks is not known.  M o s t o f the existing information related to mechanisms o f 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). calcium metabolism in mature birds is different than i n mature mammals.  Also,  For example, these  birds can develop a temporary reservoir of calcium in the form of medullary bone in their long bones.  C a l c i u m is rapidly mobilized from the medullary bone to the developing egg shell.  Possibly some aspects of intestinal calcium transport i n rapidly growing broiler chickens may differ from those i n 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. describe  This is the first report to  these aspects of intestinal calcium transport i n 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 ( H C D ) (-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 o f calcium were used to supplement dietary calcium because o f their high solubility relative to calcium carbonate salts used as the primary source o f dietary calcium i n commercial diets. One consideration involved i n the preparation o f the H C D was that dietary calcium concentration does not become excessively high since concentrations approaching 2% may interfere with nutrient utilization i n broiler chickens (Shafey et a l . , 1990). The calcium and total-phosphorus concentrations ( % D M ) i n the diets were analytically determined (Table 4.1).  Table 4.1. Calcium and phosphorus concentrations in the experimental diets Diets  Calcium  Total phosphorus  (%DM)  (%DM)  Control diet  1.09%  0.82%  H i g h calcium diet  1.65%  0.75%  67  4.2.1.2. Duodenal Calcium Transport in Response to High Calcium Intake T w o 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 i n the duodenal lumen of birds fed the H C D .  The intralumenal test solution contained 75 m M each  of C a C h ( S . A . , 1.33 n C i pmol" ) and H - M a n n i t o l ( S . A . , 1.33 n C i pmol' ). A l l experiments 4 5  1  3  1  were terminated 10 m i n 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). T e n pens each containing 13 newly hatched chicks were used i n 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 i n 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  CaCh  ( S . A . , 1.33 n C i pmol" ) and H - M a n n i t o l ( S . A . , 1.33 n C i pmol" ). A l l experiments were 1  3  1  terminated 10 m i n after the intralumenal injection of the test solution. The details of the in situ loop experimental procedures and analytical techniques are described i n 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 . / k g wet weight) was used i n the control diet in this experiment.  The composition and calculated nutrient concentrations of the diets used in this  experiment are provided i n Table 4.2.  69  Table 4.2. Composition o f the rachitogenic and normal diets'  Ingredients  Inclusion (g/100g)  Ground yellow c o r n  55  2  Soybean meal (solvent extracted, dehulled, 48.5% protein) C o r n gluten meal (60% protein) Dicalcium phosphate Calcium carbonate  35 2  2  2  3  1.3  3  Iodized sodium chloride Canola o i l  2  3  0.24 4  4  Vitamin and mineral premix  0.4  5  '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, methionine plus cystine,  0.5%;  0.86%; vitamin A , 1500 I . U . / k g ; vitamin D 3 , (none i n the  rachitogenic diet, 2200 I . U . / k g i n control diet); vitamin E , 10 I . U . / k g ; 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 t d . , 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 o f development o f 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 i n these samples. Tibiae were collected and analyzed for ash and calcium concentration as described i n Section 3.3.  Based o n plasma total-calcium concentrations, birds became rachitic by 7 d o f age.  Gradually, the birds became severely rachitic between 12 to 16 d o f age. Since a major loss o f general well being o f chicks occurred after 16 d o f age, the following experiments involving rachitic chicks were conducted i n birds from 12 to 16 d o f 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 o f D r . Youngblut, and M s . Linda Jane.  This crystalline vitamin D  compound was dissolved i n a water: propylene glycol: ethanol (v:v:v) (5:4:1) mixture, as prescribed by the manufacturer.  The solution was made i n brown glass bottles under a  nitrogen environment and immediately loaded into 1 m L syringes with air excluded which were subsequently vacuum packed i n 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 o f calcium concentration. Tibiae were collected from chicks i n all experiments and tibia length and ash concentrations were determined. The tibia calcium concentration was not determined since it was noted i n 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 13d-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 C a C h ( S . A . , 1.33 n C i pmol" ) and H 4 5  Mannitol ( S . A . ,  1.33 n C i pmol' ). 1  1  The experiments were terminated 10 min after  intralumenal injection of the test solution.  3  the  A t termination o f the experiment, the intestinal  tissue was ashed to recover C a retained during the experimental procedures. 4 5  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 o f calcium transport reaches a peak after 6 h of a l,25-(OH)2D3 treatment.  T w o 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. therefore, were used as noninjected controls for this experiment.  The 12-d-old birds,  Due to the length of time  involved i n experimental preparations and procedures, it was logistically difficult to conduct the whole experiment on the same day.  Although, day-to-day variations i n 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 i n 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 C a C k ( S . A . , 1.33 n C i pmol" ) and H 4 5  1  3  Mannitol ( S . A . , 1.33 n C i pmol" ). Experiments were terminated 10 m i n after the intralumenal 1  injection o f the test solution, and intestinal tissue was ashed to recover experimental procedure.  4 5  C a retained during the  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.  T w o 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 i n 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 o f dietary calcium. Ten pens each containing six chicks were used i n this experiment. Five pens each were randomly allocated to rachitogenic or control diets from hatch.  The experiment began at 11 d of age.  recorded.  Feed consumption and body weight gain were  Total excreta from individual pens was collected on wax paper.  