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Effects of dairy constituents on calcium bioavailability : impact on utilization as indexed by bone mineral… Yuan, Yvonne Veronica 1990

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EFFECTS OF DAIRY CONSTITUENTS ON CALCIUM B I O A V A I L A B I L I T Y : IMPACT ON U T I L I Z A T I O N AS INDEXED BY BONE MINERAL  COMPOSITION  AND BIOMECHANICS. By YVONNE VERONICA YUAN B . S c . , The U n i v e r s i t y  of B r i t i s h  Columbia,  1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS  FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f Food We a c c e p t t h i s  Science)  t h e s i s as c o n f o r m i n g  to the r e q u i r e d  standard  THE UNIVERSITY OF B R I T I S H  COLUMBIA  S e p t e m b e r 1990 (c)  Yvonne V e r o n i c a Y u a n , 1990  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 department or by his or her representatives.  be granted by the head of my  It is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department of  Fnnrl ^ r i p n r p  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  September 19, 1990  written  Abstract Calcium intestinal  bioavailability  absorption,  mineralization calcium  and  In  calcium  as  absorption  a critical  necessary  for  its  absorption  of c a l c i u m  bone  study  luminal  action.  1, was  not  the  50%  different  f u r t h e r , a d e c r e a s e i n bone s t r e n g t h  of  secondary to n u t r i e n t malabsorption.  was  evidence to i n d i c a t e a  of  no  calcium  from m i l k  In experiment was  significantly  diets  containing  diets.  The  ( c o l l o i d a l ) or y o g u r t 2,  absorption  casein  as  of  compared  i n absorbed calcium  significance  in  of  between  genetically  containing  from c o n t r o l s ; a n i m a l s was  the  normotensive c o n t r o l differences not  due  casein  in  Wistar-Kyoto  calcium  metabolism  to d i f f e r e n c e s i n i l e a l and  soy  protein  (WKY)  sources.  from the  rats fed milk whey and  was  shown t o have  absorption  soy  transport.  protein little  biomechcalcium.  was  hypertensive rats,  ileum  protein  to  d i e t s containing high  there  bioavailability  similar  (SHR)  and  suggesting  that  b e t w e e n t h e s e two  calcium  and  found  study,  bone m i n e r a l i z a t i o n and  spontaneously  was  lactose  calcium  p a r a c e l l u l a r calcium  was  lactose  a n i c s when a n i m a l s were f e d a d i e t a d e q u a t e i n d i e t a r y I n e x p e r i m e n t 3,  it  of  intestinal  (ionized)  enhanced in_ normal W i s t a r  increase  physiological  the  and  In t h i s  difference in  of  enhanced  these  t o be  bone  enhancement  confirmed,  the  with  determinants  concentration  i n animals fed  isotopic  techniques  lactose  Despite  m i n e r a l i z a t i o n was  using  endpoint  experiment  suggested that  diet,  investigated  balance  biomechanics  utilization.  paracellular  and  was  strains  The  (2.0%),  was  e f f e c t of adequate  ( 0 . 5 % ) and  low  (0.05%) l e v e l s  bioavailability and  WKY  fed  animals  and  animals.  o f c a l c i u m , r e s p e c t i v e l y on  s u b s e q u e n t u t i l i z a t i o n was  I l e a l c a l c i u m a b s o r p t i o n was  than  those  f e d soy  a t the  calcium.  Femur c a l c i f i c a t i o n was  h i g h and  medium l e v e l s  a n i c s were n o t  experiment  4,  the  casein phosphopeptides fed  SHR  animals.  appeared to r e s u l t This  effect  (CPP)  was  CPP  added  a  greater  in  e n h a n c e d by  the  effect  s t r e n g t h , due In  h o w e v e r , on to the  experiment  p r o t e i n s was heat denatured  low  soy  but  of  dietary f o r t i f i c a t i o n with  investigated in t o c a s e i n and ileal  thermal  diets  c a s e i n and  soy  absorbed  of  calcium.  processing  of  i z a t i o n and  These e f f e c t s were i n f l u e n c e d by  In resulted ileal  e x p e r i m e n t 6, in  decreased  experienced a low food  calcium absorption  protein  fed animals.  (%  A low  (6%)  by  these  d o s e ) was level  of  bone m i n e r a l the  animals.  protein,  i n t a k e and  fed  intestinal  balance  nutrient malabsorption  as  dietary  Animals  e x h i b i t e d reduced well  had  biomechanical  digestibility.  as  calcium.  casein diet  and  soy  protein diets  absorption from the  level.  calcium absorption, calcium biomechanics.  were  dietary calcium  shown t o r e d u c e i n v i t r o c a s e i n and  the  Femur b i o m e c h -  source,  bone m i n e r a l i z a t i o n  severe  at  of  low  e x c r e t i o n of excess 5,  SHR  levels  casein diets  protein  increase i n calcium b i o a v a i l a b i l i t y  little  in  greater i n casein  a d e q u a t e and  dietary  h o w e v e r , a d v e r s e l y a f f e c t e d by In  determined  of d i e t a r y c a l c i u m o n l y .  i n f l u e n c e d by  calcium  low  animal  similar  phosphorus growth.  b e t w e e n 6%  diet  However, and  20%  dietary protein influenced  calcium  balance  biomechanical  and  not  balance  likely  study.  this  shown t o  in  effect  was  m i n e r a l i z a t i o n and  obtained  calcium  and r e t a i n i n g  casein  fed  biomechanical  indicators  of  i n calcium content  calcium  of  i t i n a soluble denaturation,  or b i o a v a i l a b i l i t y  bioavail-  Finally, were  utilization  i n the d i e t  calcium  animals.  parameters  was  (CPP) t h a t a r e  w i t h heat  enhancement  from the  f e d c a s e i n , which  of b i o a c t i v e p e p t i d e s  the  absorption  from a c a l c i u m  Reducing the p r o t e i n content  reduce in  calcium  calcium b i o a v a i l a b i l i t y  protein d i g e s t i b i l i t y  lost.  observed  sensitive  m i n e r a l i z a t i o n and  paracellular  be e n h a n c e d i n a n i m a l s  sequestering  however, d i d not ability  bone  w i t h those  Notwithstanding,  By r e d u c i n g  varied  equate  due t o t h e p r o d u c t i o n  involved form.  i n d i c a t e that  necessarily  i l e u m was  for  strength.  These r e s u l t s may  utilization  from  of t h i s  shown  bone t o be  diets mineral.  that  Table of  Contents Page  Title  page  i  Abstract Table  i i  of C o n t e n t s  List  of Tables  List  of F i g u r e s  v viii x i i  Acknowledgements  xiv 1  Introduction L i t e r a t u r e Review 1.  4  .  I n t e s t i n a l Calcium Absorption a. P h y s i o l o g y and B i o p h y s i c s b. A n i m a l M o d e l s f o r S t u d y i n g I n t e s t i n a l Absorption  4 4 Calcium  9  2.  Factors Influencing Calcium B i o a v a i l a b i l i t y a. F i b r e and P h y t a t e b. O x a l a t e s c. F a t d. P r o t e i n and P h o s p h o r u s e. L a c t o s e f. M a i l l a r d Browning R e a c t i o n Products g. M i n e r a l I n t e r a c t i o n s h. E n d o c r i n e F a c t o r s i . I m p a c t o f N u t r i t i o n a l S t a t u s on C a l c i u m A b s o r p t i o n . j . Physiological Status  15 15 17 17 17 22 24 25 27 30 32  3.  D a i r y Foods a. C a l c i u m D i s t r i b u t i o n i n M i l k b. C a s e i n M i c e l l e s c. C a l c i u m E x c h a n g e a b i l i t y d. C a s e i n P h o s p h o p e p t i d e s e. Soy P r o t e i n  35 35 36 37 38 41  4.  Methods of A s s e s s i n g C a l c i u m B i o a v a i l a b i l i t y Absorption a- JjL V i t r o S o l u b i l i t y b. B a l a n c e S t u d i e s c. N u t r i t i o n a l S t a t u s d. F u n c t i o n a l T e s t s e. M o d e l S y s t e m s v  and  42 43 43 45 45 45  Page 5. C a l c i u m U t i l i z a t i o n i n Bone D e p o s i t i o n a. T r e a t m e n t o f O s t e o p o r o s i s b. Anatomy o f t h e Femur c. Bone B i o m e c h a n i c s  46 48 50 51  E x p e r i m e n t 1: The e f f e c t o f l a c t o s e and f e r m e n t a t i o n p r o d u c t s on p a r a c e l l u l a r c a l c i u m a b s o r p t i o n and f e m u r biomechanics i n r a t s  54  Introduction M a t e r i a l s and Methods Results Discussion  54 54 61 69  E x p e r i m e n t 2: P a r a c e l l u l a r c a l c i u m a b s o r p t i o n and femur m i n e r a l i z a t i o n and b i o m e c h a n i c s i n r a t s f e d s e l e c t e d dietary proteins Introduction M a t e r i a l s and Methods Results Discussion  74 74 74 79 86  E x p e r i m e n t 3: C a l c i u m b i o a v a i l a b i l i t y fed rats  i n c a s e i n and s o y  Introduction M a t e r i a l s and Methods Results Discussion  91 91 91 95 110  E x p e r i m e n t 4: E f f e c t o f c a s e i n p h o s p h o p e p t i d e f o r t i f i c a t i o n on c a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n c a s e i n and soy f e d r a t s . Introduction M a t e r i a l s and Methods Results Discussion  117 117 117 122 138  E x p e r i m e n t 5: E f f e c t o f p r o t e i n h e a t d e n a t u r a t i o n on c a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n r a t s f e d c a s e i n and s o y p r o t e i n s Introduction M a t e r i a l s and Methods Results Discussion  143 143 144 147 169  vi  Page E x p e r i m e n t 6: C a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n r a t s f e d c a s e i n and s o y d i e t s v a r y i n g i n p r o t e i n l e v e l Introduction M a t e r i a l s and M e t h o d s Results Discussion  176 176 176 179 193  Conclusions  199  References  204  Appendix  223  vii  List  of Tables  Table  Page 1.1  Composition  of experimental d i e t s  1.2  D i e t e f f i c i e n c y of r a t s  1.3  Blood  1.4  I n t e s t i n a l c a l c i u m a b s o r p t i o n and femur d e p o s i t i o n of calcium i n rats fed experimental diets  64  1.5  Femur p h y s i c a l d i m e n s i o n s and b i o m e c h a n i c a l parameters i n r a t s f e d experimental d i e t s . . . .  68  2.1  Composition  76  2.2  D i e t e f f i c i e n c y of r a t s  chemistry of rats  fed to rats  . .  56  fed experimental diets  .  62  fed experimental diets  .  63  4 5  2.3  of experimental d i e t s  fed to animals  fed experimental diets  .  I n t e s t i n a l a b s o r p t i o n and femur d e p o s i t i o n o f calcium i n rats fed experimental diets . . . .  4 5  i n rats fed  80 81  2.4  Femur m i n e r a l c o m p o s i t i o n experimental diets  2.5  Femur p h y s i c a l d i m e n s i o n s and b i o m e c h a n i c a l parameters i n r a t s f e d experimental d i e t s . . . .  84  2.6  T i b i a c a l c i u m c o n t e n t and b i o m e c h a n i c a l parameters of t h r e e - p o i n t bending a n a l y s i s fed experimental d i e t s  85  i n rats  3.1  Composition  3.2  Body w e i g h t g a i n and f o o d animals  3.3  E f f e c t o f d i e t a r y p r o t e i n s o u r c e on p l a s m a m i n e r a l s of r a t s f e d d i f f e r e n t l e v e l s of c a l c i u m  97  E f f e c t o f d i e t a r y p r o t e i n s o u r c e on c a l c i u m a b s o r p t i o n i n r a t s f e d d i f f e r e n t l e v e l s of calcium  99  3.4  of experimental d i e t s  83  f e d to animals  i n t a k e of experimental  3.5  Femur d e p o s i t i o n o f experimental diets  3.6  Femur m i n e r a l c o m p o s i t i o n o f r a t s f e d experimental diets  45  calcium i n rats fed  viii  93 96  102 103  Table  Page 3.7  Femur p h y s i c a l p a r a m e t e r s experimental diets  of r a t s  fed  3.8  Femur b i o m e c h a n i c a l p a r a m e t e r s experimental diets  3.9  T i b i a m i n e r a l c o m p o s i t i o n of r a t s experimental diets  3.10  T i b i a biomechanical parameters experimental diets  4.1  Composition  4.2  D i e t e f f i c i e n c y of r a t s  4.3  E f f e c t o f d i e t on p l a s m a experimental diets  4.4  E f f e c t o f p r o t e i n s o u r c e and CPP f o r t i f i c a t i o n on * c a l c i u m d e p o s i t i o n t o t h e femur  129  E f f e c t o f p r o t e i n s o u r c e and on 24 h r . c a l c i u m b a l a n c e  CPP  130  E f f e c t o f p r o t e i n s o u r c e and on 24 h r . magnesium b a l a n c e  CPP  105  of r a t s  fed  106  fed  108  of r a t s r  of e x p e r i m e n t a l d i e t s  fed  109  fed to animals  fed experimental diets m i n e r a l s of a n i m a l s  120 .  fed  127  5  4.5 4.6 4.7 4.8 4.9  fortification fortification 132  E f f e c t o f p r o t e i n s o u r c e and CPP on 24 h r . p h o s p h o r u s b a l a n c e  fortification  E f f e c t o f p r o t e i n s o u r c e and on femur m i n e r a l i z a t i o n  CPP  fortification  Effect  CPP  of p r o t e i n  on f e m u r p h y s i c a l  s o u r c e and and  133 135 fortification  biomechanical parameters  5.1  Composition  5.2  E f f e c t of heat t r e a t i n g p r o t e i n efficiency E f f e c t of heat t r e a t i n g p r o t e i n a b s o r p t i o n from i l e a l l o o p  on  5.4  E f f e c t of heat t r e a t i n g p r o t e i n d e p o s i t i o n t o t h e femur  on  5.5  E f f e c t of heat t r e a t i n g p r o t e i n calcium excretion  on 24  5.3  of e x p e r i m e n t a l d i e t s  ix  125  fed to animals  .  136 145  diet 149  on c a l c i u m 4 5  calcium  151 154  hour 155  Table  Page 5.6  E f f e c t of heat t r e a t i n g p r o t e i n calcium balance  on 24  hour  5.7  E f f e c t of heat t r e a t i n g p r o t e i n magnesium e x c r e t i o n  on 24  hour  5.8  E f f e c t of heat t r e a t i n g p r o t e i n magnesium b a l a n c e  on 24  hour  5.9  E f f e c t of heat t r e a t i n g p r o t e i n phosphorus e x c r e t i o n  on 24  hour  5.10  E f f e c t of heat t r e a t i n g p r o t e i n phosphorus balance  on 24  hour  5.11  E f f e c t of heat t r e a t i n g p r o t e i n p h y s i c a l parameters  on  5.12  E f f e c t of heat t r e a t i n g p r o t e i n mineralization  on  femur  5.13  E f f e c t of heat t r e a t i n g p r o t e i n biomechanical parameters  on  femur  6.1  C o m p o s i t i o n of e x p e r i m e n t a l d i e t s  6.2  Effect  of p r o t e i n  level  on d i e t  6.3  Effect  of p r o t e i n  level  on p l a s m a  6.4  Effect  of p r o t e i n  6.5  157 158 159 161 162  femur 164 166 167  fed to animals . . .  180  . . .  181  level  on 24 h r . c a l c i u m b a l a n c e  185  E f f e c t of p r o t e i n balance  level  on 24 h r . magnesium  6.6  E f f e c t of p r o t e i n balance  level  on 24 h r .  6.7  Effect  level  on femur  6.8  E f f e c t o f p r o t e i n l e v e l on femur biomechanical parameters  of p r o t e i n  efficiency  178  minerals  186  x  phosphorus mineralization physical  and  188 .  191 192  Appendix Table 1. 2. 3. 4.  Page  I n t e r a c t i o n between a n i m a l s t r a i n i n t a k e i n femur u t i l i z a t i o n I n t e r a c t i o n between p r o t e i n l e v e l i n femur u t i l i z a t i o n  and c a l c i u m 225  s o u r c e and c a l c i u m  I n t e r a c t i o n between a n i m a l s t r a i n l e v e l i n t i b i a magnesium c o n t e n t  226 and c a l c i u m  I n t e r a c t i o n b e t w e e n a n i m a l s t r a i n and h e a t t r e a t m e n t i n magnesium b a l a n c e  xi  227 protein 228  List  of Figures  Figure 1.  Page Summary o f c a l c i u m  1.1 L a c t o s e  content  metabolism  of i l e a l  loop  5 of r a t s  67  3.1 E f f e c t o f d i e t a r y p r o t e i n s o u r c e and c a l c i u m l e v e l on c a l c i u m c o n t e n t and a b s o r p t i o n , r e s p e c t i v e l y f r o m t h e i l e a l l o o p o f SHR and WKY r a t s f e d c a s e i n and s o y d i e t s 3.2 Femur d e p o s i t i o n o f C a c a s e i n and s o y d i e t s  i n SHR and WKY  4 5  98  rats fed 101  4.1 G e l f i l t r a t i o n p u r i f i c a t i o n phosphopeptide (CPP) 4.2 I o n e x c h a n g e p u r i f i c a t i o n phosphopeptide (CPP)  of c a s e i n  of casein  123 124  4.3 I n t e s t i n a l a b s o r p t i o n o f C a f r o m i l e a l l o o p i n r a t s f e d c a s e i n , s o y and C-CPP and S-CPP d i e t s .  128  5.1 P e p s i n - p a n c r e a t i n d i g e s t i o n e s t i m a t e s o f n a t i v e and h e a t d e n a t u r e d c a s e i n and s o y p r o t e i n . . . .  148  5.2 I n t e s t i n a l a b s o r p t i o n o f C a f r o m i l e a l l o o p o f SHR and WKY r a t s f e d n a t i v e and h e a t d e n a t u r e d c a s e i n and s o y p r o t e i n  152  6.1 I n t e s t i n a l a b s o r p t i o n o f C a f r o m i l e a l l o o p o f r a t s f e d 2 0 % and 6% c a s e i n and s o y p r o t e i n d i e t s  182  6.2 D e p o s i t i o n o f a b s o r b e d C a t o t h e f e m o r a o f r a t s f e d 2 0 % and 6% c a s e i n and s o y p r o t e i n d i e t s  184  4 5  4 5  4 5  4 5  X I  1  Appendix Figure 1.  Page  Time-force deformation b e n d i n g o f bone  curve of I n s t r o n 3-point  xi i i  224  Acknowledgements I would l i k e who a s s i s t e d  t o e x p r e s s my g r a t i t u d e  t o t h e many  me d u r i n g t h e c o u r s e o f t h i s  I would l i k e  to  acknowledge the  study.  Department of  individuals  In p a r t i c u l a r , Food S c i e n c e f o r  t h e u s e o f t h e a n i m a l and l a b o r a t o r y f a c i l i t i e s . I w i s h t o e x p r e s s my s i n c e r e g r a t i t u d e Dr. D a v i d D. K i t t s , participation t i o n of t h i s  in  D e p a r t m e n t o f Food S c i e n c e , f o r h i s d e d i c a t e d  the animal  thesis.  I  s u r g e r i e s as w e l l  am a l s o  Durance,  t h e Department  William  Powrie  the p r e p a r a t i o n of t h i s I would  like  to  I would  also  mother, Mrs.  S c i e n c e , and D r s .  John V a n d e r s t o e p ,  s u g g e s t i o n s and  a c k n o w l e d g e my i n d e b t e d n e s s Moriyama  of the  help during  for their  t o Dr. T a k a s h i  assistance  w i t h the  and l a b o r a t o r y w o r k . like  t o thank  B u r e a u o f Canada f o r t h e i r Finally,  committee,  thesis.  Nagasawa and Mr. Y o s h i o experiments  of Animal and  D e p a r t m e n t o f Food S c i e n c e f o r t h e i r  animal  study.  a r e a l s o g i v e n t o t h e members o f my t h e s i s  Dr. L e s l i e H a r t , o f Timothy  as i n t h e c o m p l e -  t h a n k f u l f o r h i s encouragement  and f r i e n d s h i p d u r i n g t h e c o u r s e o f t h i s Thanks  t o my t h e s i s a d v i s o r ,  I would l i k e J a n e t M.  Dr. B e r n i c e M i l l s  s u p p o r t and f u n d i n g o f t h i s t o t h a n k my f a m i l y ,  Yuan, f o r t h e i r  d u r i n g the course of t h i s  and t h e D a i r y  thesis.  xi v  support  project.  and e s p e c i a l l y  my  and u n d e r s t a n d i n g  Introduction  well  The  i m p o r t a n c e of  as  soft tissues,  bone d i s e a s e Morris,  diet,  Numerous 1987, that the  calcium  of  intraluminal  general, specific  have t h e  absorption,  bone  of  increased in  ticular, of d a i r y calcium  and  for  general, i n calcium  and  the  gained  bioavailability.  and  ionized)  of  calcium  food  system.  Bronner et a l . . have shown  absorption.  simultaneously bind  in  ionized  A the  calcium In  the  r o l e of  influencing  calcium  bone d e p o s i t i o n ,  chemistry  p r o t e i n c o n s t i t u e n t s and (colloidal  in  interest  t o be  level  concerning  as  indexed  parameters.  Such  both  t o Food S c i e n t i s t s and  knowledge  dietary  i o n i z e d form w i t h i n  biomechanical  considerable  as p r a c t i c a l l y ,  the  diet  i s of  components  and  dairy  bioavailability.  information  utilization  mineralization  information well  and  therefore  of  product  present  and  of  1989,  optimal  p o t e n t i a l to  and  i s a paucity  bioavailability by  lumen t o f a c i l i t a t e  contents  dairy  a1..  as  75%  the  r e v i e w e d by  i n a s o l u b l e and  constituents  there  from  Heaney e t  dietary  impair  as  i m p o r t a n c e of the  adequately  (McCarron  North American d i e t ,  p r o v i d i n g as much  present  intestinal  established  In the  and  i n hard,  e t i o l o g y of c a r d i o v a s c u l a r  bioavailability  1988,  must be  m a j o r component  been w e l l  to the  its  Greger et a1..  variety  has  investigators,  small  and  is  a  i n the  role,  In a d d i t i o n  the  as  Marcus, 1987).  products play a v i t a l  in  and  prevention  1985;  calcium.  calcium  academically,  Nutritionists  concerning  the  because r o l e of  of d a i r y p r o d u c t s i n  Specifically,  the  various  as  they  the  as  par-  chemistry  forms of a v a i l a b l e  pertain  to  relative  protein  digestibility,  quantity  consumed; as w e l l as t h e  as  lactose  or  influencing  on  lactose  calcium  example, the  alteration  absorption,  in  digestion  of  casein,  by  dietary constituents.  may  study.  sequester  calcium  s t u d i e s were  t h e s i g n i f i c a n c e o f CPP on c a l c i u m  such  For  (CPP) p r o d u c e d  of i n s o l u b l e c a l c i u m  Further  and  potentially  further  of casein phosphopeptides  prevent the p r e c i p i t a t i o n  assess  processing  co-nutrients  products  require  thereby, other  thermal  presence of  fermentation  production  tryptic  with  and  complexes  required to  b a l a n c e and  utilization  f o r bone m i n e r a l i z a t i o n . The e x p e r i m e n t s determine  the  reported  significance  associated  with dairy products  absorption  and  biomechanical  utilization  the r e l a t i v e  this  of  the  were c o n d u c t e d t o  aforementioned  or processes  In.  binding  thesis  thereof,  factors  on  calcium  as i n d e x e d by bone m i n e r a l i z a t i o n and  properties.  determine calcium and  in  to  vitro  studies  casein derived  digestibility  of t h e r m a l l y  conducted  bioactive  to  peptides,  treated proteins  were  also investigated. In s p e c i f i c  experiments, the spontaneously hypertensive  (SHR) was c h o s e n as t h e reasons.  Firstly,  suggested  as  a  characteristic reported more  calcium  factor to  these  t o be p r o n e  sensitive  modifications  experimental  to  absorption  in  the  animals.  i n level  subtle  model  for several  i n t h e s e a n i m a l s has been  disturbed  calcium  homeostasis  S e c o n d l y , t h e SHR has been  to osteoporosis, the  animal  and t h e r e f o r e  potentially  changes a s s o c i a t e d w i t h  of c a l c i u m 2  rat  i n t a k e and  dietary  bioavailability.  Finally, assess  various experimental  calcium  (Greger, method  bioavailability  have  1988; Pak and A v i o l i , has  endpoint  1988).  i t s advantages  and  measurements used t o  been r e p o r t e d and It  i s apparent  potential  t h a t each  disadvantages  a c c u r a t e l y a s s e s s i n g c a l c i u m a b s o r p t i o n and u t i l i z a t i o n . present  study,  mechanical isotopic of  a  physical  parameters  and  calcium  modifications  balance in  o f bone  was u s e d t o a s s e s s  t h a t would  fed  different  products.  3  in  In the the b i o -  complement t h e c a l c i u m  s t u d i e s u s e d t o q u a n t i t a t e bone  animals  of d a i r y  method  critiqued  dietary  utilization proteins  or  Literature  Review  Introduction: Calcium the m i n e r a l is a  c o n t r i b u t e s t o 1.5 t o 2.0% o f body w e i g h t , content  o f t h e human b o d y .  m a j o r component o f t h e h a r d  present  tissues. blood  extracellular  fluids  activity,  calcium  ( 9 9 % o f body c a l c i u m i s remainder d i s p e r s e d i n  and w i t h i n t h e c e l l s  cell  t r a n s m i s s i o n and r e g u l a t i o n o f considered  membrane  of the s o f t  transport,  cardiac function.  t o be an e s s e n t i a l m a c r o n u t r i e n t  Thus,  nerve calcium  o f t h e human  diet  RDA f o r c a l c i u m i s 800 mg/day f o r a d u l t s ) .  1.  Intestinal  la.  P h y s i o l o g y and B i o p h y s i c s :  Calcium  Absorption  Dietary calcium enters the i n t e s t i n a l chyme by from  mechanical  the  junctions (Figure  lumen to  1).  intestine Bronner, The  body,  The r o l e s o f c a l c i u m i n t h e body i n c l u d e bone s t r e n g t h , clotting  (U.S.  tissues  i n t h e bones and t e e t h ) , w i t h t h e  the b l o o d ,  is  Within the  o r 39% of  by  crossing  is  an  dependent process  a passive, along  the  Calcium  intestinal  calcium absorption  and  which occurs  therefore in  upper jejunum (Pansu nonsaturable the e n t i r e  and c e l l  e n t e r i n g t h e lymph and b l o o d  K e r s t e i n , 1976;  active,  i s absorbed  mucosa  t h e sum o f two i n d e p e n d e n t p r o c e s s e s  1 9 7 1 ; B e h a r and  duodenum and  activity.  t h e s e r o s a and f i n a l l y Transepithelial  is  first  occurs  and e n z y m a t i c  lumen f r o m t h e g a s t r i c  the  ( Z o r n i t z e r and  P a n s u .et. a_l_. , 1 9 8 3 b ) . saturable  proximal  e t a 1. . 1 9 8 3 b ) .  concentration  from the s m a l l  dependent  vitamin D  i n t e s t i n e , the The s e c o n d i s process  which  l e n g t h of t h e s m a l l i n t e s t i n e , but which 4  Diet  Fig.  1  Summary o f c a l c i u m m e t a b o l i s m : ( 1 ) v i t a m i n D-dependent active calcium transport; ( 2 ) endogenous calcium s e c r e t i o n ; (3) passive p a r a c e l l u l a r calcium t r a n s p o r t ; (4) f i l t e r e d c a l c i u m l o a d ; (5) r e n a l t u b u l a r reabsorbed c a l c i u m ; ( 6 ) bone m i n e r a l i z a t i o n ; ( 7 ) PTH m e d i a t e d bone r e s o r p t i o n ; s u b j e c t t o i n h i b i t i o n by CT.  5  is  especially  predominant i n  the a c t i v e process 1971;  the d i s t a l  i s not of s i g n i f i c a n c e  component n a t u r e  was e l u c i d a t e d f r o m kinetic  studies.  ( 1 9 8 6 ) were  in. vitro Using  able  duodenal loop  to  ( Z o r n i t z e r and B r o n n e r ,  diffusional process.  of i n t e s t i n a l as  kinetic  s t u d i e s was  component  alone. in  previously hypothesized and  a  as  calcium in  absorption  vivo  absorption  a b s o r p t i o n d a t a , B r o n n e r e t a 1. the e f f l u x  much g r e a t e r This  the  than  of c a l c i u m  from  had been p r e d i c t e d  l e d to the  intestinal  I t i s noteworthy that  a saturable  well  c a l c u l a t e that  for a saturable process  hypothesis  calcium  Wasserman and  of a  absorption  Taylor  ( 1 9 6 9 ) had  t h a t d u o d e n a l c a l c i u m a b s o r p t i o n had b o t h  nonsaturable  component.  c a l c i u m a b s o r p t i o n from the i n t e s t i n a l  The  k i n e t i c s of  lumen were e x p r e s s e d  using  f o l l o w i n g equation; Jms  where flux  Jms  - flux  = J . . . TCal + D [ C a ] [Ca] + K*.  of the a c t i v e  saturable  process;  - transport  c o n s t a n t ) ; and  coefficient  et a1..  (Arnold  curve  [Ca]  constant  the M i c h a e l i s - M e n t e n  biphasic  D  1975).  =  dominant,  (Wasserman  as i n d i c a t e d et. a l _ . ,  1961;  a b s o r p t i o n as a At r e l a t i v e l y low  by t h e h y p e r b o l i c n a t u r e  6  and  diffusion  d e s c r i b e s the  the a c t i v e t r a n s p o r t  Krawitt  calcium  i s comparable t o  equation  calcium  - maximum  luminal  paracellular  This  of the r a t e of i n t e s t i n a l  c o n c e n t r a t i o n s , 0 t o 2 mM,  =  (which  f u n c t i o n of the luminal calcium c o n c e n t r a t i o n . calcium  Jmax  o f c a l c i u m f r o m mucosa t o s e r o s a ;  c o n c e n t r a t i o n ; Kt  is  where  B e h a r and K e r s t e i n , 1 9 7 6 ) . The d u a l  the  j e j u n u m and i l e u m  Schedl,  component  of the curve 1968)  until  saturation and  is  achieved  Patrick,  becomes  1970).  linear,  10 mM,  at  Above  with  indicating  concentrations  a  the  these  (Papworth  concentrations,  transition  dominance  o f 2 t o 5 mM  of  o c c u r r i n g at the  the  curve  approximately  nonsaturable  diffusion  process. The  above  kinetics and  equation  of s p e c i f i c  Rosen,  1959;  i s a l s o a p p l i c a b l e to the  segments Krawitt  and  a b s o r p t i o n of n u t r i e n t s i s a b s o r p t i o n from the  residence  of the  Thus, w h i l e  much g r e a t e r f r o m t h e than  from  the  ileum  absorbed are q u i t e 4 5  Calcium,  Marcus  small intestine  Schedl,  a function  a particular  time  Copp, 1 9 5 9 ) .  of t h e  1968).  of n o t  segment o f t h e  substrate the  and  and  In  23 and  and  62%  dietary  ileum,  dose.  tract.  c a l c i u m from  the  a liquid  ileum  (due  duodenum, Thus,  the  coupled  with  to i t s g r e a t e r  distal  different  calcium  the  longer  duodenum, 0,  i n t h e dog  7  8,  estimates 2.4,  distal luminal  residence  time  the of 15,  jejunum  4 and  t o be  and  greater  l e n g t h ) , more t h a n  regions  t o a b s o r b 0,  d o s e and  jejunum  relatively  and  radiolabe11ed  M o r e o v e r , Cramer ( 1 9 6 5 ) r e p o r t e d  8.1  concentration,  with  stomach,  and  respectively.  also  transport)  t h e amounts of  A n i m a l s were a b l e  i n t r a l u m i n a l calcium concentrations i n the  (active  studies  the  mM  r a t e of  Lengemann ( 1 9 6 2 ) were a b l e t o q u a n t i f y  respectively with  with a solid  o n l y the  the  segment ( C r a m e r  jejunum  p r o p o r t i o n of d i e t a r y c a l c i u m absorbed from the g a s t r o i n t e s t i n a l  Moreover,  of c a l c i u m a b s o r p t i o n i s  (passive d i f f u s i o n ) , different.  (Schachter  i n t e s t i n e , but  i n that  efficiency  duodenum  characteristic  88% of 2.0  ileum, calcium i n the  compensates f o r  the  slower (16%  absorption luminal  r a t e of n o n s a t u r a b l e  calcium  h o u r ; Z o r n i t z e r and The in  stages.  into  intestinal  the  luminal  a l . . 1979 ; diffusion allowing  side, Miller  across the  on  and  with  to  gradient  ( P a n s u e_t  aj_. ,  component  of  v i a brush  pole  side against As  The as  transport across  calcium  the  cell  1983b;  active  calcium  (Rasmussen  a concentration  gradient  with  a  v a r i e t y of  (Ca b i n d i n g p r o t e i n ; ( K r e t s i n g e r et a1. .  i s the cell  e x t r u s i o n of  into  the  an  transcellular  CaBP  i s vitamin  intracellular  the  cell,  poles  Roche  e_t  and  intestinal  8  i s not  D  mediator  producing  a  ( K r e t s i n g e r .et. a 1 . .  aj_. ,  t r a n s p o r t i s age  e x a m p l e , i n t h e newborn r a t , c a l c i u m  gradient  t o b o t h p h y s i o l o g i c a l as  cytosolic  the  the  extracellular  electrochemical  a result,  and  et  requires  r e l a t i o n s h i p between the  transport  1981,  an  i s subject  acting  between  the  on  next step  stage  of  regulation. CaBP  The  interact  final  calcium  b o r d e r membranes  or p a r t of o r g a n e l l e s The  per  occurring  e n t r y of  gradient  usually proteins  There i s a c l o s e l i n e a r  transmural  i n v o l v e s the  possibly  absorption  calcium  transcellular,  against  Binswager, 1980).  facilitating  1982).  cytoplasm  serosal  as n u t r i t i o n a l  is  Bronner, 1981).  1983).  of c a l c i u m  calcium  stage, cells  molecules,  the  dependent;  of  the  t o body f l u i d s  Pansu et a l . , 1983b).  a concentration  at the b a s o l a t e r a l  (Schiffl  well  and  cytoplasm  Feher,  process  down  calcium  CaBP), i n the  fluid  first  epithelial  calcium-binding  calcium  B r o n n e r , 1971;  The  transport in rats  translocated  a c t i v e t r a n s p o r t of c a l c i u m  three  1982;  content  calcium  CaBP  1986).  velocity content Another  dependency.  actively  For  transported  i n the  intestine  ( P a n s u e t a l . . 1 9 8 3 a ; D o s t a l and  This observation levels  of  induced  receptor  d a y s o f age, r a t and  the  with vitamin  sites  for this  H a l l o r a n and  Deluca,  absence  D therapy,  are not  passive  able  vitamin steroid 1981).  CaBP can  and  1983b).  t o a d a p t t o a low  transport process between  i n d e p e n d e n t o f age,  the  the  both  Reports  have t h r e e junctions;  appear  saturable lb.  mucosal  suggested  (Cassidy regions an  region  (Ueng by  o f age  intake  and  Tidball,  30  (Ueng  as  are  calcium flux  s y s t e m and  calcium  flux  calcium  K i m b e r g , 1978;  f r o m the  1967).  junction; The  rate-limiting (Bronner,  calcium  i n which  This  Pansu  tight  and  et  cellular  lumen mucosa mucosa  namely t i g h t  and  is  intake  Intestinal  f r o m mucosa t o s e r o s a ,  intermediate  the  one  t h a t a m o d i f i c a t i o n of  (Guyton, 1977).  t o be  abnormal  intestinal  low  s e e n t o peak i n t h e  cells.  N e l l a n s and  Animal Models f o r S t u d y i n g An  hormone  is a paracellular  vitamin D endocrine  B r o n n e r , 1971;  serosa  basolateral  the  However,  calcium  j u n c t i o n s w o u l d a l l o w movement o f c a l c i u m  gap  be  to  be  1981).  c a l c i u m moves  (Zornitzer  detectable  Human i n f a n t s show a s i m i l a r d e v e l o p m e n t p a t t e r n .  (Younoszai,  The  cells  due  t h e r e a f t e r d e c l i n e t o a minimum a t 3 months  Thus, they  to  of  1984).  A c t i v e t r a n s p o r t cannot  1980,  a c t i v e t r a n s p o r t and  et a1. . 1979).  al..  by  e_t a 1 . . 1 9 7 9 ) .  i n t h e newborn  e t a l . , 1979;  the  supported  CaBP (Ueng  number o f  adults  was  Toverud,  lastly gap  factors controlling  a  and  wider  junctions the  non-  1987).  Intestinal  metabolism  absorption  and  Calcium  Absorption:  potentially  disturbed  have f r e q u e n t l y been r e p o r t e d 9  in  the  spontaneously  genetically  related  Hypertensive weeks o f ations  hypertensive normotensive  blood pressure  age i n  rat  WKY  homeostasis  in  the  these  absorption;  serum  total  has and  border  into  variously as w e l l and  ionized  as g r e a t e r  have  intestinal transport  used  by  SHR  a  animals  a g e - m a t c h e d WKY  i n v i t r o Ussing  techniques  Kitts  et  in  different  intestine  i n SHR a n i m a l s  investigate to  (1986)  whether  animals.  A greater  compared  to  WKY,  calcium positive  as  from,  1986).  These  methodologies  duodenal  in situ calcium  ligated  were u n a b l e  ileal  to detect a  c a l c i u m from  the d i s t a l  compared t o WKY c o u n t e r p a r t s . a  the development  examined  been  (Toraason  chambers and  an i n s i t u  a l . (1989)  has  controls  measuring  Using  intestinal  histomorphometry.  v a r i e t y of a b s o r p t i o n study  studies,  calcium  circulating  factors;  as bone  i n a b s o r p t i o n of r a d i o l a b e l l e d  contributed a1 .  as w e l l  i n m a t u r e SHR a n i m a l s .  technique,  To  calcium levels,"  endocrine  absorption  than, i n  perfusion  difference small  intestinal  1 9 8 1 ; L a u e t a l _ . , 1984; G a f t e r et_ a_l. ,  i n c l u d i n g , balance  loop  on  r e p o r t e d t o be l o w e r , n o t s i g n i f i c a n t l y  Wright,  studies  calcium  alter-  the r e g u l a t i o n of calcium  focussed  membrane f l u i d i t y ;  Intestinal  with  o f t h e SHR when compared t o a g e -  p a r a t h y r o i d hormone and c a l c i t r i o l brush  (WKY) c o u n t e r p a r t .  changes a r e c o u p l e d  Research SHR  to i t s  i n c r e a s e s b e g i n t o a p p e a r by a b o u t 10  t h e SHR;  animals.  compared  Wistar-Kyoto  i n the calcium metabolism  matched  (SHR)  disturbed of g e n e t i c  balance calcium  evidenced 10  in  calcium  metabolism  h y p e r t e n s i o n , Lau et prehypertensive  balance  by d e c r e a s e d  in fecal  SHR  SHR  animals,  and u r i n a r y  c a l c i u m was The  suggestive  of  an a b n o r m a l  f i n d i n g s o f L a u and c o w o r k e r s  (1986) helped  changes i n t h e c a l c i u m m e t a b o l i s m o f by  an e l e v a t i o n i n b l o o d Plasma  similar 1981;  levels  of  ejt a_l_. ,  c o n c e n t r a t i o n has  total  age-matched  WKY  in extracellular Supporting  found i n e a r l i e r smooth  muscle  intracellular rise and  to  work cell  1988).  an a l t e r e d d i s t r i b u t i o n  It is  r e s t o r e SHR  noteworthy that  ionized  i n t h e SHR  transport.  The  membrane c e l l s compared t o  calcium  animal lipid  altered  an  increase  calcium,  calcium  in  a diet levels  model, fluidity  high to  profile  i n vascular  with  decreased  extracellular  i n calcium  leading to of  and c a l c i u m  11  of  giving  et a l . . ( 4 % ) can  WKY c o n t r o l s  p e r m e a b i l i t y t o c a l c i u m may be changes i n c a l c i u m  intestinal  c o n t r o l s (Lau  i n the  can be  SHR a n i m a l s  that  an  (McCarron  f o r this hypothesis  o f c a l c i u m between  Cellular  a g e - m a t c h e d WKY  lipid  suggesting  binding a c t i v i t y  was i n c r e a s e d i n p r e h y p e r t e n s i v e  change i n membrane f l u i d i t y an  to  m a t u r e SHR i n  c o m p a r t m e n t s ( A o k i e t a 1 . . 1976 ," S c h e d l  (McCarron et a l . . 1981). altered  reported  permeability  in  controls,  calcium  o r g a n e l l e uptake of  intracellular  ( M c C a r r o n e t a 1. .  decreased  evidence  which  t o be  However, p l a s m a i o n i z e d c a l c i u m t o be  irregularity  1981) .  that  are not caused  have been r e p o r t e d  counterparts  been f o u n d  to  a1. ,  SHR a n i m a l s  calcium  1989).  comparison  et  to e s t a b l i s h  pressure.  b e t w e e n m a t u r e SHR and WKY Merke  renal calcium r e t e n t i o n .  brush  border  SHR a n i m a l s  et a l . . 1986).  p e r m e a b i l i t y was  membrane c o m p o s i t i o n ,  when This  due t o namely a  decrease  in  the  amount  of  saturated  lipid  in  favour  of  unsaturates . The (PTH)  role  and  animals levels  of  endocrine  calcitriol, has  not  i n the a l t e r e d  been  clearly  o f i m m u n o r e a c t i v e PTH  icantly  increased  i n SHR  result  11 week o l d SHR to PTH  did  not  animals  appear  which  r e s p o n s e t o PTH PTH  were  earlier is  age  The  be  balance  a  t h a t o f WKY  animals.  WKY  animals  in  of  an  signif-  counterparts may  as  may  of  SHR  of mature  i f serum  Support f o r t h i s and  animals  of SHR  end-organ levels calcium  have been p r o m i n e n t  Lau  in  opposed  difference in urinary  1981).  have  levels  impaired  at  an  hypothesis  coworkers  u r i n a r y c a l c i u m , and  of p r e h y p e r t e n s i v e  be  (1989),  Alternatively,  the  (McCarron et a1..  decrease  coworkers  indicative  SHR  h y p e r p l a s i a as o b s e r v e d  the h y p e r c a l c i u r i a  elevated, and  to  elevation in circulating  found i n the o b s e r v a t i o n s  observed  over  by Merke and  these  not  l e v e l s b e t w e e n SHR  reported  of  Circulating  i n c r e a s e d s e c r e t i o n o f PTH  to a f f e c t  may by  been  p a r a t h y r o i d gland  hyperparathyroidism.  animals,  of  of  have  The  calcium homeostasis  e l u c i d a t e d as y e t .  animals  ( M c C a r r o n e_t_ a_L- , 1 9 8 1 ) . been t h e  f a c t o r s n a m e l y , p a r a t h y r o i d hormone  (1986)  who  increased  calcium  t o WKY  counter-  been  similarly  compared  parts . The  r o l e o f v i t a m i n D and  undefined; increased (Schedl  there are serum  reports  calcitriol  et_ al_. , 1984;  Administration  of  Lau  et  vitamin  i t s metabolites of d e c r e a s e d ,  levels  i n SHR  aj_. , D3  and 12  1986 ;  has  similar,  as  well  compared t o WKY Lucas  et_  25-hydroxyvitamin  as  animals  al_. , 1 9 8 6 ) . D3  had  no  effect  on d u o d e n a l  calcium  (Toraason  and  metabolite  o f v i t a m i n D,  in  both  icant SHR  Wright,  m a t u r e SHR  i n the  SHR  animals  1981).  and  at a  S i m i l a r l y , Gafter  tissue  from  chamber. an  so, net  increase  and  coworkers  SHR  animals  F u r t h e r , these intestinal  the  niques  age  the  (3.5  and  breeder  calcium  d u o d e n a l sac  SHR  of  u s i n g an  levels  weeks,  but  t h a t SHR  calcium i s and  that  Wright, duodenal  a g r e a t e r , but in vitro animals  compared t o  A conflicting  i n the  not  Ussing  exhibited  age-matched  r e p o r t f r o m Merke  t o be  decreased  not  a t 24 weeks o f  had  greater  in age.  numbers  animals  variation metabolism as  r e p o r t s of i n t e s t i n a l  l e v e l s may used  be due  of  to d i f f e r e n c e s  the  As w e l l ,  potential  SHR  well  and as  13  WKY  animals.  measuring  tech-  Schedl  contribution  to the d i s p a r i t y of r e p o r t e d of  calcium  as w e l l as d i f f e r e n t  calcium flux.  investigated  studies  had  in  homeostatic  (Toraason  weeks o l d ) SHR  plasma c a l c i t r i o l  (1988)  calcitriol  transport  animals  signif-  receptors.  controversy  the  animals  not  calcium  serum c a l c i t r i o l 11  of  (1986) observed  treated  of m e a s u r i n g i n t e s t i n a l  coworkers rodent  of  active  workers reported  Much o f t h e  effect  disturbed  calcitriol  calcitriol  t r a n s p o r t and  a  e_t_ a_l_. , 1 9 8 6 ) .  5  i n c r e a s e was  decreased  et a l .  (1989) found at  the  f l u x of c a l c i u m ,  i n serum  c o n t r o l s (Lau  albeit  The  Prehypertensive  WKY  in  WKY,  calcitriol  significantly  WKY  absorption  near-maximal l e v e l  1981).  and  d i d i n c r e a s e duodenal calcium  intestinal  already operating  SHR  the a c t i v e  indicate  mechanism, or t h a t  in  However, c a l c i t r i o l ,  animals.  may  transport  and of  findings in From  plasma  everted  levels  of  vitamin D  m e t a b o l i t e s , they  transport of  i s decreased  breeder  source  differences breeder  in  may  t r a n s p o r t by  and  the  plasma  independently  tissue  to  be  receptor  sites  together,  may  be  i n a hypocalcemic  calcium  despite  calcitriol enhanced  these  which  calcium  for  suggest  PTH  that  respect  levels  are  to  calcium  state with  elevated  receptors,  renal  data  The  subject  SHR.  Taken  calcium  concentration.  appeared  Increased  animal  the  levels  WKY,  response to the decreased  tissue  ameliorate  calcitriol  that  a homeostatic  ionized  increased  to conclude  compared t o  plasma  calcitriol  be  t h e m a t u r e SHR to  i n t h e SHR  variability.  calcitriol  were a b l e  and  unable  leak reported  in  to  these  animals. Despite o f SHR  numerous r e p o r t s on  animal  potential  link with  Disturbances may  models,  in  coworkers  to  bone  WKY  (1985),  bone h e a l t h  and  counterparts. of a d e c r e a s e i n  cent  cortical  dry weight,  respectively in support  specifically  quality The  shaft  of  the  tibia  o f SHR  findings  femur c o r t e x  m a t u r e SHR  animals  hypothesis.  i n SHR  animals,  increase  in osteoclastic  activity 14  may  PTH  of Izawa  as a p e r  Moreover, have b e e n  in and  to  cent WKY  increased observed  i n combination  be  levels  animals  compared  s m a l l d e c r e a s e i n bone m i n e r a l i z a t i o n r a t e ( M e r k e e t The  the  t h i c k n e s s , bone  a r e a , as w e l l as bone a s h  this  metabolism  osteoporosis.  particularly  numbers o f bone r e s o r b i n g o s t e o c l a s t c e l l s the  calcium  a p a u c i t y o f i n f o r m a t i o n on  m e t a b o l i s m and  l e n g t h , per  counterparts  is  disorders,  calcium  i m p a c t upon t h e  comparison  there  the abnormal  in  with a  a l . . 1989).  a reflection  of  the  elevated that  PTH  the  l e v e l s reported  SHR  animal  osteoporotic 2.  Factors  bone  and  calcium  i n c l u d i n g the  subject.  The  and  form  Avioli,  1988).  calcium  in  calcium  intake  dietary  for  (78%  in  remaining  5%  (Allen,  important,  as  are  F i b r e and I t has  calcium  i n food  calcium  Canada;  the of  many that  acid),  otherwise  the  nutri-  and  grains and  Kaup,  and  of  just  can  namely  In  20%  providing  fat,  influence  fibre  of  total  of d i e t a r y c a l c i u m  which  calcium,  soluble  1989).  supply  the  other  d i e t a r y source  poultry  level  factors  oxalates,  in a  p r o v i d i n g u p t o 75%  The  of  i n t e s t i n e (Pak  the major  fish  1982).  released  from  and  meat,  the  i s complexed w i t h  must be  Shahani  fruits  of the is the  components  p r o t e i n , phosphorus,  p r o d u c t s and  vitamin  D.  Phytate:  been r e p o r t e d  of wholewheat, c e l l u l o s e , consistently  of  numerous  states, respectively  l a c t o s e , M a i l l a r d Browning R e a c t i o n 2a.  c o n s t i t u e n t s ; and  absorption  with  uronic  food  American d i e t ,  vegetables,  (phytates,  development  d i e t i s d e p e n d e n t on  p r o d u c t s are  the N o r t h  bioavailability  f o r the  suggest  Bioavailability  from the  The  Dairy  calcium;  These f i n d i n g s  at r i s k  physiological  constituents.  contrast,  be  a c t i o n of  m a j o r i t y of  ionized  SHR.  fragility.  of  t i o n a l , metabolic  dietary  model may  I n f l u e n c i n g Calcium  Absorption factors  i n the  that  the  fruits  and  resulted i n negative  adequate c a l c i u m  K e 1 s a y e t a 1 . , 1979;  Allen,  intake 1982). 15  a d d i t i o n of r e a s o n a b l e vegetables  calcium  balances,  (McCance and This  to a  area  amounts  normal d i e t inspite  Widdowson, of r e s e a r c h  of  an  1942a; remains  highly have The  a c t i v e and c o n t r o v e r s i a l . been  implicated  structural  contributor  ionized  calcium  However, o v e r microbes i n colon. of  contains  at  80%  pH  of  residues calcium  uronic precludes  with  unavailable  dietary  them  from  ionized  binding  Berlyne  consumption  with  the development of o s t e o m a l a c i a  have  argued  contain  of  foods  in  Reinhold the  Supporting  decreasing  of f i b r e  calcium that  i n the lower i n t e s t i n e  et a l . . 1973).  calcium  Digestion  f o r absorption  constituents  because  Phytates  a l . (1973)  phytates  report  that  associated Reports  invariably  also  p e r se w h i c h i s i n h i b i t o r y  evidence f o r a smaller bioavailability  phytates  can  be  et role  i s found i n digested to  (McCance and Widdowson, 1942a; of the  from the c o l o n .  i n the i n h i b i t i o n  16  bind  making the c a l c i u m  (McCance and Widdowson, 1942b; R e i n h o l d  human s t u d i e s w h i c h i n d i c a t e some e x t e n t  i n the  of the a c i d  i n Bedouin people.  containing  1973; G r e g e r , 1988). phytates  been c o n s i d e r e d  et  bind  groups.  f o r absorption  calcium.  i t i s the f i b r e  absorption  may  f e r m e n t e d by  u n l e a v e n e d , w h o l e w h e a t b r e a d was  f i b r e and t h a t  to calcium al..  that  of  calcium  are  however m e t h y l a t i o n  the  t o be a  in vitro.  residues,  phosphate m o i e t i e s  f o r absorption.  cations  acids  have a l s o  content,  absorption.  i t s charged c a r b o x y l  freeing  pectins  acid  uronic  acid  their  does n o t b i n d  with  of f i b r e  i s not considered  uronic  7.4  the i n t e s t i n e ,  Nonstructural  their  cellulose  pure c e l l u l o s e  which  components  the impairment of c a l c i u m  polysaccharide  since  Hemicellulose,  in  Various  phytates  would  Thus, w h i l e  of calcium  release the r o l e  absorption i s  significant, relative 2b.  considerable  effects  controversy  of i n d i v i d u a l  fibre  still  exists  as t o  the  components.  Oxalates : Oxalates  are  vegetables. chocolate the  found  Other sources  c o c o a powder.  calcium present  (Kikunaga may  in  Erdman,  The  if  capable  the d i e t a r y  of  acid  effect  making i t p o o r l y  calcium  content  on  and  absorbed  Moreover,  oxalates  dietary  1987 ;  sources  Poneros-Schneier  of c h o c o l a t e  calcium  intake  leafy  b i n d i n g a l l of  of c a l c i u m from o t h e r  oxalic  green  oxalates include tea  same m e a l (Weaver e_t a l . .  1989)  in  Doane ejt_ a l . . 1 9 8 9 ) .  been f o u n d t o have l i t t l e diet  are  i n leafy vegetables  reduce the a v a i l a b i l i t y  and  amounts  of d i e t a r y  Oxalates  e_t. a l . . 1988;  consumed i n t h e  large  milk  has  a b s o r p t i o n from  the  i s adequate  (Roberge,  1986;  R e c k e r ejt. a_l . , 1 9 8 8 ) . 2c . F a t : Under normal c i r c u m s t a n c e s , a  factor  Kaup e t  in al..  malabsorption, to the calcium  calcium  is  However,  intestinal  precipitated as  by  P r o t e i n and  in  fatty  have  not  fatty acid  and  been  conditions  17  marked  can  soaps i n  chain  be  the  1951; by  fat  reduced  due  lumen.  length increases,  (Allen,  observed  Phosphorus:  Mitchell,  The  a c i d s w i t h the a v a i l a b i l i t y  increases  absorption. 2d.  (Steggerda  calcium absorption  of s a t u r a t i o n  Triglycerides  amount o f d i e t a r y f a t i s n o t  of i n s o l u b l e c a l c i u m  calcium decreasing the degree  absorption  1990).  formation  the  1982; to  Greger, affect  and  of as  1988). calcium  Protein  i n t a k e does n o t a f f e c t  range of the normal d i e t . urinary dietary is  calcium calcium  not  is  associated  (Zemel,  1988).  increase  calcium  balance  Linkswiler,  Protein  in  1979;  of  induced  glomerular Schuette  Whiting  amino a c i d c o n t e n t  and  ism of the s u l f u r (Amy,  absorption,  I n c r e a s i n g the  amino  renal tubular of  affect  1988) l e a d  to  has  been  reabsorption  calcium  ( K i m and  reporting protein  by  sulfur  a d d i t i o n to  calcium balance.  sulfate  The  action, further  protein, in  to increased and  condition  1 9 8 1 ; Chu e t a l . , 1 9 8 5 ) .  of d i e t a r y  acids  this  Catabol-  hepatic  sulfite  r e n a l a c i d e x c r e t i o n and  reabsorption  of  calcium  by t h e  ( L i n k s w i l e r et a1.. 1981).  The  hypercalciuretic  s u b j e c t s f e d d i e t s of studies  with  complex  effect  of  proteins.  dietary  proteins  ( S p e n c e r e_t_ a_L. , 1978; 1 9 8 3 ) .  to  that  despite  calcium  observations  a  was  The  observed i n  results  o f human  s u c h as meat a r e q u i t e These  w o r k e r s were  h i g h meat i n t a k e o v e r s e v e r a l  e l i m i n a t i o n was  were  protein  purified  different  urinary  calcium  t o be r e l a t e d t o a c a l c i u r e t i c  changes i n the f i l t r a t i o n  show  in calciuria  hypercalciuria  Draper (1981)  t h e q u a n t i t y f e d , can a d v e r s e l y  kidneys  increase  in  filtration  et. a l . ,  demonstrated t h a t the source  oxidase  from the l e v e l of  results.  to a decrease i n f r a c t i o n a l  an i n c r e a s e  findings  This  protein intake,  c a l c i u m has n o t been shown t o a l l e v i a t e  attributed and  an  a high  independently  e_t_ a_l_. , 1979) .  with  therefore a negative i n t a k e of  However, w i t h  increased  (Allen  calcium absorption w i t h i n the  attributed  not s i g n i f i c a n t l y to 18  the  fact  months,  altered.  that  red  able  These  meat i n  addition  to being  high  Dietary  phosphorus  in protein,  has  hypocalciuric  effect  activity  phosphorus  of  been  known  reported  that  protein alone, diet  is  is  especially  that  phosphorus  activation increased  and  have  1988). priori to  overall  An  improvement  bone  or  from animal  other and  body  calcium  fed  high  of  Zemel e t  Draper  the a1.,  hypothesized  by  parathyroid  calcium resorption  and  and  calcium. that  phosphorus  metabolism  (Greger,  c a l c i u m r e t e n t i o n does n o t  utilization  ( Z e m e l and  of  calcium  mechanisms. as  to  and  coworkers  animals  increases i n  renal calcium  from  exhibited  whether or  reports  not  excess  content  disturbed.  19  while  to  Yuen and  Draper,  (1987) r e p o r t e d  that i n  calcium  enlarged  a.  in deposition  Conflicting  L i n k s w i l e r , 1981;  utilization  supplements,  were n o t  in  balance  high  t o i n c r e a s e d bone r e s o r p t i o n s e c o n d a r y  Moreover, Greger  s t u d i e s on  1981;  metabolism  on bone  human s t u d i e s e x i s t  hyperparathyroidism  a  human s t u d i e s  subjects  possible effect  homeostatic  phosphorus c o n t r i b u t e s  1983).  the  calcium  i n increased  with  and  r e n a l t u b u l a r r e a b s o r p t i o n of  on  result  in  e t a 1.,  calcium  surrounds  t o have a  This h y p o c a l c i u r i c  T h u s , Yuen and  subsequent e f f e c t s  fractional  time  when t h e p h o s p h o r u s c o n t e n t  w e l l (Hegsted  affects  i n phosphorus.  prominent  hypercalciuria  Draper, 1983).  Controversy may  the  i n c r e a s e d as  Yuen and  some  Numerous a n i m a l  i s ameliorated  1981;  for  rich  (Spencer et a l . . 1965).  p r o t e i n h i g h phosphorus d i e t . have  i s also  phosphate d i b a s i c  kidneys  w i t h >20  bone c a l c i u m  fold  contents  Normal affect  calcium  research nor  dietary  has  calcium  general,  phosphorus  absorption  adversely.  been c o n f o u n d e d by occur i n i s o l a t i o n  a  high  levels  protein  the  have  Controversy  been shown t o  in this  fact that neither  i n foods or  intake  not  i n the  D r a p e r , 1983; In c o n t r a s t  o f p r o t e i n on information on  calcium  seems t o i n d i c a t e an  the  protein  of  osteoporosis. also  low  collagen  with  of  calcium  concern are  synthesis  the  calcium  A d i e t low  and  This  a  is  paucity  of  b a l a n c e and  the  may  e l d e r l y who  components  skeletal  c h i l d r e n (Garn  Moreover, previous  section  be  of  susceptible  which are  in the  that  necessary  to is for  t h e y r e l a t e t o bone  components, which c o n t r i b u t e  to  ( F r o s t , 1988). has  been  cortical et  al..  reported  to  have  bone t h i c k n e s s  i n both  1964;  et a l . .  