The excreta was  dried i n 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 o f high calcium intake on body weight, and plasma and excreta calcium concentrations i n 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 o f 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 o f age, the plasma total-calcium concentration o f  chicks fed the H C D was not significantly different from that o f chicks fed the control diet. A t 14, and 21 d o f age, however,  the plasma total-calcium concentration  was significantly  ( P < 0 . 0 5 ) less i n 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 o f 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 o f 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 o f chicks fed the H C D was significantly less compared with chicks fed the control diet ( P < 0 . 0 1 ) .  The average percentage reduction i n calcium transport i n  75  >n  o  O  NO  d  d  d  d  +l  +l  +l  o  .2 B  Q U X  ON CN  00  co  CN  o CD  I '-3  NO  o  8 V a.  o  CN  +1 ON  8  o V a.  CO  o  uo  o o o V a.  d  d  +i  +i  +i  +1  in  NO NO  o  r-H  IT)  CN  o d  o  q  d  d  d  +l  +i  +i  o  ON  r-~  CN  co  16  Q U  CO  II &,  CN  CN <*>  Qs  +1  o  m  >n ts.  c>  II a.  >o  m o o V a.  CN  O  <o q  d  d  d  +1  +1  +l  +1  o  co in  CO  m  CN  CN  co CO  o  q d  CN  ON  +1  o r--  +1  +1  r-~ oo CN  r—( T—H  00  NO  o d V ft.  CN O  CD  +-»  c  m o o V a.  NO  +1  '53  I'-3  o o o V a.  d  1—1  CD  NO  CN d  00  Q U  CO  vq  r-H +1 T—H  i5  ON +l  NO O i—i  +1 ON CN  CO  i—i  +1  o ON  CN  76  o I  u  73  O •  4  — I f_  a  *  TO  H:  r-- cCU u <N  —  —  © —  2  ca  a S  H  S 03  CU  *  23 (=1  CO  <D 03  CU 03  S3 CU  ^ si 43 5  co  «  a  CU  •§ 2 a > (mm ox « ! asop jo %) 93UBJB3ddesip lojmueiu pm? uinpre^  77  o Q  eg  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 o f 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 o f in situ duodenal loop preparations was significantly less than that of calcium transport ( P < 0 . 0 5 ) , 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 o f 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 i n calcium transport i n 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 i n 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 o f chicks fed the control diet, after the cross over (results not shown).  78  0> s s  3  ^  00  o  CO  13 c  cu  U  ro tti  5  O  P  •a a  2 < B 00  (uira o i ui asop JO %) 33UBJBaddBsip JOJIUUBUI pire uinpir>3  g  79  u  o  ^  4.3.1.3. Ileal Calcium Transport in Response to High Calcium Intake Figure 4.3 shows the effect o f high calcium intake on calcium and mannitol transport from the lumen o f in situ ileal loop preparations i n 4-, 7-, and 14-d-old chicks. Calcium and mannitol transport are described as the percentage loss o f 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 o f chicks fed the H C D was not significantly different compared with calcium transport i n ileal loops i n chicks o f the same age fed the control diet. A t all ages under investigation, mannitol transport from the lumen o f in situ ileal preparations in chicks fed the H C D was also not significantly different compared with that i n chicks o f 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 o f the rachitogenic diet o n body weight, and plasma and tibia calcium concentrations i n 7-, 12-, and 21-d-old broiler cockerels.  A t 7 d o f age, body  weights o f chicks fed the rachitogenic and control diets were not different; however at 12, and 21 d o f age, body weights were significantly (P < 0.01) lower i n birds fed the rachitogenic diet.  In chicks fed the control diet, there was an almost 100% increase i n body weight from  12 to 21 d o f age. In contrast, during the same time period, there was only a 41 % increase i n body weight o f chicks fed the rachitogenic diet.  80  3  2 .= 2 u o u  ox  I  +1  u  a  2  2 u  4  CD 1 1  2 no  © M  C8  O • i  «  ^  (UIUI ox ui asop jo %) 33UBJB3ddBSip JOJIUUBUI pUB Ultipp33  81  o  ° CT3  H  CD <U  .ca  in —. i  00  75  +l  r-~1  +1  +l CN ON  >—i  o  in  o  oo CN  o  CN CN  o  q  oco  d  d  ON  ON  o  +1  +l  r~-  G  d  CN  00  d  u  d  r-  d  diet  73, ~o  d+l  00  CN  P<  S B 2 "J3 ?^  _>  oo  p<  +-* -i—<  bn  P<  cu  Rachito nic die  1-1  CD  CO  CN CN  o  oo O  oo o  +l  +1  i—i  o '3  >n  CD  Rac  O CD .Si '-rt  d  co CN  in O  u  d  § V ft.  d  75 -t—' t-l eg  d  +i  o  q o 00  o  V  ft.  d  o  O  CN  •st  O  o ON  O  V ft.  o  CN CO  ON  co  q  +1  ,—i  u  o  d  +l  +1  o oCN  achit ;en:  o  O  .5? '53  <u  +1  +1  CN  CN CN CN  — r «•> <N  ontr  75  +1  o CO  82  o  o o  ft.  ft.  V  t+1 co in  CN  U r--  CO  q  II  a.  CD  +1  in Tl  C>  6-0  O CN  CN  V  ON T—(  +1 co O  in  CN  A t all ages under investigation, the plasma total-calcium concentration was significantly less (P< 0.001) i n 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 o f age.  Based on the  differences i n 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) i n chicks fed the  rachitogenic diet gradually and continuously decreased from 7 to 21 d o f age (P< 0.001). A t all ages under investigation, the tibia calcium concentration was significantly less i n birds fed the rachitogenic diet than i n birds fed the control diet.  Tibia calcium concentration at 7 d o f  age was not significantly different from tibia calcium concentration at 21 d o f age i n 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 o f 12-, 13-, 15-, and 16-d-old rachitic and control chicks. A l l parameters had lower values i n rachitic chicks compared with control chicks o f the same age. A t all ages under  83  +1  NO  00  +1  <N  0.29  in  NO  in  0.018  0.020  +i o  +l r-  +i in  d  d  0.07  o  P<i  00  00  d  00  oo  T-H  o  +l  +l  d  d  +l  T-H  o  0.408  +i  d  0.393  Co  *3  d  o  0.301  >  .SH  o  CN O  0.307  rol  T-H  d  p<\  oo  II  in  +i co  P<<  •S ,29 a ccs  Tibi  CO  T-H  wei  bfl  04  CN CN  0.019  -t—»  a.  0.017  diet  •3 o u CN in  in  P<|0.01  II  a.  in  ± 1.2  CO  m  0.04  CN  P<  oo  ± 1.2  2H  +1  0.98  — (- »  T-H  +1  1  ± 1.3  ontrol  Tibi  es G  T-H  0.02  oi  i  T-H  P<  bb  CO  T-H  ± 1.2  •3  co  r-H  CO  o  o  q  o  d  +l  d  d  +l  d  Control  Body ^ 3  TH  +1  CN  T-H  +1  ON CN  NO ON CN  CN  CO  T-H  T-H  ON  84  V a,  CN CN T-H  +1  T-H  CN  0.01  0.810 +1 in  p<  P<  1.137 ±  V a.  T-H  +1  CN  P<  CN  +1  T-H  0.01  CN CN  ^ 'So  '•3  0.756 1.097 ± CN  T-H  +1  — t- »  0.01  co 00  04  P<  d  CN  TOO  '53  o  d  TOO  43 W>  o  d  0.864 ±  H  ro  d  0.01  /-\  •<* o  +1  0.847 ±  .3  diet  C+H  Cont rol diet  U Of) el) w  0.702  0.620  04  +1 100  dry wei  bfl  p<  >  0.01  ON  CN  V a.  +1 T-H  CO  CO  m  NO  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  Control  Chicks  Total-Calcium  Concentration  and Hematocrit  Values  In  Rachitic  and  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  a  '3  CD W) _ O CD  o cd  NO  d  rd  +i  +l  +!  ON  CN  q  CO CO  J-l  CO  •-I C5  1*1  co  co O  oo  a.  V  a. 3 -3  CN  co  CN  V  a.  d +1  +i  •<* NO  d  U  +1  d  +1  +l  d  >n co  V  d  d  o  oo  d  ON  CO  o  o d  CD O  >n  NO  '3 CD  o d  +i  +l  >n  oo  O  o d  +1  +i  o  oo  NO  o d  r-  ca  V NO  O  o 3! ;3 O  U  ^  d +i o ON  CN  CN  86  ft,  q d +l  V a.  NO  o d  +l  V a.  in  o d  +i  o  o  co oo  CN  CN  CN  i-O  NO  V a.  chicks fed the rachitogenic diet compared to chicks fed the control diet ( P < 0 . 0 5 ) .  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 i n 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 . 0 5 ) .  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 . 0 0 1 ) ; surprisingly however i n control chicks, the plasma calcium concentration slightly but significantly decreased 4% below the pre-injection value.  87  ca O  GO  -T->  O  CO  •  in ca J3 G ca JH CN«• ca <*-> 2 g S3 o T3 O cG S3 - <u hi GO  •EgH c ca  3$ 2 - - o |> SP-£ || 2 ®  CA  IS C M  O  & •> H  in  -S  s  (11  8 s "  XJ  (SB  '—^ «-> r-H f i ,  s >  cu >H CO  <D O <u  S  °-9 §  O ea  ° f"  TS  JH  "2 * T3  1—1  'co  o <p  2 O  n c ,"3 s  o o  T)  2  •a's ca  a  3  o  ca I<C in c a  2  o  (90  a  v CL  00  O-  «"  TH  C+H J H  -a  •-< — ca ^o fa O T - H  CU  (U  ao  •sic ™Scca -Sa c  ft-a  "  <4H  •H  CU IH  CO  <u ca  88  -a  CO  •S S G CO  <4-H O  M  I  S CD  1  £ SP  ca  CU  -SP  O to OX) <*> ca <*- «  3 3 -2  H  >H  a  .•-i  a § -a _a a o vo 7 N G -J3 ^ " — '  2  .CD  TH  6 -S TS  III  a  P  * -^ 2ca &  .4>  [j\[iu] uinp[BD-iB;o; euiss]j  NO,  s *? i  ^  CO  O X5  r §  ?  § £ |  O i  < £  ft-  O  o tS *s > c«s u  4.3.2.5. Duodenal Calcium Transport in Rachitic and Control Chicks With or Without 1,25(OH) D3 Treatment 2  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  lumen o f in situ duodenal  loop  preparations in 12-, and 13-d-old unfasted rachitic or control chicks, with or without  1,25-  (OH)2D3 treatment.  shows  calcium transport from the  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 i n control chicks. A t 13 d of age, the rachitic as well as the control chicks were treated with l , 2 5 - ( O H ) 2 D 3 .  The difference between the rachitic and  control chicks i n terms o f 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 i n 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 , 2 5 - ( O H ) 2 D 3 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 . 0 5 ) 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 C3  h^  ^  ^  ^  ^  ^  a a ca  o ? ID  X) |  ca U  cu I  l  l  s a cu s s  S  ca  ca O  ts  (UIUI oi  (v|  ui asop jo  —  -H  %)  33UBJB3ddBSip [OJIUUEUI piIB I i m p i B 3 i m  .5  g  > 32 q, C X o • cd cu co 90  .3  control chicks.  In 13-d-old unfasted rachitic chicks that were treated with l , 2 5 - ( O H ) 2 D 3 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 , 2 5 - ( O H ) 2 D 3 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, i n 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, i n 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  co  s  ©  c cu  s  13  cd ca U  ON  (mm oi ui asop jo %) 33UBJB3ddBSip (OJIUUBIU piIB U i n p j B 3  92  4.3.2.7. Calcium Retention by Duodenal and Ileal Loop Tissues in Rachitic and Control Chicks With or Without l,25-(OH) D Treatment 2  3  Tissue calcium retention was defined as percentage o f calcium injected into the loop lumen that was retained by the tissue at 10 m i n after injection.  Figure 4.7 shows calcium  retention by duodenal and ileal loop tissues i n 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 , 2 5 - ( O H ) 2 D 3 -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 o f intake) by intact chicks are shown by Figure 4.8. The feed intake (g bird" 24 h" ) was 29% less i n rachitic than i n control chicks ( P < 0 . 0 0 1 ) . 1  1  The body  weight gain (g bird" 24 h" ), during the experiment period was 40% less i n rachitic than in the 1  1  control chicks ( P < 0.001).  Feed utilization was, therefore, adversely affected i n 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  <n  CU  4^xxxxxxxx^  3  CA CA  o  V©  H  a o o  ^  ca  ^  ^  ^  IT)  CU  1  1  1  1  1  3 M  o +3  3  1? cu  CU  3  cn a o  73 e cu © 3  o  2  >n  (uiiu 01 ;B 3S0p JO %) U0IJU3J3J umpn?3 o  94  o JS  O  -H  CO  3 •r; is  B  ^  cu cu c u > =• £  £  u  C  £  ~ 2 .3 S cu x> .3 Q  fi  B  n. u ©  ^3  U "S  B -  B  cu .a 3 Q 60 60  o  u  X) (95[Bjui  jo %) uopdjosqe  OA  •SP "5  "  c£ B  ^  cu  cu '53  CD  .B  1-1  2  ca  •B >  <4-^  &  •  3  CD *4-l CD CD CO  CD  o •W  95  a  OH  ca  +1 cu  -B-  ca CD  B  s3 ^O  CU  '•o3 o o  H B  t-H  CD  60 co ca  M  cw  o  o  3  CN i—i c3  ftl) co>—i fa •§  173 -  c3  ^ CO  ^  53 in  T3 « 3 "3  B  cu  1-1  iJ 3 «  OH  4-1 CU  o  C 'B 60 lo  g cu .2 <u I-H "3 ^  CD  CO C D , " 00  rr .2 T3 ^  B  rt  J-H  B  fa  P  2 p  ^  .•a B -i->  B -° *3 -»-> B °  ( l - W Z T-PJm §) W  S3  "2  3  CD  Q  3  3 cN ^ cu  7 3  •c JS .3 .> c^u > B  T3  cu cu  O O  r-; >, O § g> c00u Jo •a a -g . oB S3  CD  CO  S  o  ca  1  B  IS  cu  y  3  |  rt  CD  O  CU  co  cu -  ~  CU  -B  2 $ 8 S •§  ca S Ta O B ^  UiniOIBD }3JsI  7! .B  8  CU - B CD  „  CU  >  C/3  es  £ 5i  a .a CU  -Q  .22 o CO V  .a $ CD  H  CD g 60  '•a ^  CD ca ° ^ 60 - ° O  CU 1  3  >-  cU  3 ca 1-H  2 JB  OH  Calcium intake during the course of the experiment (g kg" body weight) was 1.56 ± 1  0.05 g k g ' i n chicks fed the rachitic diet, compared with 2.01 ± 0.05 g k g ' i n chicks fed 1  control diet.  1  Net calcium excretion (g kg" body weight) during the course of the experiment 1  was (1.19 ± 0.05 g kg" ) in chicks fed the rachitic diet compared with 0.79 ± 0.05 g kg" in 1  chicks fed control diet.  1  A s a percentage of intake, the net absorption of dietary calcium in  intact rachitic chicks was 2.5 fold less than that observed i n 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 i n the chick small intestine.  