Crosby  s t u d i e s have r e p o r t e d  i n humans s u f f e r i n g f r o m a p r o t e i n  20  especial  retention.  i n p r o t e i n i s c o n s e q u e n t l y one two  of  protein  a d o p t a d i e t low cross  malnutrition  bone l e n g t h  l e v e l s of  low  homeostasis; a  Protein-calorie  1983).  or  r e t e n t i o n may  t i s s u e content  y o u n g a d u l t s and  is  bone m i n e r a l i z a t i o n as  on  dietary levels  there  calcium  a r c h i t e c t u r a l matrix  effects  (Yuen  p o s s i b l e d e l e t e r i o u s e f f e c t s of  bone s h a p e , s i z e and  deleterious  increased  calcium  impact of h i g h  utilization.  p h o s p h o r u s on  i n phosphorus;  g r o w t h and  and  reports  to maintain  population  the  e f f e c t of d e f i c i e n t ,  p r o t e i n and  People concerned  on  bioavailability,  bioavailability past  In  1988).  to r e s e a r c h  calcium  on  concern given dietary  Greger,  of  phosphorus,  human body.  r e q u i r e m e n t f o r b o t h d i e t a r y p h o s p h o r u s as w e l l as and  area  a reduction  in  d e f i c i e n c y (Adams  and  Berridge,  negative  and  bone c a l c i u m deficiency  can  lead  ( L o t z et  retention The the  as  i n the  by  dairy origin, are  subjected  or  during  nutritional absorption. treatment  the are  to improve  from the  diet. and  processing  milk  K-casein;  of p h o s p h o s e r y l  and  can  denatured  rather,  reduce  affect from  the  calcium thermal  whey p r o t e i n s  and  of  consumption,  polypeptides  residues;  concept i s  of  (p-  proteins;  M a i l l a r d browning  1985). sterilization  denaturation  interest  i s ( J - l a c t o g l o b u l i n ,which  of  thiol-disulphide a  metabolism  to  cross-linking  in  as  treatment  of  results  a  reports  phosphorus i s  form, but  either prior  casein  interaction  P a s t e u r i z a t i o n or the  general  e s p e c i a l l y those  c a s e i n p r o t e i n s and  (Swaisgood,  phosphorus  these  This  value  with  to a f f e c t  a contributing factor  Heat  include  cross-linked  be  s e l d o m consumed i n t h e n a t i v e  of the  a  elderly alike.  A l t e r a t i o n s to the  via  calcium  pasteurization.  dephosphorylation  casein  Dietary  together,  that p r o t e i n s ,  to thermal  lactoglobulin)  reactions  Taken  of c a l c i u m  fact  not  bone m e t a b o l i s m and  o f d i e t a r y p r o t e i n may  bioavailability  but  o f d i e t a r y p r o t e i n and  a therapy  young and  quality  highlighted  1968).  content,  1973).  disturbed  restriction  I t i s noteworthy that  been shown t o r e s u l t i n a l o w e r body  (Bollet,  to  al..  the  contraindicated  1983).  r e d u c e d bone c o l l a g e n  composition  suggest that  in  Parfitt,  p r o t e i n b a l a n c e has  weight gain  malaise  1969;  result  of  whey p r o t e i n s .  of  fluid  Of  milk  particular  f o r m s an  association with  K-  interchange.  Proteins  be  the 21  processing  reaction  of  can  dehydroalanyl  residues with  (from  lysine  B - e l i m i n a t i o n of c y s t i n e , phosphoserine or s e r i n e )  residues  lysinoalanine  at  the  (Swaisgood,  1985).  r a n g e 110 t o 140°C r e s u l t s casein polypeptides  (Howat  Maillard  formed from the c o n d e n s a t i o n with  reducing  mineral  together,  these  may  bind  q u a l i t y , Weeks effects milk.  bioavailability e f f e c t s of  the  Ho and  effect  and  King  carboxyl  y l a t e d by h e a t t r e a t m e n t  2e.  of the  B e l e c and  products  can be  amino a c i d r e s i d u e The  under these  ionic  i n the  calcium,  availability conditions. potentially  1987).  processing  of milk  digestibility  e-  Taken and o t h e r  and b i o l o g i c a l  ejt. a 1 . . 1 9 8 7 ) . thermal  (1985)  treatment  d i d not  on c a l c i u m  who r e p o r t e d  a s s o c i a t e d with not only  it  their  evidence f o r these  Waugh ( 1 9 6 5 ) ,  possibly with  1934;  (Rendleman,  thermal  of  of heat p r o c e s s i n g Supporting  to  of milk  due t o s c i s s i o n o f  Wright,  i s reduced  (Mauron, 1973; D e s r o s i e r s Despite  and  of polypeptide  d i e t a r y p r o t e i n s , may r e d u c e value  residues  l a c t o s e t o form m e l a n o i d i n s .  melanoidins  t o form  dephosphorylation  Browning R e a c t i o n  o f amino a c i d s s u c h as l y s i n e Moreover,  position  Heat t r e a t m e n t  at phosphoseryl  Jenness, 1962a,b).  group  i n extensive  an o x y g e n - p h o s p h o r u s bond  amino g r o u p s  e-amino  observe  highly bioavailable. Lactose:  22  any a d v e r s e  from  pasteurized  observations  is  t h e work o f  that milk m i c e l l a r calcium.is r e s i d u e s , but a l s o  Thus, m i l k p r o t e i n s  may s t i l l  protein  absorption  casein phosphoseryl groups.  on m i l k  bind  ionized  dephosphor-  calcium  keeping  Various  hypotheses  mechanism of absorption  the l a c t o s e  (Kline  pH  and  been  effect  et. a l . ,  and S a l t m a n , 1 9 6 3 ) . luminal  have  suggested  on  1932;  S c h a c h t e r e_t a l _ . , 1961;  of  the r e d u c t i o n  calcium  fermentation  o f l a c t o s e by  intestinal  1932);  formation  a  the  absorption  (Charley  intestinal  mucosa  cell  patency  (Schachter light  and  allow  Filer,  and  milk  bioavailability  leakage  of  These  et a1. ,  1959;  shown t o enhance t h e proximal  as  system  of  (1959), enhanced  1978;  initially calcium  inhibition  to  the  not unique  effect  of  as  reported  This  distal  age  and  Behar  Armbrecht,  identified  small  Armbrecht,  intake,  this of and  t o enhance t h e (Lengemann  d i s a c c h a r i d e has of c a l c i u m intestinal 1987).  The  been  i n both segments  effect  and K e r s t e i n , 1976; Lengemann and as  Further, 23  to  was  the v i t a m i n D endocrine  1987).  the ileum  absorption.  serosa  (Vaughan  and p o s s i b l y o t h e r m i n e r a l s  (Lengemann e t a l . . 1959;  Kimberg,  is  paracellular transport  calcium  co-  luminal concentrations  has been  ( A r m b r e c h t and Wasserman, 1976; independent  for  1976).  e t a 1 ., 1987) .  well  ( K l i n e et a1. .  the  calcium  that optimal  sugar, l a c t o s e ,  Greger  to the  t h e o r i e s have been r e f u t e d i n  and Wasserman,  of c a l c i u m  intestine  complex  and  l a c t o s e a r e r e q u i r e d f o r an  I960; Armbrecht  The  and  1963);  of  and  a e r o b i c metabolism to d i s t u r b mucosal  a_l_. , 1 9 6 1 ) .  and f u r t h e r  both calcium  the  microflora  of e v i d e n c e t h a t the l a c t o s e e f f e c t  sugar a l o n e ,  the  Charley  s o l u b i l i t y due  calcium-lactose  Saltman,  cellular  and  e_t_  of  explain  calcium b i o a v a i l a b i l i t y  These i n c l u d e d  enhancement  to  the Pansu  Nellans  coworkers  site  of l a c t o s e  et  a 1 . ( 1979)  reported  that  calcium  lactose, despite  the  p r o t e i n , as w e l l as in  the  proximal  importance process  enhancement o f Lactose reported well  as  be  lower  since both  small  by of  or  this  binding  c a l c i u m were  reduced  calcium  ileum,  calcium  calcium fluid  mucosal c e l l  (Bronner, 1987). w e l l as  intestinal  by  absorption  l a c t o s e mediated  has  shift  from the  from  mucosa  shift  from the  of  ( L e i c h t e r and  serosa  ileum  with  reducing  been c a r r i e d galactose, and of  the  patency i n c r e a s i n g  the  in  a  post-weaning  it  Tolensky,  and  so,  to reach  theory do  the  out  distal  Products:  form m e l a n o i d i n s .  w i t h numerous s u g a r s ,  amino a c i d s  l y s i n e (Powrie  i n the  et a1. . 1986). 24  pentoses,  f r e e form or the The  are  residues  I n v e s t i g a t i o n s have  namely g l u c o s e ,  m a l t o s e , l a c t o s e , rhamnose and  secondary  not  1975).  c o n d e n s a t i o n o f p r o t e i n amino a c i d  sugars to  is the  Non-enzymatic browning r e a c t i o n s , or M a i l l a r d r e a c t i o n s , c h a r a c t e r i z e d by  as  within  activity  allowing  been  ileum to  lactose  Studies  lactase  2f. M a i l l a r d Browning Reaction  absorption  a d u l t humans s u p p o r t t h i s  disaccharide,  intestine intact  fed  demonstrated the p o t e n t i a l  a fluid  The  disturb  model as  lack  calcium  of  animals  bioavailability.  efflux  would  rodent animal  amount of  o s m o t i c a l l y a c t i v e load  space between c e l l s  hydrolyze  intestine,  K e r s t e i n , 1976).  which  in  paracellular  characterized  d e p e n d e n t upon an lumen  This  passive,  increased  ( B e h a r and  intestine.  calcium  enhanced  active transport  m e d i a t e d enhancement o f  to an  the  the  the  was  f a c t t h a t both the  small  of  in  absorption  fructose,  with  primary  e-amino g r o u p  browning r e a c t i o n  products  (melanoidins)  contribute  flavour  of  (1986)  reported  cooked f o o d s .  components provided glycine  a  further  that  was  well  as  These  activity  of m e l a n o i d i n s  s y s t e m s and 2g.  studies  binding  must be and  metal  calcium  of  showed  no  chelating to  derived  measurable  glucose  of m e l a n o i d i n s may  potential  of  browning  from  a  coffee binding  toast.  quite  of  number  derived be  -  potential.  the  and  a1.  (1973)  calcium  i n pigments from c o f f e e  respectively  et  Tappel  chelating  and  browning  g r o u p of  related  pigments  potential  Thus,  from  food  different.  Interactions:  Because n u t r i e n t s , isolation  Homma  f o o d pigment, i n b o t h a model  model r e a c t i o n s ,  Mineral  and  functional  t o be  browning  brew.  calcium  the  thought  donor groups i n the as  the  by  potential  Adhikari  possessed  examined  m e l a n o i d i n s , which  the  coffee.  ( p i g m e n t s ) , aroma  conducted  chelating  evidence f o r  (1987)  colour  Studies  in  melanoidins  reaction,  the  copper  present  Rendleman  acidic  to  in  either  considered  nutrient  such  relation  to  food  i n any  metabolism.  constituents  and  or  play as  discussion  a role  calcium.  interactions reviewed  in  m i n e r a l s , do  physiological  Interactions  may  d i s c u s s e d and  therefore  i n the  systems,  of n u t r i e n t  with  other  relative  the  zinc  in  interactions  bioavailability or  bioavailability  and  literature  exist  minerals,  C a l c i u m a b s o r p t i o n and with  not  food of  metabolism  magnesium ( F o r b e s et  a in  have been a1.,  1979;  G r e g e r , 1989 ; G r e g e r et. a l _ . , 1 9 8 9 ) . An and  a r e a of  calcium  p a r t i c u l a r c o n c e r n i n the  i s the  e f f e c t of p h y t a t e on 25  bioavailability the  of  a b s o r p t i o n of  zinc these  minerals.  Research  interest  in  soy  studies, phytate to  be  less  in  products  area  as  has  protein  increased sources.  along  with  From i n v i t r o  c o m p l e x e s o f c a l c i u m and z i n c t o g e t h e r a r e n o t e d  soluble  (Oberleas,  this  1973;  than  complexes  Champagne  and  of  the i n d i v i d u a l  Phillippy,  1989).  metals  Moreover,  F o r b e s e t a1 . ( 1979) r e p o r t e d t h a t a d d i t i o n o f c a l c i u m as c a l c i u m carbonate z i n c by  to  soy products  rats.  d i e t a r y phytate  i s well  levels  i s noteworthy i n l i g h t  phytate  and  effects  (Greger  essential  minerals  of  supplements magnesium. ability some  which  on  findings  being  of  respective  1987;  susceptible to  Fordyce  due  also  and  to  the  contain  coworkers  This  less  mineralization 26  These two  amounts  of each  relationship i s  significant  of calcium amounts  of  (1987) s t u d i e d the b i o a v a i l -  supplemental  that  t o have p o s s i b l e  proliferation  available  contained  were  l e v e l s and  1989).  relative  1989).  commercially  preparations  ejt a l .  b i o a v a i l a b i l i t y and  Greger,  the  (Greger,  can  that  This m u l t i p l e i n t e r a c t i o n  been r e p o r t e d  affect  demonstrated  f o r bone  by t h e f a c t  i n the d i e t .  their  can  also  u t i l i z a t i o n of  r e l a t i o n s h i p b e t w e e n bone z i n c  e_t_ a l _ . ,  Greger  which  containing calcium  the  consideration  of v a r i o u s  of  Their  observations  from the d i e t  worthy  as  of  magnesium have  metabolism  absorbed  recognized 1989).  x calcium/zinc ratios  antagonistic  decreased  (Greger,  reported a  Calcium  in  These o b s e r v a t i o n s a r e s u p p o r t e d  zinc b i o a v a i l a b i l i t y  ( 1 9 8 7 ) who  resulted  rats able than  calcium  supplements,  magnesium, i n r a t s . fed  the  to absorb counterparts  magnesium  and u t i l i z e fed milk.  Alternatively,  s t u d i e s i n humans have shown t h a t t h e  excess calcium  can  other  minerals  dietary  regard  in  of  et  effect  O'Dell,  Intakes  intestinal  (Greger  calcium  ( M o r r i s and  impair  1963;  calcium  al..  absorption 1981).  increase  of magnesium  Thus, h i g h  the  magnesium  and  magnesium a r e  and  However,  (1981) d i d not calcium  and  in  detect  levels  of  requirement  of f u r t h e r c o n c e r n  adequate c a l c i u m  the development of r e n a l c a l c u l i 1987).  and  G r e g e r e_t aj_. , 1 9 8 1 ) .  to i n t e r a c t i o n s w i t h d i e t a r y phosphorus.  magnesium d e f i c i e n c y  i n g e s t i o n of  and  In c o n d i t i o n s phosphorus  is a possibility  (Greger  s t u d i e s w i t h humans, G r e g e r and a  significant  phosphorus  levels  on  effect  with  of  retention  of  intake, e_t a 1 . .  coworkers  varying dietary of  calcium  and  magnesium. Lactose calcium,  but  can  affect  the  a l s o magnesium  intestinal and  absorption  z i n c (Greger  calcium  calcium  (calcium  c h e l a t e and  nonfat  greater deposition and  from dolomite  However,  d i e t a r y sources, when  compared  milk, respectively.  may  be  Thus, the  and  zinc  i n both  workers  In  forms  amino a c i d reported  a  dolomite  calcium b i o a v a i l a b i l i t y  differed  a b s o r b e d and  calcium  calcium  utilized  phosphate  to note,  l a c t o s e enhancing e f f e c t  a l t e r e d by m i n e r a l  only  t o bone f r o m  being  to  various  dolomite,  these  It is instructive  a d i e t a r y supplement r i c h ates.  dibasic,  dry m i l k ) ,  o f magnesium  m i l k based d i e t s .  between these  phosphate  not  et a1. . 1989).  s t u d i e s w i t h r a t s f e d d i e t s c o n t a i n i n g l a c t o s e and of  of  and  less  dibasic  and  that dolomite  is  magnesium  carbon-  on  calcium  absorption  i n t e r a c t i o n s (Greger  et a1..  1989).  27  2h.  of  Endocrine  Factors:  Calcium  absorption  endocrine  and  hormones,  calciferol),  metabolism are  namely  parathyroid  hormones  levels  of c a l c i u m r e l a t i v e l y  The  function  to  reviewed  (Deluca,  Vitamin  D largely  has  specifically  t h e duodenum and active,  i n the  transcellular  be  sun  or u l t r a v i o l e t  obtained  (UV)  product  and  microsomal  D3  to are  for  D.  circulated  conversion  metabolite to  the  1,25-dihydroxycholecalciferol hydroxylase.  The  of c i r c u l a t i n g  calciferol  is  final  o r by  UV  of a  converts then  active  absorp-  intestine,  exposure  termed  light  where  derived  the  precursor,  not  ergosterol. 28  7-  skin.  hepatocyte  them  translocated form of the  to  fish,  precursor, in  D  vitamin  to to  25the  vitamin,  by m i t o c h o n d r i a l  i m p o r t a n c e of s u n l i g h t exposure i s a p p a r e n t serum 2 5 - h y d r o x y - v i t a m i n  the  Vitamin  milk, fatty  liver  (calcitriol)  1988).  D regulates  (calciferol)  to the  Eraser,  small  1982).  conversion D3  exten-  absorption.  from f o r t i f i e d  the  been  calcium  D i e t a r y v i t a m i n D,  vitamin  This  has  1986;  the d i e t  D3-25-hydroxylase  vitamin  hydroxyvitamin  of  serum  1).  intestinal  (Allen,  (CT).  plasma, or  Thus, v i t a m i n  liver  a number  calcitonin  r e g i o n of the  routes,  light.  v i t a m i n D i s the  D2  proximal  i s obtainable  dehydrocholesterol,  on  component o f c a l c i u m  eggs and  84%  (Figure  Delvin,  jejunum.  cheese, b u t t e r ,  kidney  and  circulating  constant  1981;  t h r o u g h two  (ergosterol)  Vitamin  keep  i t s effects  upper  can  D2  (PTH)  by  (1,25-dihydroxychole-  r o l e of v i t a m i n D i n c a l c i u m m e t a b o l i s m  sively  tion,  vitamin D  hormone  These  regulated  D i s d e r i v e d from  1-aas the  Calcitriol calcium  at the  acts level  to  the  initiation  of  calcitriol  calcitriol the  not  induce  intestinal  receptor  yet  enhanced  lumen  D  is  increase  in  in  c o n d i t i o n s of  l a c t a t i o n where t h e  calcium  PTH  blood  other  and  balance the  by  PTH  by  takes  filtrate location levels  to  place  (Aurbach,  PTH  tubule which  is  tissues.  intestine  inhibited  the  from  increased the  bone c a l c i u m  of  importance  of  l e v e l s of  1982).  i s a c t i v e a t a number o f  levels  i s high  pregnancy  ( P a t t and  occurs  Luckhardt,  calcium-phosphate balance  The  and  of  under  1942).  In  between  the  r e g u l a t i o n of e x t r a c e l l u l a r  calcium  i n the k i d n e y s ,  bone and  secondarily,  1988).  calcium  excretion is  to  Renal receptor  to  evidenced  by  sites  of c a l c i u m  (Agus et_  skeleton  as  calcitriol  (Allen,  increase The  the  Calcium  transcalcium  bone r e s o r p t i o n  numbers and  ( P e c k and  distal  glomerular  extracellular  r a t e of  increased  in  from the  a l . . 1981).  o f o s t e o c l a s t s , t h e bone r e s o r b i n g c e l l s Since  levels  requirement  which binds  PTH  the  from  and  i s a n o t h e r f u n c t i o n o f PTH.  e n h a n c e d by  of c a l c i u m  of  calcium deficiency  signals reabsorption be  role  administration  S t i m u l a t i o n o f s e c r e t i o n o f PTH  regulates  signal  This  cytoplasmic  circulating  c o n d i t i o n s marked by h y p o c a l c e m i a t h i s way,  as  of  intestinal  (Norman, 1 9 7 9 ) .  Nevertheless,  P a r a t h y r o i d hormone (PTH) calcium metabolism.  the  f o r t h i s hormone t o  to i n c r e a s e d  that  absorption  of  active transport  prior  clear  Cells  clarified,  CaBP ( S p e n c e r e t a l . . 1 9 7 8 ) . vitamin  sites  o f CaBP s y n t h e s i s  is  may  intestinal  o f CaBP s y n t h e s i s .  mucosa have c y t o p l a s m i c for  stimulate  activity  Klahr,  1979).  i s a s s o c i a t e d w i t h phosphate, plasma l e v e l s 29  of  phosphate i n c r e a s e w i t h that  of plasma  p h o s p h a t e i s c o m p e n s a t e d f o r by phosphate  excretion  intestinal  calcium  on  (Agus  Thus,  PTH  et.  absorption  renal hydroxylation  of  increases  of  the  the  intestinal  T h e s e f u n c t i o n s o f PTH calcium the  pool  from  activity  secreted  by  (CT),  the  f u n c t i o n o f CT o f bone  are  a  o f PTH  thyroid  skeletal 2i.  the  The  of  renal  increase  in  D  (Fraser,  renal  has  1-a-hydroxylase  its  increase  1980).  e f f e c t s on  calcium  f o r the  the  absorption.  m o b i l i z a t i o n of  contents  to  equilibrate  calcium. acid  gland  i s hypocalcemic  on  the  intestinal  polypeptide  (Aurbach,  in  nature,  1983).  hormone, i s  1988).  due  The  primary  to i t s i n h i b i t i o n  therefore, inhibition  f r o m t h e bone ( T a l m a g e e t a l . , effects  of  responsible  32-amino  r e s o r p t i o n and  excess  i s secondary to i t s e f f e c t s  which then  and  of e x t r a c e l l u l a r  1981).  PTH  mucosa t o  body p o o l s  Calcitonin  a1 ..  by  The  m e d i a t e d s t i m u l a t i o n of  25-hydroxyvitamin  enzyme t o p r o d u c e c a l c i t r i o l cells  PTH  calcium.  of c a l c i u m  Thus,  CT  release  inhibits  bone o s t e o c l a s t r e s o r b i n g c e l l s  to  the  preserve  integrity.  I m p a c t of N u t r i t i o n a l Individual  absorption  and  nutritional  Calcium  c h a r a c t e r i z e d by  an  calcium  absorption  cellular  t r a n s p o r t of  on  status  metabolism.  vitamin D deficiencies, syndromes.  Status  Calcium can  also  impact  on  calcium  These c o n d i t i o n s i n c l u d e c a l c i u m  respectively,  deficiency, increase (Pansu  Absorption:  the al..  calcium w i l l 30  well  or n e g a t i v e  in et  as  be  as  1981).  malabsorption  calcium balance,  efficiency The  up-regulated  and  of  is  intestinal  active, transi f the host  is  vitamin PTH  D replete  secretion  (Bronner, 1987).  in  conditions  of  calcitriol  synthesis  shown t h a t  the  absorption  in  s t a t e s of c a l c i u m  The  can  play  colon  observed  by  an  deficient  diet  not  is  t o an  have  and  increased  role in  resections.  extent  stimulate  role  calcium  in  have  calcium  absorption  ( 1 9 8 0 ) i n s u b j e c t s who Adaptation  over  that  by  d e f i c i e n c y (Favus et a l . . 1980).  coworkers  possible  to  Furthermore, studies  an  important  Hylander  intestinal  albeit  may  mechanism i s m e d i a t e d  hypocalcemia  (Norman, 1 9 7 9 ) .  colon  undergone  This  an  to  a  had  calcium  extended p e r i o d  would prevent  as  of  a negative  time,  balance  ( S p e n c e r et_ a l . . , 1 9 6 9 ) . Classically, insufficient (Allen, causal  vitamin  sunlight  1982).  D  deficiency  exposure  Vitamin  and  the  D deficiency  a g e n t o f bone m a l f o r m a t i o n and  children  and  istration  of  adults. the  CaBP  within  vitamin  D i n the  of f l u i d  6  to  will  8  prevention vitamin  of D2  the  toxicity  in reference  supplemental hypercalcemia, soft tissues  vitamin  bone  has  rickets  D  lead  IU p e r  a  litre;  D.  vitamin  e x c e s s bone c a l c i f i c a t i o n  and  of  e f f i c a c y of fortification  Allen,  This  admin-  synthesis  The  t o the  supplements  Excess  to  rickets  t h o u g h t t o be  hyperalimentation  to c a l c i u m  ( F r a s e r , 1988).  been  in increased  of e x c e s s v i t a m i n  D.  disease,  (Norman, 1 9 7 9 ) .  (400  linked  D-dependent r i c k e t s ,  result  hours  been  deficient mineralization in  vitamin  note about v i t a m i n  because of pertinent  vitamin  milk with  cautionary  In  has  1982).  A  i s necessary is especially  which also D intake  can  calcification  contain lead  to  of  the  M o r e o v e r , s t u d i e s have shown t h a t 31  vitamin  D,  sufficient  with only al . .  a few  f o r s e v e r a l months i s p r o d u c e d  hours of  absorption  malabsorption alcoholism,  discussed  and on  above.  Chronic  status  and  and  (Hepner  ( P o s k i t t .et_  demonstrated that the a c t i v i t y  of  liver  calcium  particular,  in  effect  vitamin  result  i s secondary  malabsorption time,  can  1976).  o f f a t and  to  may  1965). fat  vitamin D  these  have a n e g a t i v e  workers  impact  D3-25-hydroxylase.  on  Surgical  of n u t r i e n t s ,  gastrointestinal  to a  been  through  c a l c i u m and  Furthermore,  fat  skeletal  (Saville,  i n malabsorption  are  and  tract resections.  reduction i n absorptive area,  v i t a m i n D (Compston et a1. . 1980).  calcium malabsorption  colonic  on  of  have  been l i n k e d mass  by  chronic  effects  absorption  bone  effects  cirrhosis  hepatic  that  impaired  mediate c a l c i u m malabsorption  a_l_. ,  procedures  The  calcium  decreased  be  fat malabsorption,  a l c o h o l i s m has  subsequent  et  to  may  procedures.  intestinal  a l c o h o l i s m may  malabsorption  metabolism  secondary  surgical  demineralization Chronic  and  syndromes  malabsorption  and  exposure  skin  1979). Calcium  The  summer s u n l i g h t  i n the  i s c o m p e n s a t e d f o r by  increased  c o n t r i b u t i o n s to calcium absorption (Hylander  and Over  ileal  et a l . .  1980). 2j.  Physiological Calcium  requirements  vary at s p e c i f i c p r e g n a n c y and In i n f a n c y ,  Status:  stages  lactation, both  animals  and  a b s o r p t i v e c a p a c i t y can  of  life,  and  menopause and  and  namely  humans 32  infancy, o l d age  exhibit  a  be  seen  to  adolescence,  (Allen, reliance  1982). on  the  diffusional,  paracellular  component  of  calcium  absorption  ( B a r l t r o p et_ a l _ . , 1977 ; G h i s h a n et_ a l _ . , 1 9 8 0 ) .  In  saturable,  i s not  until for  the a  transcellular third  observed  week o f l i f e  diffusional  i n f a n t s was  transport  a linear  Adolescence  by  represents  increase  in  length, width  1989).  The  requirement  1 t o 1.5g  calcium  calcium  rat  transcellular  well  milk  model,  skeletal  production  growth  calcium  requirements,  surface  indicated  paracellular  during  et a1. that  the  second during  (1969)  both  the  of  calcium  vitro  everted  ( 1 9 8 0 ) were a b l e  c a l c i u m t r a n s p o r t by  decreased 33  Nagel  in  with  c a p a c i t y of  components  S i m i l a r l y , u s i n g an  demonstrate increased i n t e s t i n a l  p e a k e d a t p a r t u r i t i o n and  f o r bone  recommendations  S t u d i e s by K o s t i a l  m e t h o d o l o g y , W r o b e l and  initiated  Kaup,  1990).  absorptive  animal  a b s o r p t i o n were i n c r e a s e d . sac  and  as  and  nutrients  rapid  and  bones  demand f o r c a l c i u m i s c o u p l e d  transport.  using a lactating as  and  increased  who  long  p l a c e a d d i t i o n a l demands f o r  fetal  human  (1977),  the  l e a d i n g to  Hanley,  efficiency  was  time  and  and  changes i n the  by  formula.  time,  (Grimston  The  a1.  as w e l l as d e n s i t y ( S h a h a n i for  respectively.  increase  this  to f a c i l i t a t e  to  Evidence  absorption  B a r l t r o p et  day  t h e m o t h e r , due  intestinal  calcium  During  lactation  operative  p e r i o d of a c c e l e r a t e d growth  i n c r e a s e d at t h i s  P r e g n a n c y and  intestinal  a  r a t , the  a l • . 1980).  c a l c i u m per  in adolescence  on  et  dose r e s p o n s e from m i l k - b a s e d  development.  of  in  s t u d i e s of  bone t i s s u e  development i s  (Ghishan  component  provided  of c a l c i u m  the  rats.  to  This  week o f g e s t a t i o n , lactation.  