The two pathways were  clearly and consistently separable in the duodenum i n 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 o f the vitamin D-dependent component o f intestinal calcium transport has not yet been clearly delineated i n the broiler chick.  The fractional contribution of the vitamin D-dependent component of calcium transport calculated under rachitic conditions (38%), and after l , 2 5 - ( O H ) 2 D 3 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 , 2 5 - ( O H ) 2 D 3 treatment.  After treating 13-d-old control chicks with 1,25-  (OH)2D3, there was a 16% increase (overstimulation) i n calcium transport, over that of the 12d-old control chicks not treated with l , 2 5 - ( O H ) 2 D 3 ; 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 i n 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) i n 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 i n the ileum has been questioned i n a number o f 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 i n 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 o f 10 °C.  Moreover, a low calcium intake was found to stimulate calcium  transport i n the ileum, thereby further supporting the presence of a cellular calcium transport component i n 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 , 2 5 ( O H ) 2 D 3 treatment of rats induced a significant increase i n 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).  F o r example,  Karbach and Feldmeier (1993) reported that the saturable component in the rat cecum contributes nearly half of the total calcium transport.  A l s o , Nellans and Goldsmith (1981)  reported that calcium transport i n 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 i n 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 i n vitamin D-sufficient chicks than in the rachitic chicks. M o r e recently, Takito et al. (1992) were able to stimulate calcium transport i n 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, i n 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 i n 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 i n response to l , 2 5 - ( O H ) 2 D 3 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 i n 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 i n the control chicks was absorbed in a vitamin D-dependent manner.  A  discrepancy i n the outcome, i n terms of the fractional contribution of the vitamin D-dependent mechanisms i n 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 i n 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 i n 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 2 9 % . 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 i n 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 i n 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 i n rats fed the milk sugar lactose (Pansu et a l . , 1981) and phosphopeptides derived from, casein (Kitts et a l . , 1992). inclusion of casein phosphopeptides  Mykkanen and Wasserman (1980) reported that  in intralumenal test solutions resulted i n a significant  increase i n 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 i n 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  transcription o f collagen type II in chick limb bud mesenchymal cells.  stimulates  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 i n the present study does not seem to have been seriously decreased by vitamin D deficiency.  A moderate increase in hematocrit values i n 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 i n 4-d-old broiler cockerels was significantly decreased in response to high calcium intake, the vitamin D-dependent component of calcium transport i n this species appears to exist at least by day 4 o f age.  Pansu et al.  (1983a) and Dostal and Toverud (1984) reported that the vitamin D-dependent component i n 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 o f 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 o f 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 i n the present research appears to  provide a valid estimate o f the relative contributions o f the cellular, putatively vitamin D dependent, and the paracellular, putatively vitamin D-independent components o f 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 i n 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 i n 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 i n 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 i n in situ duodenal loops ranges from 15 to 25% of the total. The vitamin D dependent mechanisms o f calcium transport are present at 4 d of age i n rapidly growing young broiler cockerels.  106  CHAPTER 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 i n 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 i n 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 i n in situ loop experimental model also exists i n 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  T o 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 l o w , 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) i n the duodenal mucosa decreases with an increase i n net calcium absorption, they concluded that the fractional contribution of the saturable (cellular) component of calcium transport was diminished in rats fed calcium i n 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 o f 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 i n part co-determined by calcium solubility and intestinal sojourn time.  L o n g 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 i n rats by either providing C a C h as liquid dose via stomach tube, or by providing the same dose mixed i n 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 i n the diet, indicating that calcium solubility enhances absorption. However, a  108  proper interpretation of their data i n terms of a relationship between solubility and absorption is difficult since the dose mixed i n the diet supplied 38 mg of calcium whereas the liquid dose supplied only 6.6 m g .  The experiments reported i n 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 i n 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 i n 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 i n the in situ intestinal loop preparations.  A l s o , the physiological relevance o f 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 i n in situ duodenal loop preparations i n 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 p m o l ) , 100 (1 n C i pmol" ), 150 (0.75 n C i pmol" ), 200 (0.5 n C i 1  1  1  pmol" ), 250 (0.41 n C i pmol" ), or 300 (0.33 n C i pmol" ) m M H-mannitol. The solutions were 1  1  1  made iso-osmolar with N a C l , where necessary. treatment.  3  Four or five birds were used in each  A t the termination of the 10 m i n 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 i n 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" ) (n=14), 1  or 100 m M (1 n C i pmol" ) C a C h (n=24). 1  with N a C l .  4 5  The 2.5 m M C a C h solution was made iso-osmolar  A t the termination o f each 10 m i n experiment, the duodenal loops were removed,  ashed at 600°C for 8 h, and the amounts of determined.  4 5  C a i n the lumen+tissue were individually  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 i n 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 ( H C D ) 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. period.  Alternately, a 40 m M C a C h solution was provided during the experimental  A preliminary trial indicated that water intake significantly decreases when the  calcium concentration i n drinking water is increased to more than 40 m M C a C h .  The calcium  concentration i n 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 i n birds at 4, 7, and 14 d o f 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  ill  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 o f 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. control diet from hatch throughout the experiment.  These birds were fed the  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" 10 min" ) from in situ 1  1  duodenal loop preparations i n 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, i n 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 o f 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 i n in situ duodenal loop preparations i n 21-d-old chicks containing 2.5 m M or 100 m M intralumenal calcium. The results indicate that intestinal calcium transport i n in situ duodenal loop preparations is a saturable phenomenon, i n contrast to intestinal mannitol transport.  The amount of calcium disappeared (pmol g" 10 min" ) 1  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 i n the concentration. In other  113  140 120  H  y = -1.29  0.19x  +  0d  100 80 60 40 20 0  T3  cu Ui cs cu  0  50  100  150  200  250  300  350  a  03  2®  7  4d  S o  S o  ° i c 3  O  i — i — r 0  50  100  150  200  250  300  350  21 d  i 0  50  100  i 150  r 200  250  300  350  Intralumenal Mannitol [mM] Figure 5.1. Mannitol disappearance (Mean + S.E) from in situ duodenal loop preparations i n 0-, 4-, and 21-d-old broiler cockerels (n=4 or 5), as a function of intralumenal mannitol concentration.  114  TS rH  rN  i  r  >o  V  co  crj  -=  T3  =  ^ ^ ^ ^ ^ ^ ^  E =  T  cu eg u  cu "t3  8  o  H^\\\\\\\\\\W  (UIUI 01 UI 3S0p JO %) 3DUBJB3ddBSip lojiuuepv  115  1=1 CD CD l-< CD  O  •t—»  .a  OH  a  03  i-H CD  O  fci r. CD  £ ^ £ •o +, o  i  o3 03 CD  o  O O  3  ii a  CD  C eS _ c ca c3 «  a  03  C  u »  CD O  ( m u i oi  i n a s o p j o %)  pajcaddesip uinpieo jiraojaj  s 03  13 cs  E 13  «  OH ™ OH -  ca  * l  8 CD of) rg .-78 3 o  T  SH  — S ° o a. ^ 1  3  - M - °  ^  O  CD  CD  1  O  V  53  ft £ o3  CD  2 .a a  "o £ co  u  „  CD  co s S 03  CD +-» - O CD 4-H  >  s .s  X! O  3  a  •a < -a  <^  s  C3 3 CD 03 O co « O ca T3 OH £03  ,  O  CD  OH  W  §  2 • >^  1 =§o ^*  § a  3 Tci o c ^-2? U - 5 .CD 3 cd T3 CD § cs o ?S r-" <4-H 3 CD w ,3 h O 13 C .D  x  Z P  •8 . 2 2 7D a -a g "53 .2? w  ^  •§  T  ^  to  u  ( .UIUI oi  <N  - § loiurl)  pajBaddesip uinpieo jo j u n o u i y  116  a  words, the amount o f calcium absorbed from in situ duodenal loop preparations did not increase as a linear function o f concentration. Unlike mannitol, the percentage o f calcium that disappeared from the in situ duodenal loops (% o f dose i n 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 i n 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 i n 4-, 7-, and 14-d-old birds. A t 4, and 14 d o f age, there was no significant difference in feed intake (g bird" 24h ) between birds 1  -1  fed the H C D and those fed the control diet. A t 7 d of age, however, feed intake i n birds fed the H C D was significantly (P < 0.05) less compared with the intake i n controls birds.  Figure 5.5 shows the effects o f drinking 40 m M C a C h on feed and water intakes. In 4and 7-d-old birds, there were no significant differences i n feed intake (g bird" 24h"') between 1  birds consuming 40 m M C a C h i n drinking water and the controls which were given distilled water.  However, i n 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 14d-old birds, there were no significant differences i n water intake ( m L bird" 24h"') between 1  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  Q CD  <  m a t :  CU  ca  '•3  CO  CO  CU  P-i  CU OS  .Xr cu cu i2 -C cu  h Os 1  VH  C D  1)  °  3  " 8 cu  CD  ca  118  CD  CO  x) -o cd  CN cd  13  OA  3 5  cd  5 o rx3 u^ CO  cd d  1)  B  on  +1 CN  in  119  O  >*  O S ?  ^5  q  Table 5.1 shows the amounts o f calcium consumed and calcium absorbed (g kg" 24 h" ) 1  1  by 4-,7-, and 14-d-old birds i n which calcium intake was increased by either feeding the H C D or by provided 40 m M C a C k i n drinking water  In birds fed the H C D , calcium intake  significantly ( P < 0 . 0 1 ) exceeded controls by 48 to 6 0 % .  While birds which drank 40 m M  C a C h , consumed 31 to 5 3 % more calcium than control birds ( P < 0 . 0 1 ) . The amount o f 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 . 0 1 ) .  A s may be expected,  therefore, the amount of calcium absorbed increased with an increase i n 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 o f calcium retained (percent o f 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 o f age.  A t all ages  under investigation, the percentage o f dietary calcium retained by birds fed the H C D was 16 to 22% less compared to birds fed the control diet ( P < 0 . 0 1 ) .  In contrast, the percentage o f  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 i n drinking water, since percent calcium retention did not differ with intake.  120  co o d  .3 |o "ca o 43 00  o d  +l  +1  o d  rm  CO 00  +i sO CN  8 CN CD  o  o O  a o  V OH  +1  V  CN  o d  V ft.  +1  +l  SO  CN o  « 43 CD CU  O d  OS  CN  m o d  d  +1  +l  +l  00  as  ca  o  o  CO  >  TH  3 £L  oo  „  1)  CN  _ cu <A 3  o d a o  3 8 ca  U  3  e 3 "ca o  T3  CU  oo  ftS  e 3 os c o U  is U .2 ^  "c3 bO  CU  cu '> o  ca o CO  co ON  CO  sO  V  +l  +1  121  +l  o CN  v a,  +l  in o d  •  +1  +l  o  Os CO CO  o d  +1 CN CN  8  CN  d  CN CN  ro d 3 O CJ  T-H  sO  o d  +l  "ca o  CO  V  co o d  co  coj  rCN CN  co §  co o d  s  +1  +1  co  ft.  ft.  