Further,  changes i n observed  to occur;  transport studies  the a b s o r p t i v e s u r f a c e area  of  the  length  calcium  indicate  was  l a c t a t i o n provided Calcium  and  women d i s p l a y a d e c r e a s e d of 60 y e a r s .  a b s o r b one  t h e ages o f 20  transcellular  female, occurs  the  a p p e a r s t o be  the  decrease  in  in  suggested  in  calcium  i n e i t h e r o f two  e f f e c t of  PTH  on  a direct  and  on  thus,  men past  to  90  between  Age-related  the  of  case  t o be  (Nordin  metabolism  and  calcium 34  of e s t r o g e n  that  responsible for  homeostasis  o f bone  1986).  the  The  role  has  been  In the  first,  r e s o r p t i o n which would  t h e r e f o r e , an  would  synthesis.  postmenopausal  et a1. . 1976).  theories (Allen,  absorption  the  levels  stimulatory effect  intestinal  Both  counterparts  Kaup, 1 9 8 9 ) .  circulating  calcitriol  w o u l d have  elderly,  e_t_ a_l_. , 1976 ;  a d e c l i n e i n serum c a l c i u m and calcium  i n the  (Nordin  lead  Intestinal  pregnancy  component  a c t s to reduce the r a t e  PTH.  are  a decrease i n  estrogen to  mechanisms  r e s u l t of  calcium absorption  estrogen  these  b e t w e e n t h e ages of 70  and  In  active  together,  impaired  c a l c i u m as  absorption  also  t o a b s o r b c a l c i u m once  a t t h e menopause, i s c o n s i d e r e d  reduction of  ability  (Shahani  A r m b r e c h t e_t_ a l _ . , 1 9 7 9 ) .  for  the p o s t - m e n o p a u s a l f e m a l e .  as much  t o 59 y e a r s  calcium malabsorption the  in  be  F u r t h e r , those  third  Taken  are  i n t a k e i s adequate.  appears to  more  years  particularly,  available  calcium absorption  that calcium  absorption  intestine  i n c r e a s e d c a l c i u m demands o f  and  t h e age  jejunum  increased.  that maternal  a b l e t o compensate f o r the and  of  of the  be  increase in  enhanced  In the second, on  calcitriol  absorption.  by  the  estrogen synthesis  Compounding  the  effect  of  decreased  menopausal renal  female  calcium  (Heaney e t  intestinal are  the  r e t e n t i o n and  a 1 . . 1978).  calcium  absorption  coexisting finally  a  The n e g a t i v e  i n the post-  c o n d i t i o n s of decreased negative  calcium  calcium balance  balance  that  i n t h e p o s t - m e n o p a u s a l f e m a l e makes h e r e s p e c i a l l y p r o n e resorption levels. mass  the  eventually,  (Aloia  Dairy  of  can l e a d  to  calcium  to maintain  to accelerated  osteoporosis  in  t o bone serum  l o s s o f bone  the post-menopausal  et a l . . 1985).  Foods  Dairy products concentrations  LaCroix,  represent  ranging  mg/100 g i n f l u i d and  mobilization  Bone r e s o r p t i o n  and  female 3.  in  occurs  of calcium  f r o m 50 mg/100 g i n c o t t a g e  m i l k and 716 mg/100  1980).  only a r e l a t i v e l y  an e x c e l l e n t s o u r c e  Dairy products  g in  c h e e s e , t o 113  cheddar cheese  (Wong  are unique i n c o n t a i n i n g not  l a r g e amount o f c a l c i u m ,  t h a t enhance t h e b i o a v a i l a b i l i t y  with  but  also constituents  of that calcium,  namely the m i l k  s u g a r l a c t o s e ( d i s c u s s e d a b o v e ) and t h e c a s e i n p r o t e i n s . 3a.  Calcium The  salts  Distribution  calcium  i n Milk:  i n milk  distributed  i s present  i n soluble  (32mM),  two-thirds  form, the remaining is  free  ionized  third Ca *  (22mM)  are  and c i t r a t e  phases (Pyne,  the d i s t r i b u t i o n  (10mM) i s (3mM).  2  phosphate  and c o l l o i d a l  McMahon and Brown ( 1 9 8 4 ) r e v i e w e d milk  as  of  The c o l l o i d a l  polypeptides,  or  bound 35  colloidal  p o r t i o n of which calcium  c o n s i s t s of complexes w i t h phosphate e s t e r s o r c a r b o x y l casein  calcium i n  i n an amorphous  soluble, a  1962).  fraction groups of  w i t h p h o s p h a t e s and c i t r a t e s i n  association fraction  with  is  the  comprised  phosphate, c i t r a t e exists  between  the  Casein  micelles.  of  the  o r serum  The  freely  and  hydrated  colloidal  and  casein  between  polypeptides  the  of  d i s p e r s i o n of  proteins  stabilized  a s s o c i a t i o n by  bridges.  The  casein  their  ation.  The  sensitivity  and  s  role of  as  solubility  cr  of  the  well and  Bingham et  relatively  sites the  as  a l . . 1972 ; and  Aoki  has  s  third  strating  that  the  organic  binding  the  colloidal  calcium  i n t e r a c t i n g casein calcium  phosphate  outer  of  been  dephosphorylated (Yamauchi  e_t. a_l_. , 1 9 8 5 ) . decreased  species  will 36  of  K~ the  caseins  et  calcium  using  both  caseins al..  their  calcium  caseins,  8  coagulate  of  binding  t h u s demon-  i s important  for  Calcium s e n s i t i v i t y  a i-casein  in  1967;  Dephosphorylation  t h a t of n a t i v e  and  i n the  investigated  p h o s p h o r u s component  d e p h o s p h o r y l a t e d whole c a s e i n  the  with  surface  calcium-sensitive  (Yamauchi et a l . . 1967).  casein  The  p o s t - t r a n s l a t i o n a l phosphoryl-  studies  a -caseins  one  two  calcium  hydrophobic core  polar  enzymatically  to  these  micellar  phosphate e s t e r m o i e t i e s  precipitation  casein  are of  caseins  capacity  calcium  a relatively  p-caseins  numerous  The  native,  equilibrium  c a s e i n p r o t e i n s of the m i c e l l e s are  forming  c o n t r i b u t i n g to a  to t h e i r  whole  in  constituent  fl-caseins  micelle. due  An  and  the m i l k m i c e l l e s .  c a s e i n m i c e l l e s form a c o l l o i d a l  and  cation  Micelles:  phosphate  a  calcium  1985).  A close r e l a t i o n s h i p exists  a  soluble  p r o t e i n complexes.  soluble  phosphate (Swaisgood, 3b.  casein  i s decreased at a lower  of  since  critical  calcium al . .  concentration 1967 ;  micelles three  Bingham  suggesting  larger  that there  phosphate  micelles  their  native counterparts  .et. aj_. ,  1972 ;  f o r m e d by d e p h o s p h o r y l a t e d  times  organic  than  a l~,  and  fewer  are  calcium binding  i n number t h a n  ester moieties  sites  i s found  ently  dephosphorylated.  l , when  B  dephosphorylated counterpart  a 2 casein  was  attributed  to  the  phosphate groups which n e u t r a l i z e d facilitating  hydrophobic  support  aji-casein  and c o w o r k e r s quite  pH  compared  to  charge of  and p r o t e i n  neutral  e v e n when  charge  is  maintained  phosphate m o i e t i e s suggest  that  the  casein polypeptide potential, is  internally  respectively  Calcium  charge a t  groups by  of  the  species  protein;  the dephosphorylated  organic  conferred of  2  phosphorus i s essential  by s o l v e n t and  the c a s e i n  molecules  Exchangeability: 37  this  negative  p r o t e i n when t h e  Taken t o g e t h e r ,  these  data  component o f t h e v a r i o u s for their  i n micelle formation.  phosphate. 3c.  S  p h o s p h a t e g r o u p s a r e n e u t r a l i z e d by C a * ,  have been r e m o v e d .  and s t a b i l i t y  charged  a 2~casein  8  to ionized carboxyl  8  precipitation.  hand, n a t i v e a l - c a s e i n r e t a i n s a n e g a t i v e  pH,  differ-  its a 1  l o s s of n e g a t i v e l y  On t h e o t h e r  due  than the  The i n s o l u b i l i t y o f  the net  interaction  were  foralternate  behaves  -  S  at n e u t r a l  -  S  other  i n t h e work o f A o k i a 2 casein  2 +  The  native micelles,  sites  Further  that  a  and C a  i n dephosphorylated  (.1985), who d e m o n s t r a t e d from  et. al_. , 1985).  K-casein  a  ( B i n g h a m .§_t. a 1 . . 1 9 7 2 ) .  calcium binding  Aoki  (Yamauchi e t  calcium-binding  Micelle  hydrophobic and  stability  interactions,  the c o l l o i d a l  calcium  The  relatively  contributes  to  open  the  equilibrium  c o n s t i t u e n t s between the equilibrium reduction  is  easily  micelle.  K-casein  colloidal  will  3d.  an  of  Casein  the  ionized  of  reported  t o enhance t h e  protein  of  caseins  (80%  milk  The  (3.6%  of m i l k  serum  relative  the  phases.  and  pH.  This Thus, a  phosphate from temperature,  increase pH  of  the milk  and  the  as  in  amount o f (pH  products  phosphate  6.8),  (yogurt), lead  to a  w/v)  proteins  (2%;  pointed  the  efficacy  calcium  (1956),  of  to the  two  of  main  of  of t h i s milk  mineral has  The  proteins  albumin,  (18%)  the and  immunoglobulins, et  a1.  d i g e s t i o n phosphopeptides i n  skeleton  of  rachitic  from v i t a m i n  c h i l d r e n (1950)  D treatment.  phosphopeptides, derived  d i g e s t i o n , t o keep c a l c i u m 38  been  f r a c t i o n s , namely  E a r l y s t u d i e s by M e l l a n d e r casein  play  dairy calcium.  t h e whey  serum  independently  of c a s e i n d i g e s t i o n  pancreatic  component  p r o t e i n ) , and  transferrin).  d e p o s i t i o n of  has  i n d a i r y products  bioavailability  bioavailability  and  vitro  calcium  calcium  protein  lactoferrin  ability  calcium  micelle  reversible dissociation  fermentation  forms of  r o l e i n the  products.  chicks  pH  of  calcium.  between  and  causes  A l t e r i n g the  colloidal  physicochemical  to  temperature  micelles  Phosphopeptides:  important  lastly,  soluble  irreversibly  low  casein  exchangeability  increasing  phosphate.  manufacture  solubilize  The  by  the  and  colloidal  (71.7°C), w i l l  calcium  the  release  and  Alternatively,  pasteurization  in  shifted  of  and  colloidal  i n t e m p e r a t u r e n e a r 0°C  of p - c a s e i n ,  as  structure  f r o m an  The in  phosphate d i b a s i c i n  solution 1958.  f r o m pH  7  t o 10  was  Support f o r these  data  the phosphate e s t e r s of associated with Casein  their by  calcium  trypsin.  protected  milieu.  to  a 2  (Manson  and  p-casein  Baumy e t a l . . 1 9 8 9 ) . in  its  primary  64  to 68);  clusters  Thus, the 70):4P,  the  major s p e c i e s  binding  binding a f f i n i t y  are  pK  2 f  to of  within  casein  Annan,  the  eight  intestinal  relative  and  o f CPP  are  clusters value, a 1. .  single  t o CPP,  have  By  to  demonstrated  at s i t e s  19.  distribution  these the  of the  oligo-  of  to  these  the s a t u r a t i o n  w o r k e r s were a b l e  cluster  binding  residues.  groups  that  r e s i d u e s , due  studying  phosphoseryl  39  five  s  charge  phosphoseryl  two  has  p o s i t i o n s 15  B  occurs  saturates  with  p-casein  of phosphoseryl  and  (residues  a i-CN(59-79):5P, a 2-CN(35-  1989).  first  at  1971;  residues  residues,  t o 61;  Studies  calcium  calcium  Waugh et. a_l_. , phosphoseryl  t o 10  cluster  chains,  a  1971;  56  from  p r o t e i n s , namely a i - ,  phosphoseryl  of  the  are  cleaved  casein polypeptide  eleven  calcium binding  determine that C a before  by  (Baumy e t of  and  p-CN(1-28):4P.  favourable  kinetics  has  binding  that  residues  s t r u c t u r e , f o u r of w h i c h form a c l u s t e r  occupied  groupings  peptides  active  various  r e s i d u e s , w i t h one  preferential peptides  the  positions 8  and  in  r e l e a s e d upon d i g e s t i o n of  the  s  s  phosphoseryl  are  a i - c a s e i n has  a 2-casein  at  Latour  knowledge  seryl  i n v e s t i g a t o r s have e x a m i n e d t h e of  -  (CPP)  become  binding potential s  and  phosphate w i t h i n the m i c e l l e .  positions within  Several  from the  polypeptide  These b i o a c t i v e  d i g e s t i v e enzymes,  by R e e v e s  i s derived  casein  phosphopeptides  casein with  reported  f o r metals  to  sites  Thus,  the  is also  dependent  on f a v o u r a b l e  residues. CPP  Further  characterization  demonstrated  insoluble further,  effective  calcium that  phosphate  importance of peptides  is  original  intact  a  of  et a l . .  pigs  intestinal  and  of  contents  (Multinger  et. a l . , 1983;  Naito  and  coworkers  Naito  and  Suzuki,  1974)  a  greater  proportion  proteins.  rats  calcium  1:1  1989).  The  of these  sensitivity  value  of  of the  Frister,  compared  CPP  1989)  calcium  phosphate  CPP  has  been  counterparts the  (Naito  i n the reported  et. .al_. ,  1972,  t o demon-  i n the d i g e s t a  from  reported  other diets  an i n t r a l u m i n a l dose  which  by S a t o e t a l .  Similarly,  in vitro  5  of a crude t r y p t i c  s t u d i e s have shown i n c r e a s e d 40  of  dietary  d e p o s i t i o n of * C a to the femora  rats given  of  Furthermore,  able  fed  calcium  upon d i g e s t i o n , was  o b s e r v e d enhanced  Naito  the  inhibition  salts  1986).  of s o l u b l e c a l c i u m to  the  been w i d e l y  1983;  consistently  of  and r a t s  Moreover,  by  bioactive  lumen c o n t e n t s  1974).  ( L e e e t a_l_. , 1980; have  these  which demonstrated  S a t o e t aX. , 1983a;  Utilization  potentially yield who  on t h e  activity  the i n t e s t i n a l  Suzuki,  insoluble  luminal  fed  in  ( M e i s e l and  Naito  of p r e c i p i t a t i o n  ( 1986)  the  physiological  phosphopeptides  1972;  a_l_. ,  the b i n d i n g  of  o f lOOmg/L, and  i s dependent  ( B e r r o c a l e_t_  to  p o t e n t i a l of  polypeptides.  casein fed mini  casein  a concentration  can be s e e n f r o m i n . v i v o s t u d i e s  presence of  strate  binding  amino a c i d  of the p r e c i p i t a t i o n  of a c t i v i t y  ratio  reflection  potential  peptides  at  stoichiometry  t h e Ca:P  of the  inhibition  the e f f i c i e n c y  c a l c i u m : phosphorus  The  charge e f f e c t s of n e i g h b o u r i n g  casein  of  digest.  calcification  of  e x p l a n t e d embryonic CPP  (Gerber  and  enhancing the  r a t bone when c u l t u r e d Jost,  1986).  intestinal  the e f f e c t  of i n t e s t i n a l  the b a s i s  t h a t b o t h r a t s and  alkaline  phosphatase  in place  activity  which  consuming  activity  on t h e  This i s proposed  significant  may  be  on  intestinal  effective  i n the  alternate protein  source  (Van d e r M e e r ,  of soybeans  of d a i r y  p r o t e i n s has  ($4.50/kg  soy i s o l a t e  functionality  1988).  sources  Weaver, 1989;  the manufacture  complexes  between  potentially the d i e t  the  globulin proteins, resistant  to  are  to t h e i r  to  i s o l a t e by and  solubility  Hartman,  have p o o r e r  acid  phytate  For  example,  precipitation,  are  formed  which  and m i n e r a l a v a i l a b i l i t y  of  1979).  digestion  which  make  (Raghunath  and amino 41  to  and p h y t a t e s  high molecular weight, highly  Protein d i g e s t i b i l i t y  Plant  t o a n i m a l p r o t e i n s due  L i e b m a n and L a n d i s , 1 9 8 9 ) .  protein  lower  similarity  V o o r t , 1988).  s u b s t a n c e s s u c h as f i b r e  characteristics  enzymatic  de  considered  i n comparison  affect protein  proteins  van  been  of soy p r o t e i n  ( E r d m a n , 1979;  Soy  been g r o w i n g due  and  have  the presence of i n h i b i t o r y and  as an  v e r s u s $ 7 . 0 0 / k g c a s e i n ) and  (Barraquio  mineral b i o a v a i l a b i l i t y  1984).  phosphatase  humans have  utilization  derived protein  in  at  Protein:  The  (Kahn  be e f f e c t i v e  i n humans  i n t r a l u m i n a l Ca-CPP c o m p l e x e s .  d e p h o s p h o r y l a t i o n of c a s e i n  in  of c a l c i u m  alkaline  of  cost  may  containing  s t u d i e s s h o u l d be c o n d u c t e d t o a s s e s s  stability  3e. Soy  T h u s , CPP  uptake  d a i r y p r o d u c t s , but f u r t h e r  i n a medium  acid  them  structured relatively  and N a r a s i n g a content  Rao,  can p l a y a  role  i n calcium b i o a v a i l a b i l i t y  a p r o t e i n source, phosphorylated acids  absorption  products,  4.  or  the  represent  ejt a l . ,  calcium  peptide  acid  relative  a limiting  The  calcium  status  balance.  Calcium  balance  absorbed  from  ascertained  the  to  is  dependent t o  mixtures  of  of  individual  i s achieved  diet  is  be  Fecal  calcium  from  individual assays calcium,  (Allen,  losses  to  could  and A b s o r p t i o n termed  calcium  plus  the  in  order  calcium  i n the  have been  endogenous mg/day.  fecal Urinary  important, food  42  a zero intake  approximately  the b i o a v a i l a b i l i t y of  namely t h e u s e f u l n e s s of or supplements to the  a considerable  bioavailability  are, solubility  maintain  l o s s e s of  items,  There i s  to  f r o m t h e U.S. RDA  Therefore,  i s very  1988).  determine  examples  of  to replace  meals, s i n g l e  (Greger,  protein  amount o f c a l c i u m  t o 130  i s required  1982).  from the d i e t  nutrients  some e x t e n t on  is  when t h e  calcium  Therefore,  30% a b s o r p t i o n  mg/day  Since  and p e r s p i r a t i o n l o s s e s a c c o u n t f o r 150 and 15  r e c o m m e n d a t i o n o f 800 mg/day 250  calcium  the equivalent of that l o s t  unabsorbed  mg/day, r e s p e c t i v e l y . a  amino  bioavailability.  Bioavailability  c a l c i u m e x c r e t i o n , w h i c h amounts t o 100  balance,  the  Calcium an  from  of p r o t e i n p o s t - d i g e s t i o n  digestibility  sweat.  calcium clearance  intestinal  f a c t o r of d i e t a r y calcium  Methods o f A s s e s s i n g  of v a r i o u s  1956 ; N a i t o e t a 1 . , 1972) .  absorption  amino  f e c e s , u r i n e and  i n enhancing  value  of casein d e r i v e d  as w e l l as t h e a b i l i t y  and L - a r g i n i n e )  (Wasserman  paracellular the  as d e m o n s t r a t e d by t h e v a l u e  peptides,  (L-lysine  as w e l l as n u t r i t i o n a l  b a t t e r y of  o f a n u t r i e n t s u c h as  tests;  balance  studies;  monitoring  of l e v e l s  functional  tests.  4a.  In V i t r o  the  s u p p l e m e n t s and  solubility  food  of a c a l c i u m s a l t  t o an  a t a pH  of c a l c i u m a b s o r p t i o n .  that  solubility  laboratory  absorption  milieu  influence  the  (Pak  calcium.  A  absorption nutrients  further  varying  is 4b.  not  in  in  and  substantial  identifies  Heaney and  substantially finding  a  the  in calcium absorption  f i n d i n g s of sources  salts,  (pH  magnitude  of  f u r t h e r emphasizes the  premise  is  a  These  simple the  the a v a i l a b i l i t y  of  on  co-ingested Recent calcium  not  differ  i n healthy adults. in vitro  of  calcium  a l . . 1989).  do  as  which i n  have shown t h a t  concept that  pre-  duplicate  sugars),  solubility,  absorption  the  the  n u t r i e n t s (such  importance  (1990),  2.0)  > 6.5),  meal-effect  (Heaney e t  coworkers  fractional  thus  vitro  solubility  lumen  cannot  and  in  in  s t o m a c h (pH  1988).  to co-ingested  solubility, of  Avioli,  however,  with respect  report  However,  intestinal  and  tests,  the  inexpensive  small intestine  amino a c i d s , d i g e s t i o n p e p t i d e s , turn,  contained  T h e s e t e s t s a r e b a s e d on  of c a l c i u m i n  bench-top  intestinal  calcium  to t h a t of the  i n the  site  to  the  individual.  similar  insure s o l u b i l i t y  requisite  of  s y s t e m s i s a q u i c k and  of the a v a i l a b i l i t y  does n o t  lastly,  Solubility:  Assaying  test  o f n u t r i e n t s i n serum o r p l a s m a ; and  This  solubility  a p r e d i c t i v e measure o f c a l c i u m a b s o r p t i o n i n v i v o .  Balance Calcium  Studies: balance  apparent absorption  studies of a  can  be  n u t r i e n t from 43  used  to  determine  experimental  diets,  the and  under v a r y i n g has  reviewed  studies.  physiological states, the v a r i o u s t e c h n i q u e s  Apparent a b s o r p t i o n  d i f f e r e n c e between the (Allen,  1982).  calcium  The  balance  studies  are  inaccuracies  i n the  contaminated  by  occur  in  f e e d , or are  important,  is  the  the  result  This  i n an  p r o b l e m can  incompletely of  of b a l a n c e  study  using  the  the s i m u l t a n e o u s calcium isotope  in  the  the  partially  oral  and  urine  diet,  is  separated,  Consequently,  the  errors  absorbed.  into  the  by are  will More the  lumen, w h i c h  by p e r f o r m i n g  absorbed. a  balance  (IV) The  proportional  body  appearance of the to  the  two oral  absorption  In a d d i t i o n , the  calcium  pools.  Howe v e r , t h e  Thus, the b a l a n c e  given  data  a d m i n i s t r a t i o n of  that  i f the  contribution  can  adequate f o r c a l c i u m balance  animal is  study  and  appearance  i s o t o p e i n the u r i n e c o r r e c t s f o r e n t r y  into  choice,  endogenous  Calcium  I f samples  calcium  alleviated  of c a l c i u m i n the body.  isotope  of  intake.  of the t r u e q u a n t i t y  1982).  Method i s o n l y q u a l i t a t i v e . method  commonly  s t u d i e s to d i s t i n g u i s h  intravenous  (Allen,  IV a d m i n i s t e r e d  oral  the  " U r i n e - R a t i o Method", a procedure which i n v o l v e s  isotopes  distribution of the  underestimation be  d e f i n e d as  of c a l c i u m  excreta.  c o n t r i b u t i o n of endogenous c a l c i u m s e c r e t e d can  balance  calcium excretion  calcium  of the  amount  failure  (1982)  i n the p r e c i s i o n of measurements  collection  estimating  fecal  absorption of the  Allen  for conducting  nutrient is  i n t a k e and  t o 40%  limited  i n use  of a  apparent  r a n g e s f r o m b e t w e e n 20  respectively.  of  Urine—Ratio is still  the  i s adapted to  the  relatively  constant.  be u s e d t o d e t e r m i n e i f a b s o r p t i o n i s (Allen, 44  1982).  4 c. N u t r i t i o n a l An can  index  Status: of the  be o b t a i n e d  levels  of  nutritional  t o some  nutrients  extent and  s t a t u s of s u b j e c t s under  by  metabolites  situation  however, the e n d o c r i n e  constant  calcium  calcium  intake w i l l  levels, n o t be  that  obtained  c a n be m o n i t o r e d  longterm  effects  of d i e t a r y  (Greger,  1988).  These  4d.  i n d i c a t i o n of Skeletal  determinants  of the  and u t i l i z a t i o n  a r e , of n e c e s s i t y , slow  f o r adaptation of the animals  Functional Tests: tests  m o n i t o r i n g growth e f f i c i e n c y and bending  (Greger,  include  parameters  h e i g h t ; bone  strength,  activities  such  as t h o s e  1988).  endpoint  and bone  These  the  such  following  determinations,  body  gain,  as  parameters such  with minerals  tests are  determinants  difficult  to  of  as bone b r e a k i n g o r well  as enzyme  o r v i t a m i n s as c o - f a c t o r s  fundamentally  the  feed  utilization  perfect  as t h e y  of d i e t a r y  These t e s t s  com-  c a n be  s t a n d a r d i z e , however, s i n c e a system of i n t e r e s t i s  seldom i s o l a t e d  but r a t h e r ,  or p h y s i o l o g i c a l Model  weight  h i s t o m o r p h o m e t r y ; as  p o n e n t s f o r g r o w t h and m a i n t e n a n c e o f h e a l t h .  4e.  relatively  1988).  absorption  determinations  In a normal  the d i e t s .  Functional  are  an a c c u r a t e (Greger,  calcium  or plasma  maintain  as e n d p o i n t  b e c a u s e a d e q u a t e t i m e must be g i v e n  serum  of concern.  system w i l l  such  calcium content  to  monitoring  study  factors  i s confounded  (Greger,  by a s s o c i a t e d d i e t a r y  1988).  Systems:  Experimental  techniques  t o determine 45  calcium absorption i n  animal  models  organelles; intestinal  i n c l u d e the  intestinal sacs,  and  use  brush in  of  isolated  border  situ  intestinal  membrane  loops  (Allen,  vesicles; 1982).  isolated  intestinal  cells,  organelles,  or brush  vesicles  alleys  strict  c o n t r o l and  definition  conditions  the  such  t e m p e r a t u r e and can  be  derived  methods vivo.  do  situ  or  the  5.  give  presence  (Gray,  small  deposited  1974).  been  f o l l o w e d by d e f i n e d as  repair  the  risk  (Niewoehner, 1988). skeletal loss with  mass age,  are  calcium  risks  life,  in  of  specific  information  absorption.  body c a l c i u m  undergoes  increase  as  matrix  cycles  (Niewoehner, 1988).  of  Osteoporosis  is a  significant  i n bone  fragility  t o m i n i m a l trauma i s enhanced generally  have  i n a d d i t i o n to a g r e a t e r osteoporosis 46  content  fibrous collagen  bone  females  of  flux  from a  for calcium  organic  associated  Because  and  in_ v i t r o o r i n  limiting  of the  o f f r a c t u r e due  than males, the  of  a c o n d i t i o n i n which there  i n bone mass w i t h an  such that  an  Throughout  these  Deposition  99%  in  models  constituents  Similarly,  entire intestine  contains  kinetic However,  dietary  pattern  i n Bone  strength,  t h e measurement o f t h e  i n t e s t i n e , but  Utilization  of  about c o n f o u n d i n g e f f e c t s i n  other  allow  use  experimental  ionic  absorption.  gradients.  secretion  skeleton  resorption  of  everted  The  of  methods, s p e c i f i c  calcium  preparations  the  salts  these  and  b o r d e r membrane  concentration,  information  electrical  the  Calcium  inorganic  loss  Using  c a p a c i t y of the  The  has  calcium  simulate  the  intestinal  segment o f on  to  and  calcium,  pH.  not  s u c h as  chemical  as  cells  are  a  smaller  r a t e o f bone  increased  in  the  female  (Avioli ,  appears  to  be  wherein the  an  by t h e  T h u s , bone  outer  (periosteum)  yield  in  human f e m a l e s ,  approximately prior  to  and  rise  contributing  to  (Niewoehner,  1988).  towards  increase  an  continues  for  surfaces (Gray,  a  a  small  gradual  1974).  thinning  the  period  rate  lasting  an e n h a n c e d  r a t e of  in overall  decline  i n renal  a b s o r p t i o n and  bone l o s s ,  change  changes  20  respectively  t o 30  continues at  can of  in  of 2  o f bone years of  a rate  i n the y e a r s  occur  i n bone mass  compact  bone  cortical  but t h e r e a f t e r  bone  shifts  resorption,  to 5 years (Marcus,  metabolism  of  Thus,  menopause i s  bone  of  at the  A t menopause, bone m e t a b o l i s m b a l a n c e in  down  layers  Some d e g r e e  about  decreases  the decade f o l l o w i n g  The  fresh  (endosteum),  subsequently  Classically,  once more.  to  1% p e r y e a r (Anonymous, 1 9 8 8 ) .  menopause,  is laid  which deposit calcareous  l o s s f r o m t h e s p i n e b e g i n s t o o c c u r by age  Bone  to replace  and r e s o r b e d t h r o u g h o u t l i f e  inner  skeleton  cells  loss  remodelling schedule,  i s not s u f f i c i e n t  giving  i s formed  a mature  a g e - r e l a t e d bone  unbalanced  osteoblast  and  of  (Marcus, 1987).  