O d  O d  +l  o o d V  +i  +i  o d  CN  O d  in o  _3  »  ca c o O "Ho  o d  o\ >n CN  43 00  -2 <2 ft 73 CU  +i  V a,  T-H  co co CN  73  ca -4—» T3  V a.  ros  O d  u la  i  o  CN  43  T3 u '> ca O U  .3 'oo 5  1—I  43  ft ^3 §  CO  p< 0.00  T3  CD  p< 0.00  u  O  rCN CN O d +l  CN 00  P< 0.00  CN  •= c  cs  o co 2 *-* -i< 3 SB " "S 2  3  ^-N  1  t; f! 3 M  O & T3 a, ,  C  cd  I -  "2  H  CD — c  '  cd » OH -3 H Cd © -5 3 -g  43  Tt  co jg cd W co > a oo co ex ^ 3 xi  :  +1 *3 .£3 3 « .3 CO IS jo cd ^ OH "o 33 u c S CO  QJ  ^ w  OX  cs  CD  3 o '3 co •3 £ B 5 » '3 CO  w  C  2  '•5 2 *>  "3 u B « to  i>  S  Q. <u  re  a. os s S o E u  _3  J3  a  in CS  'o  c^ aj O, co 3 O | < _g *co o CD  E 3  a  S ° I H  2 J3 ° ' O ft £H .3 CO  T3  a>  l-i  co S S  re H  8 £ .S  ^ ^ ^ ^ ^  •°  Tab  „ 3 4-H to 3 K « o to» O r  H  1  1  1  1  1  1  iioiju9j3.i mnpn?3  <£j  CO  33  CO  CO  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  122  5.4. Discussion The present results are consistent with those presented by Krugliak et al. (1994) showing that intestinal mannitol transport i n 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. the extracellular space.  Mannitol is generally considered to be a molecule restricted to  W i c k 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 o f polyethylene glycol ( P E G , a marker of paracellular transport) oligomers i n 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 i n 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 D a , Pappenheimer and Reiss, 1987) and P E G - 2 0 0 0 (2000 D a ; 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" 24 1  h" ) decreased i n the range of approximately 17 to 22% of controls, while calcium intake (g kg" 1  24 h" ) increased 48 to 60 percent of controls. 1  response to consuming the H C D .  1  Thus a net calcium retention increased in  A l s o , calcium retention in these intact birds appears to be a  saturable process since percent calcium retention i n birds fed the H C D would not be different from controls, i f the retention was a nonsaturable process.  O n 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 i n control birds. A l s o , the net calcium balance achieved i n 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 i n 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 i n 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 o f calcium carbonate is nearly 4 m m o l / L (Washburn, 1928), whereas the solubility of calcium gluconate is nearly 118 m m o l / L (Dean, 1979). In comparison, C a C h is many fold more soluble than gluconate or lactate salts.  T o 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 i n intralumenal test solution is routinely provided as the chloride salt.  It was  concluded i n 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 i n the in situ duodenal loops of broiler cockerels was small. Moreover, the distal part o f the small intestine i n chicks was nearly devoid o f the saturable component, a finding confirmed i n other animal species by other researchers (Behar and Kerstein, 1976; Pansu et al., 1983b; Y u a n and Kitts, 1992; Boass and Toverud, 1996).  In the present study, when  intact birds were provided calcium i n the form of a highly soluble salt i n 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 i n the distal small intestine of chickens where a relatively high p H o f 7.96 (Coates and Holdsworth, 1961) favors calcium precipitation under normal conditions (Allen, 1982).  The  bioavailability o f 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 a l . (1993), " i f a rat intestine is capable, when studied as a ligated loop in situ, of readily absorbing calcium concentrations of 100-150 m m o l / 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  126  of calcium transport is down  regulated with increased calcium absorption (Pansu et a l . , 1981; Boass and Toverud, 1996). It is possible that the down regulation o f the saturable component was relatively complete i n birds which drank the calcium solution, since they retained more calcium (g kg" 24 h" ) compared to 1  1  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 i n 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 a l . (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 i n the caudal direction (Duflos, et a l . , 1995),  some calcium may re-precipitate i n 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 precipitate (Moore and Verine, 1985).  will  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 i n 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 i n 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 s o , 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 ( C P P ) may stimulate intestinal calcium transport in both normal and rachitic chicks. Y u a n and Kitts (1994) showed that calcium absorption and bone utilization in spontaneously hypersensitive rats was decreased in rats fed heat-damaged casein, i n which the C P P production becomes limited.  128  Gerber and  Jost (1986) demonstrated that C P P inhibit the formation complexes in vitro  of insoluble calcium phosphate  indicating that the effect of C P P on stimulating  intestinal  absorption may be caused by an increased solubilization of dietary calcium.  calcium  Provision of  dietary calcium i n the form of oyster shell i n 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.  O n 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  CHAPTER 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 o f 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 v i a cellular or paracellular pathways. Absorption v i a 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 o f 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 i n 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.  F o r example, Pappenheimer (1990) reported that 50 to 65% of  creatinine ingested by rats is recovered from urine compared with 75 to 85% recovered i n urine after intraperitoneal or subcutaneous  injection.  131  These results demonstrate  a high  paracellular permeability of the intestine to this molecule which is restricted to the extracellular space. Pappenheimer and V o l p p (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 o f the author to explore the role of the paracellular pathway i n 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 i n diseases affecting different regions o f the intestine. Abnormal permeability of various regions of the intestine may also be important i n the pathogenesis and pathophysiology of various diseases.  