i n a collagen matrix  bone.  basis  bone f o r m e d  by r e s o r p t i o n  d u r i n g growth  to  The  inherently  amount o f  t h a t removed  material  1981).  which 1987).  characterized the r a t e is  by  declines  accompanied  by  c a l c i u m m e t a b o l i s m and h o m e o s t a s i s marked by a  calcium retention;  decreased  intestinal  an i n c r e a s i n g l y n e g a t i v e c a l c i u m b a l a n c e  calcium (Marcus,  1987). To a m e l i o r a t e t h e s e v e r i t y o f t h e follows  that  osteoporotic  bone mass and m i n e r a l i z a t i o n 47  symptoms, i t  s h o u l d be o p t i m i z e d i n  the  first  3 decades  ( N i e w o e h n e r , 1988;  of l i f e Shah and  a c c o m p l i s h e d by k e e p i n g i n orial  function  nutrition  and  being level  adequate c a l c i u m f o r bone h e a l t h s t u d i e s based is positively  when bone Belonje,  of  in  in light  of  the  on d i e t a r y r e c a l l correlated to  important  in  intestinal  vital and  mass p r i o r  exercise  t o menopause  5a . T r e a t m e n t o f Current replacement,  ized  a rapid  by  therapy  An  w h i c h i n d i c a t e t h a t bone calcium  and  of r i c k e t s ,  as  milk  consumption  Vitamin  D nutrition  w e l l as  i t srole  Physical activity  i n i t s p r o m o t i o n o f bone  1986).  density  Thus,  regular,  is a  formation stresses moderate,  a m a x i m a l bone 1987).  Osteoporosis'. of  osteoporosis  changes,  (Niewoehner, decline  i s e f f e c t i v e at  s o o n a f t e r menopause maintained  1988).  epidemiological  ( S i m o n e n , 1986; J a w o r s k i ,  nutritional  bone mass  from  i s of b e n e f i t i n a c h i e v i n g  treatments  increase  vitamin D  bone t i s s u e due t o m e c h a n i c a l  on t h e bone ( S i m o n e n ,  weight-bearing  best  is a multifact-  and B e l o n j e ,  transport.  component o f bone h e a l t h  placed  can be  intake,  e_t a l _ . , 1 9 8 5 ) .  active calcium  d e n s i t y of  calcium  findings  past  f o r the p r e v e n t i o n  increased  This  bone mass  (Shah  i s occurring  the premenopausal years i s e s s e n t i a l  ( O d l a n d e_t a l _ . , 1972 ; S a n d l e r is  by  activity  intake  1988).  mind t h a t  affected  deposition  in  and  1988).  include exercise  bone  (Simonen, 1986).  to maintain  mass l o s s  Thus,  or  estrogen  i f initiated  Bone m i n e r a l i z a t i o n can be  i n t h e y e a r s f o l l o w i n g menopause by c o n t i n u e d  48  hormone  Menopause i s c h a r a c t e r -  l e v e l s of estrogen.  preventing  sex  estrogen  replacement; loss w i l l  however,  occur  (Niewoehner,  The p r i m a r y porosis  to  disturbed balance ular, diet  increase  calcium  which  leads  metabolism,  bone  reported  to suppression  and e n d o c r i n e  homeostasis i n  in  indicated and  Calcium  been r e p o r t e d  and i n p a r t i c -  hormone  balance;  (Marcus, 1987).  p r o t e c t i o n of  suppress  and  of the  s e c r e t i o n and  and  may  the  calcium  supplementation  i n d i c e s o f bone m o d e l l i n g  foods  osteo-  from  negative  the e l d e r l y  calcium supplementation  supplements, or calcium r i c h have  is  loss  calcium  the e l d e r l y  mineral  postmenopausal  of p a r a t h y r o i d  improved  Thus,  of  intake  the postmenopausal female.  bone mass.  diet  therapy  calcium  have been  i s d i s c o n t i n u e d , bone  1988).  nutritional  bone r e m o d e l l i n g ;  ical  i f treatment  biochem-  improve  calcium  I n c l u s i o n of c a l c i u m  s u c h as d a i r y  products  to improve c a l c i u m balance  with  i n the reduced  bone r e s o r p t i o n and i n c r e a s e d d e n s i t y t h r o u g h  m i n e r a l i z a t o n (Lee  et. a_L. ,  e t a_l_. , 1988; S h i h  et  1981; H a s l i n g  a l . . 1988).  coworkers affect  e_t_ a_l_. , 1986; H o r o w i t z  Alternatively,  (1988)  indicate  that  the  formation  c o u p l i n g of decreased resorption explain patients evidence  in  why  (Horowitz  r a t s , but  young r a t s .  bone f o r m a t i o n  response  bone  in  secondary  dietary calcium  loss  is  not  et  a1. .  completely  1988).  49  Sinha  and  i s effective in  F u r t h e r , the p o s s i b l e  to  a v a i l a b l e on t h e e f f i c a c y  of  a h i g h c a l c i u m i n t a k e does n o t  bone m e t a b o l i s m i n aged f e m a l e  e n h a n c i n g bone  findings  to decreased supplementation prevented  bone may  i n some  In view of the e q u i v o c a l  of i n c r e a s e d c a l c i u m  intake i n  the  treatment  of o s t e o p o r o s i s ,  the best  prophylactic action  be t o i n s u r e m a x i m a l bone mass i n t h e y e a r s E x e r c i s e has been r e p o r t e d t h e hormone levels,  calcitonin  i t seffects  Thus, moderate during  lifestyle  5b.  Anatomy o f t h e Femur:  strongest  or  thigh  bone,  bone i n t h e mammalian  is  essentially  a  is  greater  the  medullary  trochanter)  f u n c t i o n , are  continue  canal.  (Simonen,  t o be a p a r t o f  longest,  l a r g e s t and  The  shaft,  cylinder with walls tissue The  and d i s t a l  f o r muscle attachment.  the  1974).  bone  expanded f o r  spongy, t r a b e c u l a r  formation  hormone  s k e l e t o n , c o n s i s t i n g of a c e n t r a l  hollow  of a d e n s e , compact c o r t i c a l interior,  l e v e l s of  o l d age.  s h a f t w i t h two e x t r e m i t i e s ( G r a y , physis,  circulating  on bone  exercise should  the  The f e m u r ,  t o menopause.  and t o d e c r e a s e p l a s m a p a r a t h y r o i d  i n a d d i t i o n to  1986).  to increase  prior  may  with  proximal  a  The e x t r e m i t i e s  and  constructed hollowed  head and  nature  of t h e i r  to allow a surface  are characterized  bone t i s s u e w i t h an o u t e r  out  (femoral  e x t r e m i t i e s , by  articulation  ord i a -  covering  by a  o f compact  bone . Compact c o r t i c a l the  skeleton  with  bone makes the  remainder  1988).  Compact bone i s h i g h l y  rate.  The m i n e r a l  decade of l i f e . a  relatively  content  mineral  mineralized with  content  per  80% of  (Niewoehner,  a slow  turnover  peaks i n the f o u r t h  t r a b e c u l a r bone i s  50  75 t o  t r a b e c u l a r bone  o f t h i s bone t y p e  Conversely, lower  up a p p r o x i m a t e l y  c h a r a c t e r i z e d by  u n i t volume o f bone.  Mineralization  o f t r a b e c u l a r bone  peaks i n  the  third  decade  of  designed  to  life . The  internal  withstand and  to  well,  the  the  bones a r e  shearing body  pressure  resist  stiffness  architecture  in  i n the  tension  required tension  forces  weight,  exerted to  as  to  the  s u p p o r t of  well  the  impact  as  l o a d i n g over a p e r i o d  of time  many  experimental  estimated  f r o m an  ( K u s y ejt  al_. , 1987 ;  been p o s t u l a t e d mineral  of  as  the  a  (Kusy  as  such  1984).  as  Bones  opposed to  under  a bone can  ( S e g a r s and  et  1987).  static  its initial  with  femur,  cm , 2  calcium  or  is tibia  M o r e o v e r , i t has with  bone  Bone s t r e n g t h has  been  failure,  or  rupture,  c r o s s - s e c t i o n a l area, o r mm .  The  2  curve)  energy  required  instrumental  absorption  and  or to  analysis  utilization  1987).  h o w e v e r , by few,  per  utilization  associated  p o i n t of  d e t e r m i n e d by  for  the  be  force-deformation  a l s o be test  of  Oxlund, 1988).  a1..  for  the  Kapsalis,  Bones a r e  calcium  s t r e n g t h may  corrected  functional  materials  O r t o f f and  i n t e r m s of Newtons (N)  (area  fracture  studies,  f o r c e a p p l i e d at the  bone,  expressed  as  loads  1974).  e n d p o i n t measurement  t h a t bone  content  the  work  allow  Biomechanics:  In  defined  (Gray,  to  As  S t i f f n e s s towards  (Currey,  to sudden impact l o a d s  body  ligaments.  compressive  bending  the  resistance  commonly f r a c t u r e due  5 c. Bone  is  w e i g h t of  shear.  bear  resist  femur  by m u s c l e s and  have  a l l o w bones t o and  of  if  their  very  any,  c h a r a c t e r i s t i c s that w i l l 51  nature,  complex  biological follow  theories taken  of e l a s t i c  in  the  approach,  behaviour.  material  or  a  1987).  of f o r c e s  i n deformation  action.  Data g e n e r a t e d of  samples  magnitude of from a  The  the  involves  the  sample.  tested  analysis  under  of v i e w , the  be  a r e a and  along  length  the  moment o f  of  the  determined  by  u n i f o r m and  a p p r o x i m a t e s two  test,  -  three  assuming  or  bending a p p l i e s  a uniform  the  Variability  support.  bone c u r v a t u r e  as  seen w i t h  a p p a r e n t a d v a n t a g e s of bending  the  The  significance  model o f  deformation to  applied. femur and can  tibia,  b o t h be  material  the  properties  ellipses. be along a  cross-  observed to  c o n f i g u r a t i o n of the  apply  In  chosen. the  bone  femur and  be  shaft  is  a bending Four-point surface,  single stress point  ( K u s y et_ a l . . 1 9 8 7 ) .  52  bending  tibia, in  detract favour  vary  can  i n bone c r o s s - s e c t i o n a l a r e a  four-point  the  t h r o u g h the t h e o r e t i c a l  stress f i e l d  b e n d i n g may  centre  highly  b e n d i n g may  whereas t h r e e - p o i n t  conditions.  test  Bone the  i n comparing a  engineering  concentric  four-point  useful  well-defined  inertia  that  application  of a p a r t i c u l a r  have l i t t l e  normalized  shaft.  and  test  a pure  then  empirical  d e p e n d e n t on  i n p a r t i c u l a r the  sectional  point  being  be  (Segars the  imitative  will  o f bone r e s p o n s e t o f o r c e s  I n b o n e , and  n a m e l y an  identical  obtained  contrast,  can  be  therefore  test  i n t h i s manner a r e  a p p l i c a t i o n of  Data  bone,  engineering  t h a t may  values  In  of  e m p i r i c a l approach i n v o l v e s  physical point  conditions.  a p p r o a c h e s can  testing  well-defined  Kapsalis,  series  Two  from of  at and the  three-  I n summary, or  biophysical,  t h e r e a r e many m e t h o d s , some c h e m i c a l , for  assessing  subsequent u t i l i z a t i o n . were t o e x a m i n e t h e e f f e c t dairy  foods,  utilization  in  using  calcium  The o b j e c t i v e s of  particular,  dietary on  both i n t e s t i n a l  53  bioavailability  of the f o l l o w i n g factors  calcium  physical and  studies  associated  with  bioavailability  and  and bone e n d p o i n t  measurements.  Experiment  1  E f f e c t o f l a c t o s e a n d f e r m e n t a t i o n p r o d u c t s on p a r a c e l l u l a r c a l c i u m a b s o r p t i o n and femur b i o m e c h a n i c s  in  rats.  Introduction In  addition  excellent  to  sources  of  been  demonstrated  the  bioavailability  (Lengemann Lactose,  et  facilitate proximal  of  principal  and  Wasserman, calcium  Kimberg, the  1978;  relative  forms  of  Armbrecht,  significance  on et  were  determine  lactose  on  and  Male +_  6g  and  in  1987).  milk  milk,  has  of  Behar  and  Kerstein,  1976;  Controversy  still  or  The  ionized)  of  bioavailability intolerant  (Wong  objectives  effects  D  was  vitamin  lactose,  and  a_l_. ,  1987).  shown in  and  independent  endocrine  system  Nellans exists  and  the  present  and  to  both  (Armbrecht  the  dietary  enhance  calcium  effect  also  minerals  been  and  1987).  may  et.  segments  This  has  other  Greger  transport  it  as  and  as  to  various  in  dairy  LaCroix,  1980;  of  the  lactose  and  subsequent  present  study  fermentation utilization  in  rats.  Methods  Diets:  Wistar  were  in  absorption  the  and  1983;  recognized  1982),  possibly  intestinal  of  1989).  calcium  tolerant  Materials Animals  al.. ,  al.. ,  (colloidal  calcium  Buchowski  products  1959 ;  calcium  products,  to  age  (Allen,  and  carbohydrate  Armbrecht,  al_. ,  et.  small  intake, e_t  calcium Lee  well  constituents  paracellular  distal  1976;  (Lengemann  of  being  calcium  various  1959 ;  enhanced  products  dietary  that  al.,  the  dairy  rats  randomly  (Charles  River,  segregated 54  Montreal,  into  four  PQ) w e i g h i n g  groups  of  8  rats  103 per  g r o u p and a s s i g n e d skim  milk  Inc.,  Montreal,  SMPC  supplemented  to d i e t a r y treatments  protein  concentrate  concentrate  P Q ) ; SMPC  (Y;  with  content  by p r o x i m a t e  atomic  absorption  absorption lactose  were  analyzed  8-galactosidase  different  The SMPC  amounts  content  contain only a trace for  in  the  lactose  lactose  by  analysis.  p r o t e i n ) and i s o c a l o r i c  weights  consume  were  reached  initiated.  The  diets  yogurt  two-week  within  w a t e r was made a v a i l a b l e  Elmer,  Norwalk,  atomic C T ) ; and  assay  (Boehringer  powder  concentrates  (BDH, T o r o n t o , w/w.  diets powder  ON) t o  were  give a  The SMPC was f o u n d t o was n o t c o r r e c t e d  c o n t a i n i n g 2 0 % and 5 0 % diet  c o n t a i n e d 8.2%  (Table 1.1).  fed experimental  a  Elmer-306  A l l d i e t s were made i s o n i t r o g e n o u s ( 2 0 %  150g, a f t e r  For  f a t and a s h  p h o s p h o r u s c o n t e n t by  l a c t o s e which  supplemented  respectively.  The  calcium, therefore, diets  0.7%  amount o f  lactose,  Animals  of  powder  ON).  for protein,  and y o g u r t  supplemented w i t h c a l c i u m carbonate calcium  lactose (ML);  Mississauga,  enzymatic  of  International  20%  (Perkin  Perkin  a control  ( H L ) ; and a y o g u r t  a n a l y s e s ; c a l c i u m and  Mannheim; D o r v a l , P Q ) .  uniform  with  lactose  spectrophotometry  a  Bariatrix  Industries,  spectrophotometer; by  contained  50%  sources  (SMPC;  supplemented  Champlain  dietary protein  c o n s i s t i n g of  a  diets  ad  1ibitum u n t i l  which a meal-feeding period,  animals  schedule  were  s i x h o u r p e r i o d (9am-3pm). ad l i b i t u m .  55  body was  trained to Deionized  TABLE 1.1  Composition  Dietary  SMPC Yogurt  component  of experimental d i e t s  SMPC  23 . 5  2  powder  3  D.L. m e t h i o n i n e * Cornstarch* Sucrose Lactose Fibre* Vegetable o i l  —  HL (g/lOOg)  23 . 5 —  0.3 11.35 50.0  -  5.0 4.8  23 . 5 —  55 . 6  0 .3 11 .35 30 .0 20 .0 5 .0 4 .8  0 .3 11 .35 50 .0 5 .0 4 .8  0.3 11 .35 18 . 65  -  5.0 4.8  Ca f r e e m i n e r a l mix* Vitamin mixture* Choline b i t a r t r a t e *  3.5 1.0 0.2  3. 5 1 .0 0 .2  3. 5 1 .0 0. 2  3.5 1.0 0.2  Calcium  0.35  0 .35  0 .35  -  20 .0 20 .6 0 .2 0 .72  20 .0 50 .6 0 .2 0 .72  Final  carbonate**  c o m p o s i t i o n by a n a l y s i s (%)  Protein Lactose Milk f a t Ca 1  ML  1  fed to rats.  20 .0 0.6 0.2 0.72  20 .0 8.2 0.2 0.72  SMPC = s k i m m i l k p r o t e i n c o n c e n t r a t e ; ML == 2 0 % l a c t o s e + SMPC; HL = 5 0 % l a c t o s e + SMPC; Y = y o g u r t powder. S k i m m i l k p r o t e i n c o n c e n t r a t e - ( 8 5 % p r o t e i n , 5% c a r b o h y d r a t e , 0.259% c a l c i u m ) . Y o g u r t powder - ( 3 5 . 9 % p r o t e i n , 5 2 . 3 % c a r b o h y d r a t e , 5.0% t i t r a t a b l e a c i d i t y as l a c t i c a c i d , 1.29% calcium). ICN B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , OH. BDH C h e m i c a l s , T o r o n t o , ON.  56  Calcium ligated  absorption  ileal  loop technique  modifications. access  to  Rats  diets  experiment.  (15  withdrawal.  for a  contents  of  the  to  of  mg CaJ  ICN B i o m e d i c a l ,  4 5  Calcium  the loop. closed.  injection  of  4 5  Ca  with  were i n i t i a l l y  mg/kg)  an  1.5  at points a  loop  8 cm  closed  minor  allowed  were  intraperitoneal  hours  left  4 5  and 20  was  unwashed. S.A. =  replaced  removed t o f l u s h o u t t h e c o n t e n t s  and  using  and t h e  cm f r o m t h e  was  and  injected  t h e abdomen intraluminal  the l i g a t e d 5 mL c o l d  A 0.3 mL  10 m C i /  R a t s were s a c r i f i c e d one h o u r a f t e r by e x s a n g u i n a t i o n ,  food  The i n t e s t i n a l  I r v i n e , CA) i n 0.15N s a l i n e intestine  after  was made  loop.  ( d o s e = 5.4 u C i , C a C l 2 j  The  (1983)  abdominal i n c i s i o n  form  ligated  aliquot  age)  the i n s i t u  h o u r p e r i o d on t h e m o r n i n g o f t h e  (50  and l i g a t e d  junction,  et a l .  anaesthetized with  A longitudinal  ileocecal  sutured  1.5  injection  ileum i s o l a t e d  o f Lee  weeks o f  A n i m a l s were  pentobarbital  into  s t u d i e s were p e r f o r m e d u s i n g  ileal  loop  0.15N s a l i n e .  Analyses : Intestinal  contents  were  homogenized  homogenizer  (Brinkmann I n s t r . , Rexdale,  homogenate  digested  Oakville,  ON)  and  (Amersham, O a k v i l l e , w i t h an  mixed ON).  LKB-1215 l i q u i d  Oy, T u r k u , separate  with  NCS with  ON)  tissue ACS  radioactivity  present  i t was  polytron  an  a l i q u o t of  solubilizer  (Amersham, cocktail  m e a s u r e m e n t s were made  spectrophotometer  F i n l a n d ) w i t h a 90% c o u n t i n g  experiments,  and  a  scintillation  Radioactivity  scintillation  using  determined  efficiency for Ca. 4 5  that  92 +_  In  3% o f t o t a l  i n t h e i n t e s t i n a l l o o p was r e c o v e r e d 57  (Wallac  i n the  saline was  flush.  The  performed  measurement o f  by  atomic  HCI/HNO3  ( M a u e r , 1977)  lactose  Content  hydrolysis limit  * Ca i n 0  absorption  determined  (Boehringer  w i t h 0.5%  by  Mannheim,  LaCl3.  p-galactosidase  Dorval ,  f o r l a c t o s e was 5 ug/mL i n t h i s  homogenate  f o l l o w i n g wet a s h i n g w i t h  and d i l u t i o n  was  the i l e a l  PQ).  The  Luminal enzymatic detection  assay.  Bone M i n e r a l D e p o s i t i o n : The d e p o s i t i o n 4 5  Ca  intestinal  determined rat.  of  Ca  i n the  f e m u r , an e n d p o i n t  a b s o r p t i o n (Mykkanen  from the r i g h t  and Wasserman,  femur i m m e d i a t e l y a f t e r  Femora were w e i g h e d and t h e n a s h e d  The bone  a s h was  taken f o r C a ively.  dissolved  radioactivity  4 5  Femur c a l c i u m  the a c i d i f i e d absorption  ashed  in  c o n t e n t measured  index of 1980)  and bone  was  s a c r i f i c i n g the  a t 550°C  f o r 24 h o u r s .  3 mL o f 4N HC1 and a l i q u o t s  were  mineral content, respect-  and magnesium c o n t e n t s were d e t e r m i n e d i n  samples d i l u t e d  spectrophotometry.  bone a s h was d i l u t e d  al.  4 5  with  with  A  0.5%  LaCl3  further aliquot  deionized  water  and  by a t o m i c  of a c i d i f i e d  the phosphorus  s p e c t r o p h o t o m e t r i c a l l y by t h e method o f Chen e t  (1956).  Blood  Chemistry: Plasma m i n e r a l s  heart puncture. were d e t e r m i n e d presence  of  colorimetric  were m e a s u r e d  from blood  s a m p l e s t a k e n by  P l a s m a c a l c i u m , magnesium, s o d i u m and by  atomic  absorption  0.5%  LaCl3•  Plasma  BCA p r o t e i n a s s a y  spectrophotometry  i n the  p r o t e i n was a n a l y z e d by t h e  (Pierce  58  potassium  Chemical  Co., R o c k f o r d ,  IL).  Ionized  Zeisler  where  calcium  both  the  total  calculated  using  the  equation  of  (1954): Ca*  2  Ca*  2  concentration, using  was  = (6Ca and  - Protein/3)/ (Protein + Ca  g/dL.  mg/dL  Plasma  p r o t o c o l of  cholesterol  are  and  a-amino  Protein  is  n i t r o g e n was  Goodwin ( 1 9 6 8 ) .  concentrations  6)  Plasma  were  determined  triglyceride  analyzed  c o l o r i m e t r i c methods, r e s p e c t i v e l y ( B o e h r i n g e r  protein  by  and  enzymatic  Mannheim,  Dorval,  PQ) . Bone  Biomechanics: An  e m p i r i c a l measure o f f e m u r s t r e n g t h was  serohydraulic materials 1122, The  Canton, left  cleaned  by  of  machine  tissues  unsupported  trochanter.  and  on  for this  epiphyses  the  load  Bone d e f o r m a t i o n  dicular  to the  chosen  because  compact c o r t i c a l The  from the  long a x i s of  and  it  distal  represents  data  attachment.  Femora were  resting  m e a s u r e m e n t s were  t h e bone.  cancellous  time-deformation  Corp., Model  removed.  speed end  This a  a  measurement once  platform  a p p l y i n g a shear f o r c e at a constant 4 mm  (instron  w i t h a s i n g l e - b l a d e shear  c o n s i s t e n t l y used  soft  consistent area,  was  fitted  femur was  positioned greater  MA)  testing  performed using  (1.0  mm/  on  the  conducted min)  to a  of the bone, p e r p e n r e g i o n of  transition  the  area  femur  between  t r a b e c u l a r bone. were m o n i t o r e d u s i n g  the  JCL  6000  Chromatography Data System (Jones Chromatography L t d . ,  Littleton,  CO)  an  w h i c h was  compatible  interfaced with  personal  computer.  the  Instron,  Sample r u n 59  through  t i m e was  IBM  AT  3 minutes,  at  a sampling rate calibrated  of 5 s i g n a l s per second.  using  1.0  and  a n a l y z e d by t r a n s f o r m i n g  the  Two  strength  and bone h a r d n e s s ( S e g a r s  strength  is  of  described  as  of f r a c t u r e , d i v i d e d  t h e bone  (mm ). 2  work o r e n e r g y Statistical All of  o f bone  known  Instron  signal  weights.  signal  and  output  the f o r c e by t h e  Kapsalis,  i n t o kg bone  1987).  Bone  ( N e w t o n s , N) a p p l i e d  at the  original  cross-sectional  The bone h a r d n e s s p a r a m e t e r i s d e f i n e d  (Joules, J) required  was  D a t a were  o s s i f i c a t i o n were d e t e r m i n e d ,  area  as t h e  t o f r a c t u r e t h e bone.  Analyses'.  t h e d a t a a r e e x p r e s s e d as mean +. SEM.  variance  imental  kg  millivolt  force.  point  indices  2.0  The  was u s e d  treatments.  to t e s t f o r d i f f e r e n c e s Where d i f f e r e n c e s  the  differences  the  Student-Newman-Keuls m u l t i p l e  did  One-way  between the e x p e r -  exist,  a t a p<0.05 s i g n i f i c a n c e l e v e l was  60  range  test.  analysis  the  source of  identified  by  Results Body w e i g h t SMPC, ML  g a i n s and f o o d  and Y  fed animals,  effects  of i n d i v i d u a l  animals  exhibited  body w e i g h t intake  which the  reduced  affected  ratio.  f e d animals animals  intake,  Further, calcium  due t o t h e l o w e r  No  experienced  feed  during  period.  also  food  some d e g r e e o f d i a r r h e a and m a l a b s o r p t i o n ,  rats  0.21 by  nitrogen  indications  experimental diets plasma p r o t e i n  mg/dL). dietary  and  Plasma treatments  triglyceride  (p<0.05) r e d u c e d  i n animals  ML f e d c o u n t e r p a r t s . by  (p<0.05) r e d u c e d  HL f e d  stools  powder d i d n o t a f f e c t +_  comparison,  of  diarrhea  were  i n SMPC, ML o r Y f e d a n i m a l s .  Feeding  6.63  HL  In  palatability  soft  experimental  observed  in  were s i m i l a r i n  no a d v e r s e  (Table 1.2).  feed e f f i c i e n c y  These p a r t i c u l a r  indicated  indicating  a significantly  g a i n , and  was  intake.  diets  i n t a k e parameters  1.4.  No s i g n i f i c a n t  from  the  detected.  and f e m u r  intestinal The HL  enhanced a b s o r p t i o n significant  mineral  (range  p r o f i l e s were a l s o n o t  (Table  1.3).  concentrations f e d t h e HL d i e t ,  were  a-amino  significantly  compared t o SMPC and  4 5  Ca  4 5  Ca  loop  f e d group Ca  in  SMPC,  exhibited from  the l i g a t e d  of  4 5  i n Table  Ca  absorbed  ML a n d Y f e d a n i m a l s a  significantly  the l i g a t e d  loop.  d i f f e r e n c e b e t w e e n t h e SMPC c o n t r o l 61  from  are presented  d i f f e r e n c e s i n the percent  4 5  Plasma  l e v e l s were n o t a f f e c t e d  absorption of  d e p o s i t i o n of  of  5.64 +_ 0.14 t o  (Table 1.3).  The 1 h o u r i n t e s t i n a l loop  levels  Plasma c h o l e s t e r o l  the experimental d i e t s  ileal  c o n t a i n i n g l a c t o s e or yogurt  were  (p<0.05)  T h e r e was no  and ML  dietary  TABLE 1.2  Diet  2  Diet  e f f i c i e n c y of r a t s f e d e x p e r i m e n t a l d i e t s . 1  I n i t i a l body wt. (g) 3  F i n a l body wt.* ( g )  Dry M a t t e r Intake (g)  Feed E f f i c i e n c y Ratio  SMPC  117  +  5«  323  37*  504 .0  20 .6*  0 .269  +_  0 .012«  ML  118  +  7*  301  39  a  477 .2  16 .8  0 .253  +  0 .014«  HL  116  +  ll  a  246  +_  46  b  366 .0  +  16 .0«>  0 . 162  Y  111  +  10  a  304  •+  66  a  452 . 5  +_  25 . 6  0 .213  1 2  3 4  a  a  0 .044b +  D a t a a r e e x p r e s s e d as mean +. SEM. SMPC = s k i m m i l k p r o t e i n c o n c e n t r a t e ; ML = 2 0 % l a c t o s e HL = 5 0 % l a c t o s e + SMPC; Y = y o g u r t powder. 5 weeks o f age . 15 weeks o f a g e .  Means s h a r i n g t h e same l e t t e r w i t h i n i c a n t l y d i f f e r e n t a t p<0.05.  62  a column  0 .027  a  + SMPC;  are not s i g n i f -  TABLE 1.3  2  Blood c h e m i s t r y o f r a t s f e d experimental d i e t s .  +?  Ca  T  a  1  SMPC  8.47 + 0.08  a  3.95 + 0.06  a  10.32 + 0.88  a  201.8 + 0 . 5  a  94.88 + 0.02  a  ML  8.51 + 0.18  a  4.23 + 0.07  a  8.07 + 0.50  a  184.4 + 0 . 3  a  106.38 + 0.01  a  HL  8.48 + 0.17  a  3.63 + 0 . 2 1  a  5.13 + 0 . 5 1  144.0 + 0.4  b  103.50 + 0.02  a  Y  8.40 + 0.28  a  3.88 + 0.13  a  7.20 + 0.66  118.3 + 0 . 2  C  106.38 + 0.04  a  a  t, Data a r e expressed as mean + SEM. SMPC = skim milk p r o t e i n c o n c e n t r a t e ; + SMPC; Y = y o g u r t powder.  d  Triglycerides  t  Ca  c  -amino-N (mg/dL)  o  Diet'  a  b  Cholesterol  ML = 20% l a c t o s e + SMPC; HL = 50% l a c t o s e  Means s h a r i n g t h e same l e t t e r w i t h i n a column a r e not s i g n i f i c a n t l y d i f f e r e n t a t p<0.05).  TABLE 1.4  I n t e s t i n a l a b s o r p t i o n and f e m u r d e p o s i t i o n o f calcium i n rats fed experimental d i e t s .  4 5  Diet  2  1  Absorbed C a (% d o s e ) 4 5  I n t e s t i n a l S.A. (dpm/ mg C a ) 4 0  Bone C a Bone S.A. (% d o s e / g A s h ) (dpm/mg Ca) 4 5  4 0  SMPC  35 .2 + 4 . 7°  2 .56 + 0. 25«  0 .712  0 . 117*  161 . 1 + 26 . 1«  ML  27 .3 + 8 . 7«  2 .32 +_ 0. 13«  0 . 747 + 0 . 167«  157 . 5 + 12 . 0*  HL  46 .2 +_ 6 .5*  4 .84 + 0. 1 8  1 .678  436 .9  Y  28 .7 +_ 2 .8*  9 .68 +_ 0 .30«>  1 2  b  0 .272«>  0 .793 + 0 . 105*  219 .8 + 24 .7«  D a t a a r e e x p r e s s e d as mean +_ SEM. SMPC = s k i m m i l k p r o t e i n c o n c e n t r a t e ; ML = 2 0 % l a c t o s e HL = 50% l a c t o s e + SMPC; Y = y o g u r t p o w d e r .  