F o r example, intestinal permeability invariably increases  in Crohn's disease i n 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 i n 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 i n different regions of the small intestine o f rapidly growing broiler chickens varies with age.  T o the author's  knowledge, there are no published reports in the literature available on this subject.  132  The experiments described i n this chapter were conducted with the objectives: 1) to determine the age-related changes  i n 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 o f mannitol from in situ loop preparations o f the duodenum (n = 10 to 30), distal jejunum (n = 10 to 13), and distal ileum (n = 13 to 23) was determined i n broiler cockerels at 0, 2, 4, 7, and 14 d o f age. The birds were fasted for 12 h before each experiment.  The intralumenal test solution contained 300 m M H-mannitol ( S . A . , 0.33 n C i 3  pmol" ) which is iso-osmolar. In some o f the experiments, simultaneous determinations were 1  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 m i n postinjection, the loops were removed, i n the same order i n which they were injected. The contents o f the loops were removed by flushing with excess quantities o f 300 m M mannitol to ensure maximum recovery o f 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 i n the growth o f the intestinal tissue. The details o f in situ loop preparation and analytical procedures are described i n 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 i n 18-d-old broiler cockerels. The birds were fasted 12 h before each experiment.  Mannitol secretion was concomitantly determined i n in situ duodenal (n =  20) and distal jejunal (n = 11) loop preparations, or i n 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). 3  A 300 m M  H-mannitol solution o f high specific radioactivity (3.3 n C i pmol" ) was injected into the wing 1  vein (1.5 m L kg" ). Intravenous injection was completed within 3 to 4 sec which marked the 1  start of the experiment.  A blood sample was collected at 9-10 m i n v i a intracardiac puncture.  The loops were removed from the body at approximately 10 m i n and their contents collected by flushing with an excess volume o f 300 m M mannitol solution to maximize H-mannitol 3  recovery.  Plasma was separated from the blood and the plasma H-mannitol content was 3  determined by liquid scintillation counting procedures.  Calculations o f the plasma H-mannitol 3  concentration and the amount o f mannitol secreted into the intestinal lumen was based on the specific radioactivity of the intravenously injected H-mannitol solution. The emptied loops 3  were longitudinally opened, blotted dry with wiping papers and their weight and length was recorded. The details o f the in situ loop preparation and analytical procedures are described i n 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) o f the duodenal, distal jejunal, and distal ileal loop tissues from 0 to 14 d of age are provided i n Figure 6.1. The data for jejunal weights at 0, and 2 d o f 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 o f the duodenum was significantly greater than that of the distal jejunum or distal ileum. A t any given age, the relative weight o f the distal jejunal loop tissue was similar to the relative weight of the ileal tissue.  Absorption o f mannitol from the lumen o f 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 o f paracellular absorption is defined as the amount o f mannitol absorbed i n 10 m i n (pmol cm" 10 min" ) which takes into 1  account the length o f the intestinal loop.  1  The efficiency o f mannitol absorption i n 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  •a S» O co  o  4  «  CN  G CU J—i  G  •3 O  43  JH ,*->  o  3 T3  CL)  ^  CO CN O  T3  ca G  -s ~.SP °  V  CO  CU W G T 3 oo ca +1 | PQ G cu ca rG .•33 43 cu w  DX)  <  S3  2  —' co  s  53 ca c ca  T.  g  •a -a^ 00 CO  G  -a S «  <U  ^ 9  y CH  •*-H W / CO  ,ca  co  <4-H  CU  o cu ca o T-H  •B II  C > CU -G. o o o  -G  3 2 G o 14D  CU  T-l JTH  '-3  ^ G  •1—»  a» -si, T3 ca VT— 3 ca T3 G WD to  h  (UID/S)  137  i  t j  H -0"  DO  The efficiency of mannitol absorption i n duodenal, distal jejunal, and distal ileal loop preparations i n 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 . 0 1 ) .  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 i n distal ileal loops ( P < 0 . 0 1 ) .  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 o f 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" 10 min" ) 1  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 . 0 1 ) 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  3  CO  O  B a  ft  cd  3 cd cd a 3 •S cu o 7  cu OS *-<  cu .3  CO  5  o CO CN  3 +->  cu  CO  a  II  X)  o  s  C/3  3 a a •3?  -H  cd cu  CC3  - M  VI  co  3 U 3  O  CO CO ft  a CU  cd _cu  2i  .3 2 cu H 'S 2 a CU  Hpl  O  -t—<  cd  v  ft - S 3-1 3 ^ cd T3 - cu •—s -•—  O  x>  "cS  7 3  c^ 55  OX)  3  cd  2  1  5  ^  6 <= s  3" 73  +1 .23  3  3  . .  co cu CU  >  ft  1  § ^ co 3 0  Jd  a  a -3 'a .3° S 8 'cd 'co *a ft 3 cu II  3  3  s  cd  a .a O 3 ft "cd 2 co O 5 cu o -3 O cu 3 co 3 ^< ^ cu cu cu O ^ ft — cu ' 0 CO Cj-I o S g IS +-> cd cu -3 cu 2 a3 +2 cd £ H 3 M  -ft  7 3  (mm oi ui asop jo ssoj %) uopdaosqe  io}ium?pv[  139  fN  cd  SO  cu  cu SH  3 CUD  3  T3 O  C  Ccd U  X!  .3-  CN  3  00  co ? 73  II  +1  CU DA  C CN  t*>  ft  • J3 Safe  ccj  ( .U1UI 01 x-UI3 IOUIUI) T  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" 10 min" ) was plotted against plasma 1  1  mannitol concentration, a direct relationship existed between the concentration and the amount secreted i n a l l three regions of the small intestine (Figure 6.4). The coefficient of correlation between these two parameters was 0.72 i n the duodenum, 0.59 i n the distal jejunum, and 0.58 in the distal ileum, and a l l correlations were significant ( P < 0 . 0 5 ) .  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 H-mannitol 3  concentration varied between individual birds, it was decided to normalize the amount o f mannitol secreted for plasma mannitol concentration.  