Means s h a r i n g t h e same l e t t e r w i t h i n i c a n t l y d i f f e r e n t a t p<0.05.  64  a column  50 . 9»  + SMPC;  are not s i g n i f -  group i n e i t h e r present  the  a b s o r p t i o n of  i n the l i g a t e d  loop, which This  (p<0.05)  was  tissue  apparent  bone o b s e r v e d  i n only the  i n HL f e d  intake;  as  well  content  of  the  animals  HL  SMPC  noteworthy  that  and  ML  Femur presented dietary  (range  (range  physical i n Table  treatment  controls. hardness  No  deposition into  (Table  1.4).  The  calcium  into  of  due t o r e d u c e d (p<0.05)  of l a c t o s e at both  both  an  calcium  There of  4 5  was  Ca  despite  i n these  on o v e r a l l  from  higher lactose  1.1).  animals,  observed  11.53 +. 0.88  1.99 +_ 0.06 and  1.5.  no  i n Y fed the  higher  animals.  Itis  t h e 2 0 % (ML) and  femur m i n e r a l i z a t i o n  t o 13.51  t o 2.12  biomechanical  +_ 0.58  mg/g  as  Both  significant in  femur  s t r e n g t h parameters  ML  and 65  weight  and bone  i n HL f e d a n i m a l s ,  difference Y  Ash)  +_ 0 . 0 5 ) .  Femur bone l e n g t h d i d n o t d i f f e r  (p<0.05) l o w e r  were o b s e r v e d  Ca  resulted  (Figure  had  activities.  likely Ca  of the  350.4 +. 10.2 t o 383.8 +. 27.9 mg/g A s h ) ,  groups.  were s i g n i f i c a n t l y  4 5  radiolabelled  4 5  Ca  Y diets,  specific  animals  of  fed  activity  by c a l c i u m ( r a n g e  Ca/P r a t i o  higher  4 0  i n the i n t e s t i n a l  i n t h e femur d e p o s i t i o n  the presence  magnesium c o n t e n t  fed  loop  ( H L ) l e v e l s had no e f f e c t  indexed  a  of  activities  HL and  contents  significantly  ligated  specific  and  by  animals  a  difference  from  t h e amount  f e d the  Ca  efficiency  as  intestinal  50%  4 0  translocation  absorption  significant  lower  paralleled  enhanced  enhanced  Animals  contributed to higher i n t e s t i n a l  result  femoral  Ca , o r  l o o p ; thus, the s p e c i f i c  t r a c e r were n o t d i f f e r e n t . significantly  45  animals  between hardness  compared t o  i n femur w e i g h t fed  are  o r bone  compared t o  controls.  These p a r a m e t e r s  were h o w e v e r , s i g n i f i c a n t l y  (p<0.05) t h a n i n a n i m a l s f e d t h e HL  66  diet.  greater  N.D. i  SMPC Experimental Diets Fig.  1.1  Values  Lactose content of i l e a l loop of r a t s f e d skim m i l k protein concentrate (SMPC), 2 0 % l a c t o s e + SMPC ( M L ) , 50% l a c t o s e + SMPC ( H L ) , and y o g u r t powder ( Y ) . SMPC = N.D.; ML = 4.15 +.0.35; HL = 6.45 ± 0 . 4 1 ; Y = 2.79 + 0.31. N.D. = n o t d e t e c t e d ; * denotes s i g n i f i c a n t (p<0.05) d i f f e r e n c e f r o m ML t r e a t m e n t .  are expressed  as mean +. SEM.  TABLE 1.5. Femur p h y s i c a l d i m e n s i o n s and b i o m e c h a n i c a l in rats fed experimental d i e t s .  parameters  1  Diet  2  Femur Wt. (g)  Bone L e n g t h (mm)  Bone H a r d n e s s (x 1 0 " J ) 3  +_  0 .03*  182 . 5  +  16 .6*  32 . 12 + 0 .44'  1 .28  +_  0 .06*  161 . 9  +_  14 .8*  0 .046*  30 . 19  1 .53  +_  0 .06*  121 .7 + 13 .8*  0 .043*  31 .04 + 0 .59*  1 .55  +  0 .04*  152 .3  0 . 681  +  0 .020-  31 .99  ML  0 . 652  +  0 .047*  HL  0 . 562  +  Y  0 . 601  +_  1  2  1 .23  SMPC  2  Bone S t r e n g t h (N/mm )  0 . 14"  +  0 . 67*  D a t a a r e e x p r e s s e d as mean +_ SEM. SMPC = s k i m m i l k p r o t e i n c o n c e n t r a t e ; ML = 2 0 % l a c t o s e HL = 5 0 % l a c t o s e + SMPC; Y = y o g u r t p o w d e r .  Means s h a r i n g t h e same l e t t e r w i t h i n i c a n t l y d i f f e r e n t a t p<0.05.  68  a column  +  23 .0*  + SMPC;  are not s i g n i f -  Discussion Previous reports  have e x p r e s s e d  w e a n i n g r a t as a model f o r calcium  absorption,  lactose  ( L e i c h t e r , 1973).  observed diet,  in  this  study  et_ al_.  intolerance lactose.  lactose  No s i g n s i n rats  This  reported of  diets  increased  intestinal  R a t s f e d t h e 50%  time, lactase  lactose  nutrient malabsorption, decreased food intake retained  in  this  because the l a c t o s e  have  been  activity  diet  (HL)  as i n d i c a t e d  in  The a b s e n c e o f a  diet,  compared  contrary of  from  to  calcium  ysis  lactose  the d i s t a l to  reports  SMPC which  bioavailability  products  (Pansu et  mediated  small  and  Y  showed  These a n i m a l s  and were  controls,  to represent  enhancement  i n lactose  of  of r a t s  animals,  of  1975). calcium  f e d t h e ML  respectively, is  have o b s e r v e d s i g n i f i c a n t attributed  1971).  signs  absorption  intestine fed  o v e r an  by s o f t s t o o l t e x t u r e ,  calcium  20%  e x h i b i t an  the absence of p a i r - f e d  f o r studying  lactose  ( B o l i n e t a l . . 1969;  m a l a b s o r b i n g i n d i v i d u a l s ( L e i c h t e r and T o l e n s k y ,  absorption  to  i n t o l e r a n t r a t has been r e p o r t e d  a n i m a l model  study  containing  reported  and body w e i g h t g a i n e d .  study,  of  of l a c t o s e  however,  were  lactose  the recent  no s i g n s  M o r e o v e r , r a t s f e d m o d e r a t e amounts of  of the r a t to  intolerance  agrees with  also  post-  intestinal  f e d e i t h e r t h e 20% (ML)  or decreased d i g e s t i b i l i t y  period  the  enhanced  of l a c t o s e  finding  ( 1 9 8 9 ) , who  extended  a good  studying  due t o t h e a p p a r e n t i n t o l e r a n c e  or the Y d i e t .  of Greger  concern i n using  to lactose  enhancement  or i t s h y d r o l -  a l . . , 1979; S a t o et_ a l . . , 1983; M i l l e r e_t  a l . . 1988; G r e g e r e_t_ a_l_. , 1 9 8 9 ) . 69  Using  t h e same  .in. s i t u  ileal  loop  technique  for  S a t o and c o w o r k e r s of  calcium  likely other  in  in a  nutrients.  calcium  absorption,  l a c t o s e induced  enhancement  distal  results  prior  may  to  i n t o l e r a n t HL f e d a n i m a l s , absorption  by t h e  p r o t o c o l used manipulations, o f l a c t o s e and  m e d i a t e d e f f e c t on  decreased.  absorption was l i k e l y  luminal  of the small  explained  content  the l a c t o s e  efficiency  i n t a k e and i n t e s t i n a l  be  the s u r g i c a l  c a l c i u m a b s o r p t i o n was calcium  portion  The m e a l - f e e d i n g  reduced i n t e s t i n a l  Consequently,  enhanced  increased  the  in  especially  resulted  paracellular  a  feeding protocols.  study,  The  from  The d i s p a r i t y  difference this  (1983) observed  absorption  intestine.  in  estimating paracellular  observed  i n the l a c t o s e  t h e combined  due  to  result  the decreased  calcium content  o f an  calcium  of these  animals;  as w e l l as a l a c t o s e m e d i a t e d enhancement o f p a r a c e l l u l a r  calcium  absorption.  to  be more  i n c o n d i t i o n s of calcium d e f i c i e n c y or negative  calcium  efficient  Intestinal  ( P a n s u ejL ILL- >  balance reported  to  conditions  have an  1981).  since  role  malabsorption lactose  is  Moreover,  even g r e a t e r  of n u t r i e n t  Alternatively,  calcium transport  known  the  (Hylander  intolerance  lactose  of our  experimental  that  lactose f a c i l i t a t i o n  is  an  acute  protocol.  process  of  during the  These r e s u l t s  70  the high  i n a greater  time  support  calcium absorption  and d e p e n d e n t on o p t i m a l  a l . . 1980).  1975),  appeared to r e s u l t  c o n c e n t r a t i o n of l a c t o s e i n the ileum our  et  i s known t o i n c r e a s e  i n r a t s ( L e i c h t e r and T o l e n s k y , HL d i e t ,  has been  i n calcium absorption i n  gastric motility content  ileum  p e r i o d of  the concept  from the i l e u m ,  c a l c i u m and l a c t o s e  concentrations Wasserman,  in this  region  of  pH o f m i l k  (pH 6 . 8 ) , a m a j o r  calcium i s present i n c o l l o i d a l moieties  phosphate Lowering  intestine  phosphoseryl  linkages  acting  to  pH  to  the  colloidal  proportion  associated with  of  Kansal  and  the  calcium  ionic  f e r m e n t e d , low  calcium i s present p r i m a r i l y  to or  paracellular Smith  et  i n an i o n i c 1982).  (1985)  bioavailability  who  Our  reported  from whole m i l k  (Swaisgood,  calcium  form  no  (Wong and the  greater  intestinal  animals e x h i b i t e d  as ML  and SMPC  term  femur  4 3  diets on  agree w i t h those of i n calcium  Similarly,  calcium  from y o g u r t  f o r m ( B e h l i n g and G r e g e r ,  calcium  deposition  specific  an i n c r e a s e d  a similar  fed animals. Ca  different  and  c a l c i u m supplements, both of which p r o v i d e  fed animals corresponded to Y fed  the  LaCroix,  based  difference  and y o g u r t .  calcium i n a predominantly ionic The  1985).  bioavailability  However,  results  as  thus,  p r o d u c t s , i n which  has been shown t o be a b s o r b e d e q u a l l y e f f i c i e n t l y commercially available  and  s t u d y were o b s e r v e d t o have no e f f e c t  calcium absorption.  al.  micelle.  phosphate  calcium  pH d a i r y  Chaudhary,  this  calcium  casein  p h y s i c o c h e m i c a l forms of c a l c i u m p r e s e n t i n the m i l k animals i n  phosphate  fermented d a i r y p r o d u c t s , such  P r e v i o u s s t u d i e s have r e p o r t e d a r e d u c e d  fed to  and  (68%) of the  residues,  stabilize  produce  the  proportion  e i t h e r bound  casein  the  increases  form,  of  yogurt, s o l u b i l i z e s  1980;  (Armbrecht  1976).  At the  ester  the  Ca  femur  d e p o s i t i o n of  Previous data 71  4 5  activity  as  4 5  Ca  w o r k e r s have an  index  1988).  of the  HL  deposition. t o t h e bone used of  short  calcium  bioavailability Sato et  and u t i l i z a t i o n  a1. . 1986).  bone o f HL  hardness  since in  in  not  neither  these  equate  bone  animals.  The  i n t e r p r e t a t i o n o f bone  HL  findings and  result  these  ileum,  and  (1975) which  of  food  that  malabsorption  intake studies  young a d u l t s  and  1983).  Thus, i t i s  tion  caused  by t h e  and  i s needed i n  data. i n both which  plasma are  digestion.  conducted  p r o t e i n and  by  Leichter  Our  albeit  or in  femur  contributed  on s k e l e t a l  other HL  results  lactose  that  nonspecific  and  hardness  cortical  bone 1964;  the n u t r i e n t  will  thickness Crosby  et  malabsorp-  i n t o l e r a n t c o n d i t i o n of r a t s f e d the  significantly  tissue content.  the  a n i m a l s , d i d not  weight  c h i l d r e n (Garn et a1.. concluded  not  intestine, specifically  lactose  an  These  f a t , but  intolerant rats.  to reducing  a r c h i t e c t u r a l m a t r i x components, which s i z e and  i n bone  I t i s known t h a t p r o t e i n - c a l o r i e m a l n u t r i t i o n effects  was  n o t be a t t r i b u t e d t o  deposition  occurring  a f f e c t bone s t r e n g t h ,  to d e l e t e r i o u s  diet,  reduction  concentrations,  the s m a l l  add  al..  HL  Ca  i n the  calcification  Thus, c a u t i o n  showed b o t h  f i n d i n g s to  were r e d u c e d .  i n both  reduced  furthermore  indicators  lead  of  malabsorption i n lactose  extend  adversely  triglyceride  support previous balance  Tolensky  calcium,  and  found  1980;  e n h a n c e d bone  fed animals also e x h i b i t e d a decrease  a-amino n i t r o g e n expected  Ca  an  a  rats could  4 5  4 5  or  Moreover,  an a l t e r a t i o n i n bone m i n e r a l i z a t i o n .  Wasserman,  with  strength  lactose malabsorbing  the c o r r e c t  and  r e s u l t s i n d i c a t e that  f e d a n i m a l s does  accretion, enhanced  Our  (Mykkanen  Together, 72  bone g r o w t h  i n turn contribute  and  t o bone  t h e s e components c o m p r i s e  the  biological  mechanisms  biomechanical required the  to  required  to  respond  u s a g e s o f bone ( F r o s t , 1 9 8 8 ) . confirm  significance  this  of  hypothesis  these  findings  in  to  the  Further  specific  studies are  p a i r - f e d animals,  is  relevant  to  since  t h e bone  metabolism of l a c t o s e i n t o l e r a n t i n d i v i d u a l s . In c o n c l u s i o n , lactose,  together  the presence with  been  soluble  calcium,  have  sustained  enhancement o f c a l c i u m  bone d e p o s i t i o n . strength attributed apparent  The  observed to  shown  a  in  to  enhancement  lactose  of  and have  reduction  decreased  of a moderate d i e t a r y l e v e l of insoluble little  absorption in  mineral  paracellular  observed.  73  forms of  s i g n i f i c a n c e i n the and  utilization for  bone h a r d n e s s , b u t n o t bone  intolerant  femur  dairy  rats  could  content,  calcium  not  be  although  an  absorption  was  Experiment 2 P a r a c e l l u l a r a b s o r p t i o n and femur m i n e r a l i z a t i o n and biomechanics i n rats fed selected d i e t a r y p r o t e i n s . Introduct ion In recent  studies,  increase  bone mass  in  1988).  In a d d i t i o n  1981).  and  urinary  Taken t o g e t h e r ,  calcium  such  rats fed selected  ization  and  i n d i c e s of calcium Materials  study i n t o the r o l e  (Linkswiler  The  objectives  absorption  of  the present  from the s m a l l Further,  diets  study  intestine  bone  mineral-  p a r a m e t e r s were e x a m i n e d as e n d p o i n t  5 week o l d male W i s t a r r a t s , m a t c h e d Charles River  (25°C) into four casein,  and l i g h t i n g dietary whey  c o n c e n t r a t e and s o y p r o t e i n fed  calcium  utilization.  purchased from  temperature  protein:  e t a 1. .  a f f e c t the  (Montreal,  f o r age and PQ).  were i n d i v i d u a l l y h o u s e d i n s t a i n l e s s s t e e l c a g e s w i t h  segregated  calcium  Diets:  Thirty-two were  et a1. .  and M e t h o d s  A n i m a l s and  sex  excretion  dietary proteins.  biomechanical  (Shih  a v a i l a b l e to the i n d i v i d u a l  t h e s e two f a c t o r s w i l l  were t o d e t e r m i n e t h e c a l c i u m  been shown t o  as p r o t e i n , on i n t e s t i n a l  calcium  balance of the i n d i v i d u a l .  in  patients  has been c o n s i d e r a b l e  constituents,  absorption  s u p p l e m e n t s have  post-menopausal  t o the  through the d i e t , there of d i e t a r y  calcium  ad  libitum  groups protein  with  lOOg  started. 74  body  weight  protein  Animals was  were  were  to dietary  milk  2.1).  Animals  controlled  Animals  respect  concentrate,  i s o l a t e (Table  until  w h e r e u p o n m e a l - f e e d i n g was  ( 1 4 : 1 0 d/n c y c l e ) .  Animals  were  reached, trained to  consume  their  w a t e r was and  diets  provided  ad  within  ligated  ileal  ( 1 9 8 3 ) as  calcium  were  described in  a b s o r p t i o n was b a s e d on  experiment  weeks o f age.  A d o s e ( 0 . 3 mL)  ICN  Inc., Irvine,  intraluminally of  calcium  remaining  i n t o the  estimated  i n the  loop a f t e r  Daily  feed  m e a s u r e d by  the  of  the procedure  1, 4 5  Ca  CA)  loop  was  Deionized intakes  recorded.  loop technique  Biomedical  6 hour p e r i o d d a i l y .  1 i b i turn t o t h e a n i m a l s .  w e e k l y body w e i g h t s Intestinal  a  when t h e (  4 5  i n 0.9%  (total  of Lee  animals  CaCl2,  18.2  5.4  ejt_ a l . were  14 Ca,  injected  uCi). Absorption  f r o m t h e amount o f a d m i n i s t e r e d one  situ  mCi/mg  s a l i n e was  d o s e was  in  4 5  Ca  hour.  Analyses : Intestinal  l u m e n , and  b l o o d p l a s m a m i n e r a l s were a n a l y z e d  p r e v i o u s l y d e s c r i b e d i n experiment Bone  1.  Biomechanics: After  e x c i s e d by and  as  the  euthanasia,  bone  samples,  blunt dissection,  cleansed  epiphyses  removed.  Bone  femur of  adhering  biomechanical  assessed  u s i n g an I n s t r o n U n i v e r s a l T e s t i n g  Instron  Corp.,  single-blade bending analyzed  Canton,  shear  analysis.  test In  MA). while the  The the  tibia, soft  were tissue,  p a r a m e t e r s were  Machine (Model  1122,  f e m o r a were s u b j e c t e d t o a tibiae  single-blade  underwent  shear  as p r e v i o u s l y d e s c r i b e d i n e x p e r i m e n t  75  and  test, 1.  a  3-point  f e m o r a were  TABLE 2.1  Composition  D i e t a r y component  of experimental  Casein 1  diets  f e d to animals.  SPI MPC (g/lOOg)  -  -  Whey  23- . 5  Casein* Soy p r o t e i n i s o l a t e * MPC* * Whey p r o t e i n * *  20 .0  D.L. m e t h i o n i n e * Cornstarch* Sucrose Fibre* Vegetable o i l  0 .3 11 .35 53 . 16 5 .0 5 .0  0 .3 11 .35 53 .23 5 .0 5 .0  0 .3 11 .35 50 .0 5 .0 4 .8  0 .3 11 . 35 50 .29 5 .0 4 .8  Ca f r e e m i n e r a l mix* Vitamin mixture* Choline b i t a r t r a t e *  3 .5 1 .0 0 .2  3 .5 1 .0 0 .2  3 .5 1 .0 0 .2  3 .5 1 .0 0 .2  Calcium  0 .49  0 .42  0 .35  0 .06  carbonate**  -—  20 .0 —  23- . 5 —  S P I = s o y p r o t e i n i s o l a t e ; MPC = s k i m m i l k p r o t e i n * I C N B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. ** B a r i a t r i x I n t e r n a t i o n a l I n c . , M o n t r e a l , PQ. * U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , OH. * * BDH C h e m i c a l s , T o r o n t o , ON. 1  76  concentrate.  In  3-point  occurred, speed to  by  lowering  ( 1 . 0 mm/  be  min).  determined,  stress  (a).  1) n e c e s s a r y as J o u l e s  a  This  tibia  centrally  was  until  failure  placed point at a  constant  t e s t a l l o w s two w h o l e  bending  failure  e n e r g y i s d e s c r i b e d as t h e  failure  into  consideration  work, or  Appendix  Figure  expressed  bone  size  (N/mm ; 2  O r t o f f and  1988):  (D  and  curve;  o f t h e bone i n b e n d i n g ,  a - 8 x Maximum B e n d i n g Load  where L  bone p r o p e r t i e s  e n e r g y and maximum b e n d i n g  under the f o r c e - d e f o r m a t i o n to achieve  bent  ( J ) . The maximum b e n d i n g s t r e s s i s a c a l c u l a t e d v a l u e  takes  Oxlund,  each  Bending f a i l u r e  energy, (area  that  bending,  4  ( L - 1) D  - d ) 4  i s t h e d i s t a n c e b e t w e e n t h e s u p p o r t i n g p o i n t s ( 1 3 mm); D  d are the outer  and i n n e r d i a m e t e r s  77  o f t h e bone (mm).  Bone M i n e r a l C o n t e n t Bone m i n e r a l tibiae  used  in  Analyses:  analyses the  were  shearing  performed and  hours.  The  ash  was s o l u b i l i z e d  d e t e r m i n a t i o n o f * C a by l i q u i d Mg by  atomic  experiment Statistical All between variance. to  absorption  then ashed  f e m o r a and procedures.  a t 550°C f o r  w i t h c o n c e n t r a t e d HC1 f o r  scintillation  5  the  3-point bending  S a m p l e s were d r i e d a t 110°C f o r 3 d a y s , 24  on  spectrophotometry  counting,  4 0  C a and  and P as d e s c r i b e d i n  1. Analyses:  the data are treatments  expressed  were  tested  as  mean  +_  f o r by  one-way  The S t u d e n t - N e w m a n - K e u l s m u l t i p l e  identify'the  source of the d i f f e r e n c e s  78  SEM.  range  Differences analysis t e s t was  a t t h e p<0.05  of used  level.  Results All feeding  animals training.  experimental  T h e r e were no  groups  (Table 2.2). course  e x h i b i t e d good e a t i n g b e h a v i o u r  In  i n body  addition,  of the experimental  different  protein  treatments  (range  weight feed  total  plasma  +_  calcium  mg/dL), n o r t h e c a l c u l a t e d 0.10 t o  efficiency  were  not  0.10  m i n e r a l s were a l s o n o t d i f f e r e n t the  or d r y matter ratios  d u r i n g the (p<0.05)  affected  by  dietary  t o 6.74 +_0.27 mg/dL). between d i e t a r y groups.  (range  8.47  +_  i o n i z e d plasma  4.04 +_ 0.05 mg/dL)  intake  (Table 2.2).  levels 6.12  gained  meal-  d i f f e r e n c e s between  p e r i o d were n o t s i g n i f i c a n t l y  between t r e a t m e n t s  Plasma  significant  following  were f o u n d  Plasma Neither  0.08 t o 8.72 +_ 0.13  c a l c i u m (range  3.82 +_  t o be a f f e c t e d by d i e t a r y  treatments. The p e r c e n t c a s e i n , whey  of  (p<0.05)  isolate  (Table  animals,  intestinal  2.3).  from the l i g a t e d  MPC f e d than  With  specific dietary  i n the  (p<0.05) 4 5  Ca  ileal  r a t s was f o u n d  for  animals  t o be  fed  loop i n signif-  soy p r o t e i n  t h e e x c e p t i o n o f whey p r o t e i n f e d activities  groups.  i n the i n t e s t i n a l  was s i g n i f i c a n t l y  specific  absorbed  greater  between  calcium present  reflected  Ca  p r o t e i n , and  icantly  different  4 5  The  loop  higher.  deposition to  were  not s i g n i f i c a n t l y  specific  o f whey  a c t i v i t y of  protein fed rats  T h i s o b s e r v a t i o n was f u r t h e r t h e bone  (Table 2.3).  The  a c t i v i t y o f t h e f e m o r a f r o m t h e whey p r o t e i n f e d a n i m a l s  was s i g n i f i c a n t l y  (p<0.05)  greater  c a s e i n , MPC, and s o y p r o t e i n i s o l a t e . 79  than  that  of  animals f e d  The s i g n i f i c a n t l y  (p<0.05)  TABLE 2 . 2  Diet  1 2  2  Diet  e f f i c i e n c y of rats fed experimental d i e t s . 1  Body W e i g h t G a i n (g)  Dry M a t t e r I n t a k e (g)  Feed E f f i c i e n c y Ratio  C  143 . 0 +_ 9 . 6  528.9  +  23 . 8  0.269  +  0 .009  s  132.8  4.0  497.0  +  12 . 7  0.268  +  0.007  M  136.4  9.9  504 . 0 +  20 . 6  0.269  W  120.8  5 .5  470 . 9 +  16.3  0.256  +  D a t a a r e e x p r e s s e d a s mean +. SEM. C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; M = m i l k c o n c e n t r a t e ; W = whey p r o t e i n i s o l a t e .  Means were n o t s i g n i f i c a n t l y  d i f f e r e n t a t p<0.05.  80  protein  0.012 +_  0.007  TABLE  2.3  Intestinal a b s o r p t i o n and femur d e p o s i t i o n o f calcium i n rats fed experimental d i e t s .  4 5  Diet  1 2  2  1  Absorbed C a (% d o s e ) 4 5  I n t e s t i n a l S.A. (dpm/ mg C a ) 4 0  Bone C a (% d o s e / g A s h ) 4 5  0 . 117*  204  .3  +  19 . 9*  706 +  0 . 114*  187  .2  +  19 . 4*  +  0 . 114*  161 . 1 +_ 26 . 1*  36 . 5 3  2 .82*  2 .91  +  0 .35*  0 .857  s  22 . 6 9  3 . 17«>  3 .65  +  0 .48*  0 .  M  35 . 19 +  4 .72"  2 .37  +  0 .28*  0 .654  W  31 . 70 +  1 . 04* •• «>  5 .04  +_ 0 . 52»>  505 +_ 0 . 1 8 7 »  D a t a a r e e x p r e s s e d as mean +_ SEM C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; M = m i l k c o n c e n t r a t e ; W = whey p r o t e i n i s o l a t e .  Means s h a r i n g t h e same letter within i c a n t l y d i f f e r e n t a t p<0.05.  81  40  +  C  1 .  Bone S.A. (dpm/mg Ca)  a column  342  .4  +  33 .8<>  protein are not s i g n i f -  l o w e r amount o f animals ition,  4 5  Ca  however,  absorbed was  not  from  reflected  o r bone s p e c i f i c a c t i v i t y  The  physical  parameters  of  different  dietary  dimensions,  the  the  femora  ileal  loop  in either  (Table  in  bone  Ca  fed  depos-  2.3).  m i n e r a l c o n t e n t s , and and  4 5  soy  tibiae  groups are summarized  biomechanical  of the a n i m a l s in  Tables  2.4  from  the  to  2.6.  D e s p i t e the enhanced i n t e s t i n a l a b s o r p t i o n of c a l c i u m i n r a t s t h e c a s e i n and isolate either  and  MPC  diets,  whey  compared t o t h o s e  protein  bone m i n e r a l i z a t i o n  these p a r t i c u l a r  or  no  bone  to e x i s t  and  hardness  b e t w e e n femur  biomechanical  82  protein  properties  Significant  calcium content  ( F ( 1, 42 ) = 9 . 38 ,  ( F ( l , 4 2 ) = 1 0 . 4 3 , p<. 0 . 0 0 2 4 ) .  soy  significant differences  a n i m a l s were o b s e r v e d .  were f o u n d bone  diets,  f e d the  p<_  0.003)  in of  correlations (mg  and  fed  Ca/g  femur  Ash) weight  TABLE 2.4  Femur m i n e r a l c o m p o s i t i o n diets .  in  rats fed experimental  1  Diet  1 2  2  A s h Wt. (g/femur)  Calcium (mg/g A s h )  Ca/P  13 .350  +  0 .07  13 . 506  +  1 .068  2 .05  +_  0 .06  13 .506  +  0 .580  2 .08  +_  0 .05  +  0 .315  0 .270  +  0 .009  361 .8  +  5 .3  2 .01  s  0 .274  +  0 .008  372 .4  +_  9. 1  2 . 10  M  0 .277  0 .004  361 . 1  +_  9. 1  w  0 .256  0 .013  369 .0  +  4 .8  D a t a a r e e x p r e s s e d as mean +_ SEM C = c a s e i n ; S = s o y p r o t e i n i s o l a t e ; M =- m i l k c o n c e n t r a t e ; W = whey p r o t e i n i s o l a t e .  Means were n o t s i g n i f i c a n t l y  different  83  0 .421  0 .06  C  +  Magnesium (mg/g A s h )  9 . 948  protein  a t p<0.05.  TABLE  2.5 Femur p h y s i c a l d i m e n s i o n s and b i o m e c h a n i c a l in rats fed experimental d i e t s .  parameters  1  Diet  1 2  2  Femur Wt. (g)  Bone L e n g t h (mm)  Bone S t r e n g t h (N/mm ) 2  Bone H a r d n e s s (x 1Q- J) 2  c  0 .646  +  0 .030  32 .57  +  0 .23  1 .312  +_  0 .069  18 .07  +_  1 .91  s.  0 .655  +  0 .036  31 .68  +  0 .36  1 .315  +  0 .078  18 . 86  +  1 .69  M  0 . 684  +_  0 .017  31 .99  +_  0 . 14  1 .232  _+  0 .034  18 .25  W  0 . 626  +_  0 .020  32 .35  +  0 .40  1 . 289  +  0 .058  13 .55  D a t a a r e e x p r e s s e d as mean +_ SEM C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; M = m i l k c o n c e n t r a t e ; W = whey p r o t e i n i s o l a t e .  Means were n o t s i g n i f i c a n t l y  protein  d i f f e r e n t a t p<0.05).  84  1 .66 +  0 .87  TABLE 2.6 T i b i a c a l c i u m content and biomechanical parameters o f t h r e e - p o i n t bending a n a l y s i s i n r a t s f e d experimental d i e t s . 1  Diet*  T i b i a Wt. (g)  Ash Wt. (g/tibia)  Calcium Bending F a i l u r e Maximum Bend(mg/g Ash) Energy ing Stress (x 1 0  -3  J)  (N/mm ) 2  c  0.375 + 0.023  0.186 + 0.007  406.0 + 17.8  53.94 + 6.31  60.44 + 3.01  s  0.407 + 0.021  0.207 + 0.011  418.6 + 25.4  53.61 + 3.95  57.60 + 2.31  M  0.395 + 0.005  0.191 + 0.004  421.0 + 19.8  62.76 + 4.56  56.18 + 1.25  W 0.420 + 0.016  0.190 + 0.012  408.5 + 23.7  63.85 + 5.64  67.05 + 2.00  —  Data a r e expressed as mean + SEM C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; M = milk p r o t e i n w = whey p r o t e i n i s o l a t e .  concentrate;  Discussion The  absorption  of c a l c i u m  subsequent u t i l i z a t i o n were e x a m i n e d foods,  f o r bone  i n animals  considered  from the  to  i l e u m , as  w e l l as i t s  m i n e r a l i z a t i o n and  biomechanics  fed d i f f e r e n t  be  excellent  dietary proteins.  sources  of  calcium,  examined to  determine the p r o t e i n - c a l c i u m i n t e r a c t i o n  to  calcium  dietary  bioavailability.  c a s e i n , whey p r o t e i n s and concentrate The  were compared t o soy  presence  d i e t s was  of c a s e i n ,  the  animals  4 5  Ca  protein diet.  in a  l o o p , and  absorbed.  higher  This  has  to  digestibility intestinal 4 0  Ca  whey time  present  Accordingly,  should  of  transit  being  sources  one  a-lactalbumin  the  observed  in  lower by  proteins  l o o p , but  its and  the  relative  t h i s was  in  86  Ca  intestinal  for calcium the  i n the  amount of of  constituent proteins, in 1988).  feasibly  loop  in a at  low  decrease  the  any  The  amount of  given  of  Narasinga  activity  case.  The  lesser  digestibility  the  4 5  feeding s t u d i e s , which  ( R a g h u n a t h and  not  the  in  Bell,  specific  of  the poor d i g e s t i b i l i t y  could  ileal  respective  c a s e o f whey p r o t e i n  activity  which would r e s u l t  have a l s o i n c r e a s e d t h e  intestinal  i n the  source.  absorption  Ca  4 0  animal  of  (Keith  others  MPC  ileal  reflect  i n previous  particular  in a milk protein  overestimation  r e s u l t may  attributed  of b o t h  of  specific  whey p r o t e i n s o b s e r v e d been  p r o t e i n s , namely  In the  level  p o s s i b l y an  applied  protein, a plant protein  a higher  however, a lower  lumen r e s u l t e d ligated  soy  in  as  were  Dairy  whey p r o t e i n , and  shown t o r e s u l t  compared t o fed  a combination  Dairy  of lower  soy Rao, 4 5  Ca  time. protein 1984), i n the  absorption  of  4 5  Ca  in rats  related highly  to  fed  the  soy  the d i g e s t i b i l i t y  The  amino a c i d  derived  products  considerably  of the  of  than  intestinal  contents  and  lower L - l y s i n e content  soy  p r o t e i n compared t o c a s e i n  calcium  pathway of absorption  coworkers  little  calcium of  4 5  Ca  was  that  n e g a t e any  other  (Sato  it  are  (Raghunath  and  from  casein  that  Ju,  constant,  1961).  reported the  The  products derived  nonsaturable, fact that  i n the  ejt a_l_. , 1 9 8 3 ) .  ileal  This  fed  relative loop  of  animals,  protein  had  observation  lower d i g e s t i b i l i t y  of  response.  availability  s o l u b l e form Lee  enhances  paracellular  t h e MPC  absorption.  from  Moreover,  a lower  ligated  hand, p h o s p h o p e p t i d e s d e r i v e d  in a  recent  L-arginine  that L-arginine  s i g n i f i c a n c e of t h e  the  regardless  However,  d i g e s t i o n p r o d u c t s o f soy  this  amino a c i d  (Jacques et a l . . 1986).  observed  the  the  shown a m a r k e d l y h i g h e r  absorption.  have been shown t o i n c r e a s e maintaining  more r e s i s t a n t  digestions  is virtually  modifying  products i n e l i c i t i n g the  are  protein  considered  e f f e c t i n enhancing calcium  On  are  proteins  and  compared t o b o t h c a s e i n and  suggests  would a l s o  Soy  not  of p e p t i d e  soy  those  therefore  the y i e l d  from d i g e s t i o n  (1956)  u p t a k e by  animals fed soy, strongly  from  ( N a s s e t and  s t u d i e s have c l e a r l y  Wasserman and  protein.  consequently,  s t u d i e s , i t was  proteins digested  in vitro  was  1984).  In p r e v i o u s profile  this  diet  r e s u l t i s that  different  N a r a s i n g a Rao,  soy  of  s t r u c t u r e d p r o t e i n s and  to enzymatic a t t a c k .  ileal  protein  i n the  ejt a_l_. ( 1 9 8 3 ) 87  from c a s e i n  of i o n i c  (CPP)  calcium,  by  lower s m a l l i n t e s t i n e  reported  that r a t s fed  a  c a s e i n d i e t had  a significantly  and  insoluble  decreased  greater  calcium  amount o f  in  the  has  c a s e i n o r egg  1989). T h e r e f o r e ,  the  o b s e r v e d i n both the hypothesis  that  d i g e s t i o n of from the  a c t i o n t o the yet  been  overall  study  mineralization rats  4 5  Ca  we  and  fed  noteworthy calcium  casein  the  that  fed  (Kitts  absorption animals produced  facilitate  absorption  intestine. calcium  The  relative  balance  et  of  a1.,  calcium  supports  phosphopeptides the  observation  the  from  tryptic  of  calcium  i m p o r t a n c e of  of the  individual  this  has  a l s o e x a m i n e d bone d e p o s i t i o n of  different  the  higher  i n d i c e s of c a l c i u m  dietary  4 5  Ca,  not  This  intestinal  method  calcium  to a s s o c i a t e d bone  specific  p r o t e i n sources.  intestinal  is  specific  activity subject activity  specific  mineralization  measurements of  calcium  and  were a p p l i e d  i n deformation  s i n g l e blade shear t e s t .  the  femora  to o v e r e s t i m a t i o n  of the  diet.  biomechanical  utilization  s o u r c e s of o v e r e s t i m a t i o n .  in  which are In t h e  88  of  greater of  these  when  the  elevated  due  On  the  other  properties not  present  to i m i t a t e a p a r t i c u l a r Data g e n e r a t e d  It is  activity  is artificially  factors characteristic  bone  utilization  i n t h e whey p r o t e i n f e d a n i m a l s c o r r e s p o n d e d t o a  animals.  similar  MPC  b i o m e c h a n i c s as  d e p o s i t i o n and  hand,  laboratory  intestinal  and  This  mixture  ascertained.  In t h i s  from  casein  small  i n our  enhanced  casein w i l l  distal  amino a c i d  albumen, r e s p e c t i v e l y .  a l s o r e c e n t l y been c o n f i r m e d  calcium  l o w e r i n t e s t i n e when  compared t o a n i m a l s f e d d i e t s c o n s i s t i n g of an simulating  soluble  in this  susceptible study,  are to  forces  action in  the  t e s t were u s e f u l  in  comparing  a  conditions.  The  importance  from  were h i g h l y 1987). point  magnitude an  calculate strength  on  of  t h e sample  significant femur  be a s s o c i a t e d  be a u s e f u l  D e s p i t e the apparent containing  ionized  properties  calcium, in  or  t h e femur  and  w o r k e r s have shown a marked of c a l c i u m  intestinal  hypercalciuria renal  tubular  filtration  of  bending  and  possess  1988).  obtained  calcium  absorption has  was  calcium  may  a 1. . 1 9 8 7 ) ,  and  utilization  1988). bioavailability e i t h e r plasma  samples in  total  were o b s e r v e d . the  from  and b i o m e c h a n i c a l  urinary  Other  excretion  p r o t e i n d i e t , a l b e i t no  effect  o b s e r v e d ( A l l e n ejc. a 1 . . 1 9 7 9 ) .  been a t t r i b u t e d  reabsorption  respectively.  calcium  mineralization  i n rats fed a high  between f e m o r a l  t h a t bone s t r e n g t h  calcium  increase  and  (Schuette 89  to a an  to  In the p r e s e n t  content,  enhancement o f  tibia  model o f  the  models  c o n t e n t (Kusy et  bone  pure  3-  that  were  increased  d i e t s , no  a  in cross-section  and O x l u n d ,  Kapsalis,  test involving  of  functional test for assessing Ortoff  they  used  the p o s t u l a t i o n  bone m i n e r a l  little  and a f o r m u l a was  ( O r t o f f and O x l u n d ,  corroborate with  stress  and  had  ( S e g a r s and  engineering  are c i r c u l a r  identical  of view however, s i n c e  employed  correlations  under  obtained  application  was  weight  ( K u s y e t a l . , 1987;  on  values  point  bending  thickness  These r e s u l t s  rate  the  The  tubes that  h a r d n e s s and  and  of  tested  test conditions  used.  t h e maximum  uniform wall  casein  samples  In a d d i t i o n , a w e l l defined b e n d i n g was  study,  of  absolute  dependent  deformation to  may  series  This  decrease i n f r a c t i o n a l  increase  et a l . , 1981).  in The  glomerular findings  of  W h i t i n g and D r a p e r content  to  strated  that  quantity  be  (1981)  related  the source  fed,  that  can  showed p r o t e i n  s u l f u r amino  to a c a l c i u r e t i c a c t i o n , of d i e t a r y  adversely  protein,  affect  in  adequate f o r  calcium homeostasis.  a b s o r p t i o n o f c a l c i u m due t o amino urinary  acids  excretion  significance Further  that  in  studies  may of  regard  this to  presence  mineral,  had  to determine  90  addition  to the In t h i s  calcium at a l e v e l  of  CPP,  or c e r t a i n  p a r a c e l l u l a r movement, o r  i t sutilization  subjects.  demon-  Thus, the enhanced i n t e s t i n a l  enhance e i t h e r  are required  calcium d e f i c i e n t  the  further  calcium balance.  s t u d y , d i e t s were i s o n i t r o g e n o u s and c o n t a i n e d  acid  little  physiological  f o r bone  metabolism.  i f t h i s i s the  case i n  Experiment 3 Calcium  bioavailability  i n c a s e i n and s o y f e d r a t s .  Introduction Dietary  calcium  intake i s considered  t o be a f a c t o r i n bone  mineralization,  and i n p a r t i c u l a r ,  Bioavailability  o f c a l c i u m from p l a n t p r o t e i n sources  be i n f e r i o r , when compared c a s e i n , due and  phytate  In  the  effects  to animal  ( L i e b m a n and study,  Landis, the  of d i e t a r y calcium absorption,  deposition  and  performed  as  well  and  Khan and were  Weaver,  source  on i l e a l  as s u b s e q u e n t u t i l i z a t i o n strength.  osteoporosis  prone  1989).  to determine the  protein  These  i n bone  studies  spontaneously  (SHR) and n o r m o t e n s i v e c o n t r o l W i s t a r - K y o t o Materials  s u c h as  c h e l a t o r s , namely f i b r e  objectives  biomechanical  using  1989;  level  i s noted t o  derived proteins,  t o the presence of p o t e n t i a l  present  calcium  the incidence of o s t e o p o r o s i s .  were  hypertensive  (WKY) r a t s .  and M e t h o d s  A n i m a l s and D i e t s : Four  week-old  normotensive  male  Wistar  Kyoto  PQ) were e a c h d i v i d e d i n t o group). isolate of  Dietary  calcium  (2  s i x experimental included  20%  casein  Montreal,  or soy p r o t e i n  C l e v e l a n d , OH) c o n t a i n i n g a h i g h w/w%); of  a  calcium  medium l e v e l  low  (Table  A n i m a l s were f e d ad 1 i b i t u r n u n t i l  body w e i g h t ,  (SHR) and  g r o u p s (6 a n i m a l s p e r  w/w%); and a 3.1).  level  hypertensive  (WKY) r a t s ( C h a r l e s R i v e r ,  groups  (ICN Biochemicals,  dietary  spontaneously  a f t e r which meal-feeding 91  level  of c a l c i u m (0.5  ( 0 . 0 5 w/w%), r e s p e c t i v e l y they  was i n i t i a t e d .  reached  lOOg  Over a two  week p e r i o d , six  animals  hour p e r i o d d a i l y  made a v a i l a b l e  were  (9 a.m.  to animals  w e e k l y body w e i g h t g a i n s Intestinal described  in  calcium  animals  f e d the high  medium  calcium  p.m.).  a_d_ 1 i b i t u m .  absorption  the  contents,  and  was  throughout the  experiment.  measured  as p r e v i o u s l y  were 14 weeks o f age. dose  uCi t o t a l 5.4 u C i  0.375  water  i n t a k e s and  injected  calcium diets;  diets;  Deionized feed  was  15  the d i e t s w i t h i n a  Daily  1 when a n i m a l s  standardize  calcium  to 3  were r e c o r d e d  experiment  an a t t e m p t t o intestinal  t r a i n e d t o consume  uCi  of  4 5  d o s e was  Ca  to the  chosen f o r  f o r animals  f o r animals  In  f e d the  f e d t h e low  calcium d i e t s , r e s p e c t i v e l y . Analyses: Analyses  of i n t e s t i n a l  as p r e v i o u s l y d e s c r i b e d Bone  l u m e n , and b l o o d  i n experiment  plasma m i n e r a l s  were  1.  Biomechanics: After  cleansed  sacrifice,  of adhering  f e m o r a and  femur and t i b i a soft  t i b i a e were  tissue, subjected  previously described  i n experiment  Bone M i n e r a l C o n t e n t  Analyses:  bone s a m p l e s were  and  epiphyses  to 3 point _  for  e x p e r i m e n t 2. were a n a l y z e d  bone  mineral  content  Femora were a n a l y z e d f o r Ca and Mg  content.  92  removed.  Both  b e n d i n g a n a l y s i s as  2.  Femora and t i b i a e u s e d i n t h e 3 - p o i n t analyzed  excised,  b e n d i n g p r o c e d u r e were  as p r e v i o u s l y d e s c r i b e d i n  f o r Ca and P,  whereas  tibiae  TABLE 3.1  Composition o f experimental d i e t s f e d t o a n i m a l s .  Casein d i e t s D i e t a r y component ( 9 / 100g)  Casein* Soy p r o t e i n  +  2%Ca  0.5%Ca  20.0  20.0  Soy d i e t s  0.05%Ca  2%Ca  0.5%Ca  0.05%Ca  20.0  isolate  20.0  20.0  20.0  0.3 15.0 45.02 5.0 5.0  0.3 15.0 48.77 5.0 5.0  0.3 15.0 49.89 5.0 5.0  0.3 15.0 45.08 5.0 5.0  0.3 15.0 48.83 5.0 5.0  0.3 15.0 49.96 5.0 5.0  Ca f r e e m i n e r a l ^ m i x * Vitamin mixture Choline bitartrate" "  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  Calcium carbonate"*"*"  4.98  1.23  0.11  4.92  1.17  0.04  D.L. methionine"*" Cornstarch Sucrose Fibre Vegetable o i l  1  ICN B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , OH. "^BDH C h e m i c a l s , T o r o n t o , ON. +  Statistical All  Analyses:  data  are expressed  t r e a t m e n t s were t e s t e d paired  T-test.  Keuls m u l t i p l e differences  for  as mean +_ SEM. by one-way  Where d i f f e r e n c e s  Differences  analysis  of  between  variance  and  d i d e x i s t , t h e Student-Newman-  r a n g e t e s t was u s e d t o i d e n t i f y t h e s o u r c e s o f t h e  a t a p<0.05 l e v e l  of s i g n i f i c a n c e .  94  Results There  was  no s i g n i f i c a n t  difference  food i n t a k e , or feed e f f i c i e n c y fed the  same d i e t  0.05% Ca d i e t s intake and  exhibited  2.0% Ca d i e t s .  exhibited which  body w e i g h t ,  SHR and  SHR and WKY body w e i g h t  WKY  rats  rats  f e d the  (pi.0.05), food  (p<0.01) compared t o a n i m a l s f e d t h e 0.5%  2.0% c a l c i u m  to a  only observed  diet  (p<0.05)  level;  lower  food  l o w e r body w e i g h t  to a f f e c t  soy f e d a n i m a l s intake  and FER  g a i n than observed i n  T h i s o b s e r v a t i o n was common  f o r both  SHR and  animals. Plasma  and  Both  a lower f i n a l  a significantly  corresponded  final  (PER) i n  D i e t a r y p r o t e i n was  at the  those f e d c a s e i n . WKY  (Table 3.2).  (p<0.05) and PER  animal growth  ratio  in  WKY  effect  minerals  are presented  rats, dietary protein on  plasma  mineral  profiles Plasma  significantly  decreased  casein diet increased  o n l y , whereas in  these  0.05% Ca s o y d i e t .  3.3.  s o u r c e was n o t o b s e r v e d  medium c a l c i u m l e v e l s . (p<0.01)  i n Table  in  total  plasma  animals,  animals fed Ca, and  in P  compared  to  SHR  have an  t h e h i g h and  i o n i z e d Ca*  animals  was  For both  2  were  f e d t h e 0.05% Ca  significantly  (p<0.05)  to c o u n t e r p a r t s f e d the  T h i s t r e n d was o b s e r v e d  in  b o t h SHR  and  WKY  animals. Animal  s t r a i n d i f f e r e n c e s were n o t o b s e r v e d  the i n t e s t i n a l 3.4).  Ileal  4 0  Ca  content or  * Ca 0  content  4 5  Ca  level  (r=0.845,  p<0.0l).  also  n o t a f f e c t e d by d i e t a r y  correlated  Intestinal 95  either  a b s o r p t i o n ( F i g u r e 3.1, T a b l e  was  p r o t e i n s o u r c e , b u t was d i r e c t l y  to a f f e c t  specific  with dietary calcium activities  TABLE 3.2  Diet  Body weight g a i n and food i n t a k e of experimental animals .  I n i t i a l body wt. (g) SHR WKY  2% Ca C a s e i n 116 +  2  ax  125 +  4  ax  110 +  4  ax  118 +  2  ax  C a s e i n 116 +  4  ax  119 +  5  ax  108 +  3  ax  108 +  6  ax  6  ax  125 +  4  ax  6  ax  122 +  5  ax  Soy  0.5% Ca Soy  0.05% Ca C a s e i n 119 + Soy  118 +  F i n a l body wt.3 (g) SHR WKY  Dry M a t t e r Intake (g) SHR WKY  284 + 6  a x  309 + l l  a x  863 + 3 8  262 + 7  a b x  257 + 8  a b x  793 + 2 5  307 + 9  a x  259 + 8  a b x  982 + 1 5  282 + 5  a x  252 + 1 4  bx  888 +  l g  239 + 1 3  bx  249 + 9  b x  730 +  n  260 ± 9  b x  253 + 9  b x  800 +  Data a r e expressed as mean + SEM; SHR = Spontaneously 5 weeks o f age. 14 weeks of age.  a x  a b x  a x  ax  bx  22bx  838 +  1 0  Feed E f f i c i e n c y Ratio SHR WKY  ax  750 + 1 4 844 +  l g  ax  850 + 2 7 693 +  1 3  b x  a x  bx  718 + 1 5  b x  0. 132 + 0 . 0 1 5 0. 146 + 0 . 0 1 5 0. 173 + 0 . 0 1 5 0. 177 + 0 . 3 0 7 0. 104 + 0 . 0 1 4  a b x  a b x  ax  ax  bx  0. 120 + 0 . 0 1 0  a b x  0.157 + 0 . 0 1 9 0.102 + 0 . 0 1 3  b c y  0.180 + 0 . 0 0 8  ax  0.184 + 0 . 0 1 6  ax  0.092 + 0 . 0 1 6  cx  0.111 + 0 . 0 1 2  h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s .  = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n columns. = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows.  a b x  b c x  TABLE 3.3  E f f e c t of d i e t a r y  Diet  Ca SHR  2% Ca Casein Soy  protein  WKY  11.3+0.3 10.4+1.4 10.1+1.1 10.3+0.5  SHR  source on plasma m i n e r a l s o f r a t s f e d d i f f e r e n t  Ca"^ WKY  Mg SHR  Na WKY  SHR (mg/dL)  WKY  SHR  levels of calcium .  K WKY  P SHR  WKY  5.3+0.1 4.5+0.3  4.7+0.6 5.0+0.2  2.0+0.2 2.1+0.4 2.2+0.5 2.1+0.1  313+11 315+26 282+ 6 283+ 8  22+2 20+4 23+4 16+1  8.8+0.3 9.6+0.8  8.9+0.3 8.6+0.4  4.3+0.1 4.4+0.1  4.1+0.2 4.3+0.1  1.9+0.1 2.0+0.2 2.1+0.3 2.5+0.5  311+10 290+ 9 313+11 329+13  18+1 19+1 23+3 23+6  9.3+0.5 8.7+0.7  9.0+0.4 8.4+0.5  0.05% Ca 5.9+0.7* 6.6+0.6* 2.2+0.4* 3.0+0.4* 1.8+0.2 2.4+0.2 Casein 10.3+0.5 9.8+0.6 4.8+0.2 4.5+0.4 2.4+0.2 2.4+0.1 Soy  265+22 289+ 3 288+ 9 295+ 3  19+5 16+2 18+1 17+1  0.5% Ca Casein 9.7+0.4 Soy 10.6+0.6  9.4+0.7 9.9+0.4  11.8+0.7* 12.5+1.3* 8.6+0.8 8.6+0.9  Data a r e expressed as mean + SEM; SHR = spontaneously h y p e r t e n s i v e r a t s , WKY = Wistar-Kyoto Ca = (6Ca-P/3)/(P+6); where Ca and C a a r e mg/dL and P i s g/dL. • denotes s i g n i f i c a n t d i f f e r e n c e a t p<0.01. + 2  + 2  rats.  „  s O  C-SHR  S-SHR  C-WKY  C-SHR  S-SHR  C-WKY  S-SHR  C-WKY  S-WKY  5  o •* a  e E a u <  - o  S-WKY  100 » a  o  10 a O  •*  •o e  D <  C-SHR  S-WKY  Protein and Animal Type 2.0% C a  Fig.  3.1  0.5% C a  \  M 0.05% C a  E f f e c t o f d i e t a r y p r o t e i n s o u r c e and c a l c i u m l e v e l on calcium content and a b s o r p t i o n , r e s p e c t i v e l y f r o m t h e ileal loop of spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) r a t s f e d c a s e i n ( C ) and s o y protein i s o l a t e (S) d i e t s . *oCa c o n t e n t o f i l e a l l o o p (a); C a s p e c i f i c a c t i v i t y of loop ( b ) ; * C a absorbed from loop ( c ) . 4 5  5  98  TABLE 3.4  Effect of dietary protein  4 0  source on c a l c i u m a b s o r p t i o n i n r a t s f e d d i f f e r e n t l e v e l s o f c a l c i u m  C a i n loop  4 5  (mg/ loop) Diet  SHR  2% Ca Casein Soy  (dpm/ mg WKY  10.3 8.8  + 1.2 + 1.0  0.5% Ca Casein Soy  2.3 2.7  + 0.3 + 0.4  0.05% Ca Casein Soy  0.08 + 0 . 0 1 0.14 + 0 . 0 2  ax  2.5 + 0 . 6 2.7 + 0 . 4  a x ax  a x ax  4 0  ax ax  ax ax  0.07 + 0 . 0 1 0.11 + 0 . 0 1  a x a x  ax  3.9 + 0 . 6 3.5 + 0 . 5  ax  a x  a x  a x ax  Ca  absorbed  2  (% dose)  WKY  2.4 + 0 . 2 3.3 + 0 . 3  1.4 + 0 . 3 2.1 + 0 . 5  4 5  Ca)  SHR  13.2 + 1 . 7 U.1 + 1.8  ax  Ca Specific activity  2.1 + 0 . 3 2.8 + 0 . 3  4.7 + 0 . 9 4.0 + 0 . 7  1.5 + 0 . 3 1.9 + 0 . 2  SHR  a x a x  a x a x  a x a x  WKY  36.6 + 4 . 4 40.0 + 2 . 6  ax  35.8 + 3 . 8 20.8 + 5 . 3  ax  87.8 + 2 . 3 71.0 + 5 . 0  a x  b x  a x b x  37.9 + 1 . 8 29.1 + 4 . 0  31.9 + 0 . 9 20.3 + 3 . 4  80.G + 4 . 6 70.4 + 1 . 6  ^•Data are expressed as mean + SEM; SHR = spontaneously h y p e r t e n s i v e r a t s , WKY = W i s t a r - K y o t o 2  a  4 5  *  b  x , y  C a a b s o r p t i o n (% dose) = [ l - C a 4 5  = significant difference = significant difference  (dpm) a t 1.0 h r / dose  4 5  C a (dpm) a d m i n i s t e r e d ] x 100.  (p<0.05) between i n d i v i d u a l treatment means i n column. (p<0.05) between i n d i v i d u a l treatment means i n row.  ax b y  a x bx  ax bx  rats.  were  successfully  standardized  d i e t a r y p r o t e i n source. 4 5  Ca  absorption  Intestinal lower  Ca  4 5  in  T h e r e was SHR  and  absorption  i n soy f e d a n i m a l s  than  of the 2%  4 5  Ca  4 5  Ca  rats  fed  however,  by t h e  the  3.5).  deposited  to  The  indicated  a  tissue  levels,  significantly  to  bone  agreement w i t h significantly  (p<0.05)  the  greater f o r animals  in  animals  lower  in  calcium  d e p o s i t i o n of (Figure  amount o f  4 5  Ca  deposition  of  the absorbed  These o b s e r v a t i o n s  activities  t h e 2%  which  diet  are i n  were a l s o  calcium fed animals  f e d t h e 0.05% c a l c i u m  content  only  calcium  and  diet.  Femur m i n e r a l i z a t i o n i s s u m m a r i z e d i n T a b l e w e i g h t and  was  2%  animals.  specific  0.5% and  the  these bone  efficiency  absorption data  greater  fed  in  (p<0.05)  f e d the  (p<0.05) l o w e r  efficiency by  Ca  (p<0.05)  Animals fed  i n t h e 0.05% Ca f e d a n i m a l s  bone  lower  4 5  diet.  at both the  respectively.  intestinal  The s i g n i f i c a n t l y  the  same  casein fed counterparts  significantly  3.2, T a b l e  difference in  exhibited a greater absorption  levels.  i n t o femoral  calcium  no s i g n i f i c a n t  d o s e when compared t o c o u n t e r p a r t s  calcium  confirmed  level  e l i m i n a t e v a r i a t i o n s due t o  WKY  was  medium and l o w d i e t a r y c a l c i u m t h e 0.05% c a l c i u m  to  were s i g n i f i c a n t l y  3.6.  Femur a s h  (p<0.05)  decreased  i n animals  f e d 0.05% d i e t a r y c a l c i u m compared t o c o u n t e r p a r t s f e d  0.5%  2%  and  calcium  levels.  (p<0.05) g r e a t e r f e m u r a s h counterparts fed  animals  at both  than  w e i g h t and  had a  a  those  significantly  calcium content  t h e 2% and 0.5% d i e t a r y c a l c i u m  exhibited  calcium content  SHR a n i m a l s  significantly  (p<0.05)  levels. lower  f e d c a s e i n a t t h e 2% and 0.5% 100  than  WKY Soy  femur  calcium  C-SHR  S-SHR  C-WKY  S-WKY  1409  <  v»  0  CO  O "0  #  12-  tiiii iiii  10-  •ilflil I  86 -  IP ill  4-  <0 O 210  oC-SHR  3.2  mm®  f  8  S-SHR  S-WKY  C-WKY  Protein a n d Animal Type 2.0%  Fig.  i i i  1^  I  0.6%  Ca  Ca  0.06%  Ca  Femur d e p o s i t i o n o f C a i n s p o n t a n e o u s l y h y p e r t e n s i v e (SHR) and c o n t r o l W i s t a r - K y o t o (WKY) rats fed casein (C) and s o y p r o t e i n isolate (S) d i e t s . Bone Ca s p e c i f i c a c t i v i t y ( a ) ; Bone C a c o n t e n t ( b ) . 4 5  4 5  4 5  101  TABLE 3.5 Femur d e p o s i t i o n diets .  of  45  calcium i n rats fed experimental  1  Diet  Bone C a (% d o s e / g A s h ) SHR WKY  Bone S.A. (% d o s e / g ° C a ) SHR WKY  4 5  4  2% Ca Casein  0.52 + 0 . 0 6 *  0.18 + 0 . 0 4 *  1.29 + 0 . 1 0 *  0.41 + 0.04**  Soy  0.50 +. 0 . 0 3 *  0.22 +_ 0 . 0 4 r  1.32 +. 0 . 0 8 *  0.58 ± 0 . 0 3 '  Casein  1.26 + 0.06°*  0.62 +. 0.03°*  3.54 +. 0 . 1 2 *  1.85 ±  Soy  0.91 +_ 0 . 0 4 *  0.86 +_ 0 . 0 6 *  2.66 ±_ 0.12«*  2.02 +_ 0.09°*  d  d  d  d  d  d  d  0.5% Ca  c  c  c  0.07=*  0.05% Ca Casein  11.22 ± 0.09»* 13.25 + 0.10«* 28.30 +_ 0.34»* 26.80 ± 0.20»*  Soy 8.13 i 0 . 0 8 * 6.53 +. 0 . 0 8 * 20.92 +. 0.27 » 15.46 +. 0.16<>y Data are e x p r e s s e d as mean +_ SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r K y o t o r a t s . a,b,c,d - s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n columns. *•y ~ s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n rows . b  b  1  102  b  TABLE 3.6  Femur m i n e r a l c o m p o s i t i o n o f r a t s f e d experimental d i e t s .  Diet  Ash wt. (g/bone) SHR  Ca (mg/bone) WKY  2% Ca Casein  0.247 + 0 . 0 0 6  Soy  0.226 + 0 . 0 0 8  0.5% Ca Casein  0.251 + 0 . 0 1 1  Soy  0.218 + 0 . 0 0 8  0.05% Ca Casein  0.067 + 0 . 0 1 5  Soy  0.095 + 0 . 0 0 9  ax  ax  ax  a b x  dx  cx  SHR  0.219 + 0 . 0 0 9 0.196 + 0 . 0 0 7 0.184 + 0.006 0.173 +  WKY  ay  99.60 + 2 . 9 8  ay  88.10 + 4 . 7 0  a:y  93.09 + 4 . 3 9  ax  ab  0.108 + 0 . 0 0 5  a b x  ax  o.on y 79.72 + 0 . 8 3  0.056 + 0 . 0 0 6  bx  dx  27.75 + 3 . 1 8  dx  cx  39.00 + 3 . 6 8  cx  Data a r e expressed as mean + SEM; SHR = Spontaneously  Ca/P Ratio SHR  86.25 + 4 . 0 2  ay  2.22 + 0 . 0 5  S  n  c a n  2.00 + 0 . 2 0  ax  1.91 + 0 . 0 6  ax  a x  2.11 + 0 . 0 3  ax  a x  71.55 + 5 . 0 1 *  2.06 + 0 . 1 9  64.50 + 2 . 7 1 *  2.17 + 0 . 0 7  68.62 + 2 . 3 6  bx  2.15 + 0 . 0 3  a x  2.19 + 0 . 0 4  ax  27.98 + 2 . 3 1  d x  1.36 + 0 . 0 5  b x  i.44 + 0 . 0 4  bx  + 3.01  c x  bx  1.38 + 0 . 0 5  bx  b  b  43.11  1.39 + 0 . 0 4  a x  h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s .  a,b,c,d _ i g i f i t (p<0.05) d i f f e r e n c e between treatment means i n columns. » y = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows.  x  WKY  levels,  respectively.  with dietary icant  Femoral  calcium content  i n t e r a c t i o n s were f o u n d  animal  strain  protein  (r=0.765,  pOO.001;  ( F ( 2 , 6 1 ) = 9 .43 ,  f o r femur c a l c i f i c a t i o n .  significantly  (p<0.05) d e c r e a s e d  d i e t when compared t o t h o s e f e d  effects  There  on femur Ca/P  were  and  WKY  rats  was  significantly  calcium diet  (Table  i n t h e 0.05% energy  to  and  bending  femoral decreased  fed animals.  0.5%  and 2),  ratio  was  calcium  calcium diets,  animal s t r a i n or p r o t e i n  bone d r y w e i g h t were  source  in  significantly  animals  the  were  failure  fed  only,  animals  bones  energy  bending  these animals  weight  the  0.05%  2% c a l c i u m d i e t s .  of  found  these  not  or  Femur b e n d i n g  c o r r e l a t i o n was  and  fed  (p<0.05) d e c r e a s e d  differences  calcium  Bone d r y  i n only  calcium casein diet.  strain  3.8).  not d i f f e r e n t i n  (Table 3.7).  animal  maximum in  no  parameters  failure  Table  femur Ca/P  and  biomechanical  break  significant  t h e 2%  (p<0.05) d e c r e a s e d  t h o s e a n i m a l s f e d t h e 0.05%  source  The  and  1)  i n a n i m a l s f e d t h e 0.05%  f e d t h e same d i e t  However, bone l e n g t h was  a f f e c t e d by  Table  Appendix  compared t o t h o s e f e d t h e 0.5%  Femur  signif-  ratio.  Femur bone l e n g t h and SHR  Thus,  Appendix  p<0.001;  respectively,  respectively.  p<0.01).  correlated  t o e x i s t between c a l c i u m i n t a k e  ( F ( 2 , 6 1 ) = 7 . 33,  source  c a l c i u m c o n t e n t was  by  dietary  energy  animals.  (F(1,54)=41.28, was  p^.  104  protein  decreased less  work  Moreover,  a  calcium content 0.01).  Further,  significantly  (p<0.05)  when compared t o 0.5%  T h u s , t h e maximum f o r c e  was  indicating  b e t w e e n femur  stress  significantly  required  and  to break  2% c a l c i u m t h e bones  TABLE 3.7  Femur p h y s i c a l parameters  Diet  Bone Length (mn)  SHR  of r a t s fed experimental d i e t s .  WKY  SHR  Bone Dry Wt. (g)  WKY  2% Ca Casein  30.39 + 0 . 4 4  ax  30.10 + 0 . 2 5  ax  0.400 + 0 . 