The amount o f mannitol secreted from plasma into the in situ intestinal preparations is, therefore, expressed as pmol m M " cm" 10 min" i n Figure 6.5. This figure shows that the 1  1  1  amount o f mannitol secreted into the duodenal loop preparations was not significantly different from the amount secreted into the distal ileal loop preparations.  However, the amount o f  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  Duodenum  i  i  i  0.0 0.3 0.6 0.9  T3 c-> u -< cu Ji  r 1.2  1.5  1.  ^  Distal Jejunum  ».'s U in  3  a  1 i C M  —  O  O  a  |  o  i  i  0.0 0.3 0.6 0.9  i  r  1.2  1.5  1.  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 i n 18-d-old broiler cockerels fasted for 12 h. F o r these determinations, a 300 m M H-mannitol solution (3.3 n C i p m o l ) @ 1.5 m L k g was injected into the wing vein. Determinations in duodenal loops were made concomitant with determinations i n either jejunal or ileal loops. 3  1  142  1  en  a o  ca 2  • mm  DX)  cy u  13  a  ^-1  ca ca  *-M  CU  a 3 s  T3  (j-Uiui oi  r-Ui3 ^ l o i u r l 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 i n the distal jejunal loops during this period, whereas the efficiency remained constant i n the distal ileal loops during 0 to 14 o f 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 i n terms o f paracellular permeability.  The suggestion that the small intestine of very young broiler cockerels is functionally mature i n terms of paracellular transport is consistent with observations reported i n 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 i n 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 i n 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 i n 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, i n 4- to 14-d-old chicks, the efficiency of mannitol absorption i n the distal jejunum was not significantly different from the efficiency of mannitol absorption i n the distal ileum. A similar observation was made i n 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. W i t h 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 i n 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 i n 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 o f 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 m M " ' cm" 10 1  min" ) there was no significant difference between the in situ duodenal and distal ileal loop 1  preparations, whereas for the amount of mannitol absorbed (pmol cm" 10 min" ), it was 1  significantly greater i n the duodenal than i n the distal ileal loop preparations.  1  Data obtained by  Karbach (1991) also show difference i n mannitol absorption and secretion i n 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 i n 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 i n 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  CHAPTER 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 N y s , 1992). weakness  are  indeed  L e g abnormalities such as tibial dyschondroplasia and bone  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 i n 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 o f 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  149  to be vitamin D-independent, taking place via the paracellular pathway. present investigation, therefore,  The first part of the  dealt with determining the fractional contribution of the  vitamin D-dependent and vitamin D-independent mechanisms of calcium transport i n 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, i n 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 i n the form o f the chloride salt. T o 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. W i t h 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 i n the rations increase the incidence of tibial dyschondroplasia i n broiler chickens.  The results obtained i n 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.  Elliot 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 i n 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 i n the spot light of clinical nutritionists.  Based on the mechanisms of intestinal calcium transport in broiler chicks,  investigated i n 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 i n the ileum.  151  Whereas  some  researchers maintain that calcium transport i n 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 i n situ ileal preparation in rapidly growing young broiler cockerels occurs solely via the paracellular pathway.  Whether controversy.  or not vitamin D regulates paracellular pathway  is also a subject  of  Some of the early reports which suggested the existence of such a regulation has  not been revisited.  The present investigation show that mannitol transport i n 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 i n 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.  152  2.  A 12 h fast does not affect calcium or mannitol disappearance i n in situ duodenal preparations i n 7-d-old broiler cockerels.  3.  There is no significant circadian variation i n plasma total-calcium concentration i n 18d-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 i n 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 i n 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.  153  Chapter 5. 1.  Mannitol transport in the in situ duodenal loop preparations i n 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 i n 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 i n situ duodenal loop preparations of 0to 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 o f paracellular absorption i n the i n situ duodenal and jejunal preparations in 0- to 14-d-old broiler cockerels tends to decrease with age whereas the efficiency does not change i n 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.  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Physiol. 220:1261-1266.  170  APPENDICES  171  +->  s  >  a  O  73  %  73  3  »  5  co c o  ' S3  1 o 1—1  a  3,  T3 CU u a cu D-  a « CA a  s  'C 73 U  ( J85j . p 3 p 3 f u i u i d p ox i - i u i u i d p ) T  l  jCjIAipBOIpBJ B 3  s  s j 7  BUISBIJ  172  1=1  .— •-H  CU  O .-H  ft  1ft cu OH X CU OH O  cu 5  ft 8 -2 CU 12 '-ft — P 2 cd .3 & 2 g C CO  cd »ri  a B ©  cd W  .S " T3  cd  0  S  1 .3 3  "o S  3.  S-9  cu  =3 cu cu cd "73  T3  ft  ,  ft  Cftl  g oO si . >^ ft HM ^ CU  g « ^ .3 cu 3 .3 3 =*• § e  cu u  y-! ft cd O ft4 o U cu cu '-' o c  a a  x! a 1=1 d  T3  CS cu  a  c«  '•B  cu  ft  co  *3  •S  <=>  ft  8 .2 & < £ td X)  c  o  %  &  OH ft 3 CM S3 & cd o .2  Hi  °  £  ft -ft o "3  11 - I cx?u w  rt  . 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