0 1 1  ax  0.360 + 0 . 0 1 4  Soy  29.98 + 0 . 4 9  ax  29.54 + 0 . 5 4  ax  0.365 + 0 . 0 1 3  ax  0.331 + 0 . 0 1 2  0.5% Ca Casein  32.00 + 0 . 5 8  ax  30.78 + 0 . 6 6  ax  0.383 + 0 . 0 1 6  ax  Soy  31.84 + 0 . 0 4  ax  29.56 + 0 . 5 9  ax  0.342 + 0 . 0 1 1  ax  0.05% Ca . Casein 26.96 + 1 . 3 6  . 24.24 + 1 . 8 2  by  0.167 + 0 . 0 2 6  cx  Soy  29.61 + 0 . 3 5  ax  0.211 + 0 . 0 1 0  bx  bx  29.20 + 0 . 2 5  ax  ^ Data are expressed as mean + SEM; SHR = Spontaneously WKY = W i s t a r Kyoto r a t s .  , 0.296 + 0 . 0 1 0  x  n  c a n  a b x  by  0.270 + 0 . 0 1 4  b c y  , 0.144 + 0 . 0 0 8  dx  0.235 + 0 . 0 0 7  cx  hypertensive r a t s ,  a,b,c,d _ - i g i f i t (p<0.05) d i f f e r e n c e between treatment means i n columns. »y = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows. s  ax  TABLE 3.8 Femur biomechanical parameters  Diet  Bending  F a i l u r e Energy  (x 1 0 SHR  2% Ca  - 2  Maximum Bending  J) WKY  SHR  ax  7.87 + 0 . 7 4  ax  Soy  7.98 + 0 . 7 9  ax  6.96 + 0 . 3 1  ax  0.5% Ca Casein  8.80 + 1 . 0 1  ax  6.71 + 0 . 6 1  ax  Soy  7.31 + 0 . 6 3  6.05 + 0 . 6 1  ax  0.05% Ca Casein  3.31 + 0 . 1 5  5.70 + 0 . 5 1  ax  Soy  5.45 + 0 . 7 8  a b x  Stress  2  7.55 + 0 . 4 6  bx  diets  (N/mm )  Casein  ax  of r a t s f e d e x p e r i m e n t a l  5.40 + 0 . 5 6  ax  74.18 + 6 . 2 1 63.15 + 1.80 60.26 + 2 . 2 8 56.91 + 1 . 9 7 12.10 + 2 . 1 7 24.81 + 2 . 3 2  WKY  ax  ax  ax  ax  bx  bx  Data are expressed as mean + SEM; SHR = Spontaneously r a t s , WKY = W i s t a r Kyoto r a t s .  69.44 + 1 . 8 3  ax  70.04 + 1 . 6 3  ax  67.43 + 5 . 1 4  ax  69.74 + 6 . 0 6 14.44 + 0 . 6 2  ax  cx  37.66 + 5.76 * b  hypertensive  a,D,c _ i g i f i t (p<0.05) d i f f e r e n c e between treatment means i n columns. » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows. S  x  y  n  c a n  of  t h e s e a n i m a l s was d e c r e a s e d , a p a r a m e t e r  with the decreased (F(1,53)=106.81,  bone m i n e r a l i z a t i o n  p<0.01).  source  protein  (F(2,49)=8.74,  for  f e m o r a l maximum b e n d i n g were  a l s o seen  correlated i n these  An i n t e r a c t i o n b e t w e e n  and  interactions  which  found  with  animals  calcium intake  p < 0 . 0 0 1 ) , was  stress.  well  found  In contrast,  no  to exist  significant  r e s p e c t t o femur b e n d i n g  failure  energy. The  tibia  mineral  composition  biomechanical  (Table  parameters  and  data f o l l o w  t h e same t r e n d s as t h o s e o b s e r v e d  addition, dietary ( T a b l e 3 . 9 ) . An the groups  protein animal  fed the  have a s i g n i f i c a n t l y compared t o WKY  calcium  strain  (p<0.05)  decreased  diets  calcium level.  to  affected  by  when  d i f f e r e n c e was  i n t a k e and a n i m a l s t r a i n  ( T a b l e 3.10)  tibia  In  Mg c o n t e n t  observed  only i n  SHR a n i m a l s were f o u n d t o  decreased  i n b o t h SHR  tibia  and WKY  Mg  c o n t e n t when  T i b i a Mg c o n t e n t animals f e d the  compared t o c o u n t e r p a r t s a t t h e 0.5%  Thus, t i b i a a  physical  f o r the femora.  source d i d not a f f e c t  2% c a l c i u m d i e t s .  dietary be  parameters  c o u n t e r p a r t s f e d t h e same d i e t .  was s i g n i f i c a n t l y 0.05%  strength  3.9),  significant  magnesium c o n t e n t interaction  (F(2,54)=13.17,  3).  107  p<0.001;  was  found  between c a l c i u m Appendix  Table  TABLE 3.9  T i b i a mineral c o m p o s i t i o n of r a t s f e d experimental d i e t s .  Diet  Ash wt. (g/bone) SHR  Ca (mg/bone) WKY  2% Ca Casein  0.167 + 0 . 0 0 3  ax  Soy  0.166 + 0 . 0 0 6  ax  0.5% Ca Casein  0.172 + 0 . 0 0 7  ax  Soy  0.152 + 0 . 0 0 2  ax  0.05% Ca Casein  0.044 + 0 . 0 0 8  cx  Soy  0.068 + 0 . 0 0 6  bx  SHR  0.155 + 0 . 0 0 5 0.140 + 0 . 0 0 6  0.129 + 0 . 0 0 9 0.057 + 0 . 0 0 3 0.077 + 0 . 0 0 7  WKY  ax  62.40 + 1 . 2 3  ax  ay  61.86 + 1 . 9 3  ax  63.64 + 1 . 9 4  ax  bx  57.82 + 0 . 8 6  ax  dx  19.38 + 3 . 1 7  cx  cx  27.54 + 2 . 3 8  bx  0.133 + 0 . 0 0 4  a b y  Data a r e expressed as mean + SEM; SHR = Spontaneously  Mg (mg/bone) SHR  57.90 + 2 . 4 8 51.90 + 2 . 5 8 51.11  ax  ax  + 2.50 * a  53.14 + 4 . 5 1 22.65 + 0 . 9 5 30.28 + 2 . 7 1  a x  bx  b x  1. 58 + 0 . 1 7 1. 52 + 0 . 1 5 2. 10 + 0 . 1 3 2. 17 + 0 . 1 2 1. 58 + 0 . 1 9 1. 53 + 0 . 1 5  c  d  bx  bx  ax  ax  bx  bx  2.33 + 0.08 * a  2.42 + 0 . 1 4 ^ 2.16 + 0 . 1 0 2.24 + 0 . 2 0 1.62 + 0 . 1 1  ax  ax  bx  1.42 + 0 . 0 7  h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s .  » * = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n columns. ^ = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows. b  WKY  bx  TABLE 3.10  T i b i a biomechanical parameters  Diet  of r a t s f e d experimental  diets .  Dry wt.  Bending F a i l u r e Energy  (g/bone)  (x 1 0 " J )  SHR  Maximum Bending S t r e s s (N/itim )  2  WKY  2% Ca  1  SHR  Casein  0.260 + 0 . 0 0 4  ax  0.242 + 0 . 0 0 8  Soy  0.261 + 0 . 0 0 8  ax  0.222 + 0 . 0 0 8  0.5% Ca Casein  0.261 + 0 . 0 0 9  ax  Soy  0.236 + 0 . 0 0 5  ax  0.05% Ca Casein  0.109 + 0 . 0 1 4  cx  Soy  0.145 + 0 . 0 0 7  bx  0.206 + 0 . 0 0 8 0.198 + 0 . 0 1 4 0.129 + 0 . 0 0 5 0.148 + 0 . 0 0 9  2  WKY  SHR  6.68 + 0 . 1 5  ax  4.49 + 0 . 4 2  6.11 + 0 . 6 6  ax  3.42 + 0 . 5 0  by  6.04 + 0 . 6 0  ax  4.13 + 0 . 3 8  ax  by  6.01 + 0 . 7 2  ax  4.42 + 0 . 2 4  ax  cx  2.89 + 0 . 4 4  bx  4.19 + 0 . 3 1  a x  cx  4.33 + 0 . 6 2  bx  4.97 + 0 . 2 7  ax  ax  a b y  Data a r e expressed as mean + SEM; SHR = Spontaneously  ay  ay  s  n  c a n  60.24 + 1 . 0 6 56.19 + 1 . 9 6 59.11 + 4 . 7 8 10.66 + 2 . 3 2 20.18 + 2 . 8 6  ax  75.50 + 4 . 1 8  ax  77.38 + 5.53 *  ax  63.15 + 3 . 7 3  ax  ax  79.17 + 4 . 4 0  ay  bx  16.56 + 1 . 1 5  CX  bx  34.38 + 5 . 6 9  by  ax  a  h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s .  a,b,c _ - j g i f i t (p<0.05) d i f f e r e n c e between treatment means i n columns. = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows. x , y  67.95 + 2 . 5 0  WKY  Discussion Previous may  have  studies  a disturbed  metabolism. in  absorption  the  (Lau  loop,  In  not  to d i f f e r  SHR  The  and  differences state,  present calcium  in  of d i f f e r e n c e s  t o be  significantly  higher  1984;  McCarron  were n o t al..  et a1..  by  and  however  model calcium  intestinal  confounded  et  al. .  study,  SHR  calcium  the  1986; and  absorption  rats  present  various  and  WKY  animals  from  the  i n SHR  were  homeostasis  normotensive c o n t r o l s  study,  can  be  In the  t h a n WKY  absorpbetween  explained  animals  calcium  b e t w e e n SHR  and  and  by  post-absorptive reported  (Lau  e t a 1. .  In f a s t e d , or meal-fed r a t s ,  total  and  ligated  ionized calcium  used.  these  Toraason  i o n i z e d c a l c i u m have been  1981).  u s e d h e r e i n , serum different  the  and  i n p a r a c e l l u l a r calcium  feeding protocols calcium  those  absorption  i n plasma  i n the  serum t o t a l  as  animal  t h a t the d i f f e r e n c e i n c a l c i u m  hypertensive  animals  SHR  ages, r e s p e c t i v e l y used i n  Gafter  l a c k of d i f f e r e n c e  WKY  on  been  animal  in  suggesting  a result  tion.  and  the  between g e n e t i c a l l y is  have  t h a t the  calcium  reports  e t a 1 . . 1984;  observed  ileal  SHR  techniques  W r i g h t , 1981). not  intestinal  Conflicting  absorption  studies  have i n d i c a t e d  WKY  such  ionized  calcium  animals  (Lau  et  1984). The  decreased growth  calcium  diets  calcium  in  d i g e s t i o n and  may  the  be  parameters explained  reabsorption  absorption.  have been r e p o r t e d  to  of  by  of  animals  the  i n t e r f e r e n c e of  bile  salts  and  fed  110  fecal  bile  acid  high  excess  hence,  O r a l supplements of c a l c i u m  increase  the  fat  carbonate  excretion  by  inhibiting  the  a b s o r p t i o n of b i l e  precipitation Sillery,  of  bile  1989).  salts  Moreover,  calcium.  The  t h a t the could  to  levels  casein  of b i l e  fed  f e d soy  acid  1981).  in binding  to b i l e  a c i d s were e x c r e t e d salts  are  intestine 1977).  for  and  an  as a  study,  e x h i b i t e d decreased which  were  nutrient  observed  animals  feed  likely  caused  lumen of in  the  and  increased  calcium  in  being  and  soy  and  protein  Schneeman,  peptides  active  reabsorbed,  a1..  1979).  of  by  fat  bile Bile  in  the  d i g e s t i o n (Guyton,  of f a t d i g e s t i o n would cause f a t a  reduction in  feed  efficiency  expected. the  low  and  calcium  The high  (Roy  ( S k l a n et  fed  by  contents  i s provided  of r a t s fed  undigested  efficiency  malabsorption.  intestinal indicative  w o u l d be  lumen  evidence  hydrolysis  result,  suggested  acids, resulting  emulsificat ion  inhibition  body w e i g h t g a i n e d this  fat  of  i n t h e f e c e s , when compared  r a t h e r than  the  and  have been  (1979)  intestinal  contents  i n f e c a l matter  to f a c i l i t a t e  Therefore,  In  in  contained  a c i d s , and  essential  malabsorption, and  coworkers  Supporting  contents  the  (Saunders  r e a b s o r p t i o n may  opposed to c a s e i n c o n t a i n i n g d i e t s  Intestinal  to  protein with a high level  excreted  animals.  c o l o n , due  ions  r e a b s o r p t i o n of b i l e  r e p o r t s of i n c r e a s e d i n t e s t i n a l d i e t s , as  acid  protein  i n t e r f e r e w i t h the  i n the  calcium  f i n d i n g s o f S a u t i e r and  p r e s e n c e of soy  elevated  by  bile  f u r t h e r d i s t u r b e d i n animals  salts  low  calcium of  intestinal  111  and  growth  content  of  * Ca absorption 5  absorption  negative  also  characteristics  deficiency, rather  efficiency  deficiency  calcium diets  than the are  of  calcium  calcium  balance  (Pansu et a l . . 1981). diets  exhibited  similar  4 5  Ca  replete  an  In c o n t r a s t , animals  increased  u p t a k e as  status  is  those  the  intestine  of r a d i o l a b e l l e d animals  by  and  the  percent  absorbed  calcium  (Allen,  is  2.0% least  content  Ca  activities.  that  uptake level.  1.0  had  Potential extracellular 1981;  of  and r e l i a n c e calcium amounts by  conclusion i s  amount o f  calcium  though the a diet  relative  adequate i n  was  inversely  case of a n i m a l s high,  fed  Conversely,  in  animals  levels  and  reduced  a positive fed  the  reflecting  c a l c i u m a b s o r p t i o n and  bone Ca  reflect  bone  a c t i v i t y was  resulting  fed  the  up  the  took  bone  specific  calcium balance adequate  low  in  animals  or  in  the h a n d l i n g , or d i s t r i b u t i o n  and  above  calcium.  differences c a l c i u m by SHR Aoki  the  In the  higher  hour,  These r e s u l t s  adequate l e v e l s  This  from  by  animals.  a d e q u a t e bone c a l c i f i c a t i o n  et a i . ,  diets.  femur s p e c i f i c  i n these  diets  in  of a c t i v e  transported paracellularly  e f f i c i e n c y of i n t e s t i n a l  calcium 4 5  than  calcium  calcium d i e t s ,  femur Ca  be  a  calcium  Thus, s i m i l a r  c a l c i u m i n t a k e , even  to d i e t a r y calcium  the enhanced  1987).  calcium  less  A  but  1982).  Radiolabelled related  intestine,  that a greater absolute  i s absorbed from a h i g h  content,  down-regulation  small  (Bronner,  0.5%  fact  by  calcium  p a r a c e l l u l a r movement o f  c a l c i u m would  f e d 2.0%  supported  0.05%  proximal  c o n c e n t r a t i o n dependent  from the d i s t a l  calcium  high  f e d medium c a l c i u m d i e t s .  characterized  c a l c i u m t r a n s p o r t i n the on  intestinal  f e d the  animals  r e p o r t e d by  e t a l . , 1976 ; S c h e d l 112  others  of  (McCarron  e t a l . , 1 9 8 8 ) , were a l s o  observed  in  increased  the  amounts  counterparts. workers  who  of  4 5  calcium  the  and  4 0  ability  by  making  the  manufacture  a1. .  likely  confounded  occurred  influence 1979;  been  binding  soy  the  Erdman,  digested  releasing  calcium  1973).  Despite  calcium  absorption,  0.5%  translocation fact  that  of  by  i s diminished  phosphate  acid  from  moieties,  by  of  i n reducing  that  phytates thereby  (Reinhold  e t a1. .  the colon  to i n t e s t i n a l  isolate,  w i t h 2.0%  decreased  These f i n d i n g s  of o s t e o m a l a c i a  o b s e r v e d i n humans who h a b i t u a l l y consumed a d i e t (Berlyne 113  reported  bone  calcification.  of u n l e a v e n e d , wholewheat bread  the  (Hartman,  intestine,  exhibited  support  the  precipitation,  reports  soy p r o t e i n  respectively,  In  the d i e t  from the c o l o n  fed  bioavail-  a r e f o r m e d w h i c h may  i n the lower  contribution  animals  to the exchange  f o r absorption.  minerals  some e x t e n t  the  1988).  of t h e bone, r a t h e r  ionized  and p h y t a t e  f o r absorption  calcium,  a 1. .  However, t h e r o l e o f p h y t a t e s  can  to  et  by t h e  isolate  the p r o t e i n  1979).  of other  and i n t r a c e l l u l a r  Schedl Ca  WKY  have an a l t e r e d  to decrease calcium  with  protein  bioavailability  and  reported  availability  4 5  than  tissue.  unavailable  calcium be  for  bone,  animals  on t h e s u r f a c e  calcium  calcium  complexes between  1976;  translocated  observations  extracellular  time  Ca  the  the  SHR  short  have  of  that  animals  to  supports  than d e p o s i t i o n i n t o o s t e o i d Phytates  SHR 45Ca  between  et  SHR was  Ca  absorbed  suggested  (Aoki  Alternatively,  study.  finding,  have  compartments  femora of  of  This  distribution  between  present  development  et a l . . 1973).  Calcium mechanisms  deficiency to  parathyroid  and  maintain  hormone  Parathyroid  plasma  (PTH)  between the  1942) blood  to  calcium  tubular  reabsorption  osteoclastic increasing  and  other  balance  vitamin  of  in  production  1988).  e x h i b i t e d by  the  Elevated casein  t o be  is  w i t h phosphate  associated is  levels  of  in  soy  fed  given  d i s t u r b e d plasma m i n e r a l  profile  not  endocrine  observed.  maintain  Since  relatively  1988), the  calcemia  f e d a n i m a l s was confirm  and  constant  a1..  the  elucidate  the  increasing  Further possible  114  in  turn  intestinal  lumen  phosphorus, diet,  by  PTH  (Agus  st_  low  calcium fed  calcium  studies  soy  are  have  excess mediated  a 1 . . 1981). diet,  the  animals  was  generally  levels  calcium  as  calcium  The  h o r m o n a l mechanisms  plasma  vation.  i n t e s t i n e by  1981).  noted i n casein  d i f f e r e n c e b e t w e e n low  unexpected.  by  s i n c e bone  for  extra-  increasing  calcium  activity,  excretion  animals  the  low  compensated  balance  by  which  plasma  a  (Agus e t  s t i m u l a t i o n of r e n a l phosphate  bone  calcitriol, from  (Patt  r e g u l a t i o n of  the  the  systems.  hypocalcemia  kidneys  calcium  a r e s u l t of PTH  eventually  by  s e c o n d a r i l y at the  f e d a n i m a l s on  been r e p o r t e d  namely  endocrine  This  at  of  of  homeostatic  levels, D  the  calcium;  of  calcium-phosphorus  tissues.  bone r e s o r p t i o n ; and renal  phosphate  the  occurs  active transport  However,  variety  calcium  regulate  and  cellular  (Aurbach,  a  hormone s e c r e t i o n i s s t i m u l a t e d  Luckhardt,  increases  triggers  (Greger, and  casein  required  mechanism f o r t h i s  to  obser-  The  PTH  deficiency  mediated  will  bone  result  resorption  in  decreased  longterm, osteoporosis (Sandler et mineralization deficient content  in  animals  calcification, and  Ca/P  but  not  calcium balance 1969). early and  Belonje,  an  extent  is  emphasized  exist  diets  is  to control  evidence  excretion.  excreted  i n the u r i n e ,  the s o f t  tissues  m i n e r a l , such present study. which  has  (Aurbach,  i f a diet as i n These  would  (Greger  support d i e t can  prevent a negative (Spencer  an a d e q u a t e  in  calcium  calcium  et a1..  calcium intake  mineralization study. animals  the l e v e l s  (Shah  The f a c t fed  of  Thus, excess absorbed  or  has  antagonistic an  diets  to  bone  that  the h i g h  mechanisms intestinal calcium i s  b o n e , o r even  1988).  the high  been r e p o r t e d  calcium i n r e l a t i o n metabolism  low  calcium homeostatic at  i n the  decreased  r a t h e r than over c a l c i f y i n g  interactions,  become a p p a r e n t  that  calcium metabolism  a b s o r p t i o n and r e n a l  Mineral  a  by t h i s  increased  and  Impaired  bone m e t a b o l i s m of  calcium  These r e s u l t s  t o m a x i m i z e bone d e n s i t y and 1988),  mass,  by  which  importance  in  c a l c i u m d i e t s was due t o  respectively.  bone c a l c i f i c a t i o n was n o t calcium  the low evidenced  and d i s t u r b e d the  bone  a l . . 1985).  that adaptation to  to  Moreover, in life,  as  ratios,  previous hypotheses occur,  fed  occurring  or low were  t o have their  e_t a_l.. ,  excess  not  or  balanced  respective  1987 ; G r e g e r ,  115  deficiency  c a l c i u m d i e t s used  antagonistic  bone magnesium c o n t e n t i n a n i m a l s f e d  relationships  can  o f one i n the  f o r magnesium,  interactions  with  bioavailabilities 1989).  high calcium  The  and  decreased  d i e t s may  be  e x p l a i n e d by levels  an  increase  of c a l c i u m  ( M o r r i s and  Moreover, s t u d i e s inhibit  the  (Greger  <al_. ,  1981).  On  of  calcium suggests  an o v e r a l l  animals  bone  in  other  animals  dietary calcium other  minerals  the  decreased  hand,  fed  can  diets  deficient  in  decrease i n m i n e r a l i z a t i o n secondary  e n h a n c e d femur a s h w e i g h t f e d t h e 2.0%  c o n t r a r y to the  and  data,  of  bone  osteoporotic  observations  may  be  former study,  study.  0.5%  suggestions  histomorphometric  and  calcium d i e t s , of Izawa  t h a t t h e SHR  and  observed  coworkers  The  compared  i n the  Finally, biomechanical reflection  of  a  to  reduction  in  bone  i n animals  decreased  weight bearing  decreased  p e r i o d of the p r e s e n t  strength  calcium deficiency  with respect  time  bone  mass  (Niewoehner, 1988). s t r e s s are  also  t o bone d e n s i t y and  body w e i g h t o f t h e s e  s m a l l e r bone s i z e  and  14  of t h e s e  116  are  (1985) from development these  not  in  in this  have been  parameters  and  calcium diets  is a  bone Since  likely  in  study.  low  remodelling  animals.  herein,  weeks,  calcification  in  physical activity  components o f  animals  SHR  of t h e a n i m a l s  physical  f e d the  of  discrepancy  t o t h e much g r e a t e r age  26 w e e k s , as  content  i s prone to the  fragility.  due  calcium  T h u s , s i g n s o f a g e - r e l a t e d o s t e o p o r o s i s may  manifested  and  G r e g e r et_ al_. , 1 9 8 1 ) .  t h a t excess  the  high  calcium deficiency. The  the  1963;  when f e d  a b s o r p t i o n of magnesium and  magnesium c o n t e n t  to  O'Dell,  have r e p o r t e d  intestinal  et_  i n magnesium r e q u i r e m e n t  bone  formation  (Simonen, 1986), contributed  to  the the  4  Experiment  E f f e c t of c a s e i n p h o s p h o p e p t i d e f o r t i f i c a t i o n c a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n c a s e i n and soy f e d r a t s .  on  Introduction The  principal  milk  protein  fraction,  p h o s p h o p e p t i d e s upon d i g e s t i o n o f casein phosphopeptides precipitation soluble  of i n s o l u b l e  complexes  Latour,  (CPP)  with  luminal  a l . . 1983;  S a t o e_t a l _ . ,  present  have  ionized  contents  (Naito  s t u d y were t o f i r s t  from  of  c a s e i n and  the  distal  utilization  in  CPP  1986).  mineral  Materials In V i t r o To protein PQ)  (SHR)  and  intestine, balance,  and  4  incubated  of Casein  exchange (MPC;  extrinsically  ( »CaCl2; S.A. for  (Reeves jjn_  as  and  vivo in  objectives  and  and  Secondly,  calcium  osteoporosis  the  absorption  subsequent  bone  the  characteristics  in vitro. on  of  calcium  m i n e r a l i z a t i o n and prone  spontaneously  Methods  concentrate  was  in  forming  rats.  Purification label  well  determine the b i n d i n g  b i o m e c h a n i c s were d e t e r m i n e d hypertensive  vitro  The  supplementation  small  by  the  et. a_l_. , 1972 ; M u l t i n g e r et_  of c a l c i u m to c a s e i n d i g e s t i o n p e p t i d e s effect  These  to prevent  salts  in  as  yields  trypsin.  phosphate  1989)  1983;  with  calcium  al. .  caseins,  been r e p o r t e d  calcium  1958 ; B e r r o c a l e t  intestinal  casein  the  18.2 30 min  Phosphopeptides  micellar  Bariatrix  labelled  mCi/  mg  4 0  4 0  Ca  with  ICN  Ca,  4 5  Biomedical,  i n a 37°C s h a k i n g 117  4 5  a 3.6%  I n t e r n a t i o n a l , Inc.,  w i t h 0.545 u C i  Ca,  (CPP):  Ca/  1.1  Irvine,  water bath.  milk Dorval,  ug CA)  4 0  Ca and  Ultracentri-  fugation  at  30,000 rpm  Casein p e l l e t s concentration NaOH,  the  f o r 90  were h o m o g e n i z e d o f 2%.  casein  hr, in  itated with rpm)  for  8% TCA  20 m i n .  22 cm)  a Sephadex  and  eluted with  TCA  EDTA and  0.02%  Na  Fractions  (3.6  mL)  (280  activity.  R a d i o a c t i v i t y was  and  10  mL  ACS  using cm).  at a flow  215  nm),  CPP  was  starting  EDTA, 0.02%  Na A z i d e  NaCl) at  and  cocktail  (pH 8.0)  r a t e o f 0.17  were c o l l e c t e d and a n a l y z e d radiolabel  4 5  Ca  and p h o s p h a t e  as a b o v e . was  a  with mL)  was  5  mL/  linear  mL/  min. A  mM  min.  peptide  and  300 uL  radioaliquot  Counter.  ON)  Further  chromatography  column  Tris-HCl  an  (1.5 x  for  phosphorus,  (2.5  x  containing  salt  gradient  Fractions  s i n g l e peak  r e c o v e r e d a t 0.25  118  (9,500  (Amersham, O a k v i l l e ,  50 mM  with  precip-  column  monitored  organic  was  1:200  containing  p e r f o r m e d by i o n - e x c h a n g e  buffer  a flow  were  (0.5  m e a s u r e d by m i x i n g a  scintillation  of  r a t e o f 0.3  IN  (Sigma,  extracted  buffer  a DEAE C e l l u l o s e ( S i g m a , S t . L o u i s , M0) The  M  of  ratio  aliquot  c o u n t e d on a LKB-1215 L i q u i d S c i n t i l l a t i o n  purification  0.5  An  collected  with  trypsin  Digests  final  then c e n t r i f u g e d  Tris-HCl  A z i d e (pH 8.0)  and  pH 8.0  with  f r a c t i o n was  (4X).  50 mM  were  and  f r o m whey.  water to a  ( S i g m a , S t . L o u i s , MO)  absorbance  with  nm  4°C  soluble  G-25 a  reacted  water bath.  at  ether  to  deionized  a t an enzyme t o p r o t e i n  overnight  e q u a l volume o f e t h y l applied  was  shaking  The  casein  pH a d j u s t m e n t t o  preparation  a 37°C  separated  with  Following  Type X I I I ; S t . L o u i s , M0) f o r 23  min  (5.0  6.3 5  mM (0mL)  containing  M NaCl.  A n i m a l s and D i e t s : Four-week  o l d male  spontaneously  hypertensive  m a t c h e d f o r age were p u r c h a s e d f r o m C h a r l e s Animals under  were  individually  controlled  cycle).  maintained  temperature  Animals  were  C l e v e l a n d , OH),  Kaisha,  Science  respectively  (Table  calcium  available  to animals  A 24  were  level  +  hour balance  in  NY)  rats,  150g  collection  f u n n e l s and s e p a r a t i o n  urine,  eliminate urine of  excreta  apparatus.  Urine  frozen u n t i l  analysis.  a l s o measured a f t e r  and  24  contained  water  was  an  made  i n t a k e s and w e e k l y  to  fecal  cages  300g,  cones  immediate  to  24  hr p r i o r to  cages  were  (Nalgene,  equipped  separate  contamination and  were 10  each d i e t a r y group  Metabolic  w a s h o v e r and  was  from  metabolic  Rochester,  Separation  Deionized  Seika  (S-CPP),  was p e r f o r m e d when a n i m a l s  f o r acclimatizat ion.  to  (12:12  (Meiji  S + 3%CPP  D a i l y feed  collection  for  cages  recorded.  study  placed  (C-CPP)  A l l diets  Randomly s e l e c t e d a n i m a l s  individually  conditions  Japan),  10 w e e k s .  ad 1 i b i t u r n .  steel  c a s e i n ( C ) , soy (S) (ICN  3%CPP  (0.5%).  were r o u t i n e l y  weeks o f a g e .  20%  Laboratories,  4.1) f o r  adequate  body w e i g h t s  C  ( M o n t r e a l , PQ).  i n stainless  lighting  meal-fed  Biochemicals, Bio  and  River  (SHR) r a t s ,  complete  with  f e c e s and of feces. using  this  s a m p l e s were c o l l e c t e d , w e i g h e d and  Feed i n t a k e hours.  119  and w a t e r  c o n s u m p t i o n were  TABLE 4.1  Composition  of experimental  diets  fed to animals.  Diets D i e t a r y Component ( g / 100g)  Casein* Soy p r o t e i n CPP-III* *  C  C-CPP  20 .0  20 .0  S  S-CPP  -3.0  20.0  20 .0 3.0  0.3 15.0 46.82 7.0 5.0  0.3 15.0 44 .12 7.0 5.0  0.3 15.0 46 .82 7.0 5.0  0.3 15.0 44 . 12 7.0 5.0  Ca f r e e m i n e r a l m i x t u r e * Vitamin mixture* Choline b i t a r t r a t e *  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  Calcium  1.224  0.916  1. 176  0.881  isolate*  D.L. m e t h i o n i n e * Cornstarch* Sucrose Fibre* Vegetable o i l  carbonate**  -—  1  -  C = c a s e i n ; S = s o y p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP. * ICN B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. ** M e i j i S e i k a K a i s h a , B i o S c i e n c e L a b o r a t o r i e s , J a p a n . * U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , OH. ** BDH C h e m i c a l s , T o r o n t o , ON. 1  120  Ileal anical  4 5  Ca  absorption,  parameters  a n i m a l s were  were d e t e r m i n e d  14-weeks o l d .  4  L/min f o r  m i n e r a l i z a t i o n and b i o m e c h -  as  Animals  previously  d e s c r i b e d when  were a n a e s t h e t i z e d u s i n g a  and O 2 ' , 4% h a l o t h a n e  vapour m i x t u r e of halothane of  femur  at a  flow  rate  i n d u c t i o n , and 2.5% a t a f l o w r a t e o f 2 L/min t o  m a i n t a i n a n a e s t h e s i a d u r i n g the s u r g i c a l  procedure.  Analyses : Intestinal previously mineral performed  l u m e n , and b l o o d p l a s m a m i n e r a l s were a n a l y z e d as  described  analyses on  in  (calcium,  diet, urine  I t a y a and U i , 1966; J o n e s Bone  experiment  1.  magnesium  and f e c a l e t aj^. ,  In the balance and  phosphorus)  study, were  s a m p l e s (Chen e t a l . . 1956;  1988).  Biomechanics: Femur b i o m e c h a n i c a l p a r a m e t e r s  Universal described  Testing  i n experiment  Statistical All  Machine  3-point  bending  as  previously  Treatment  differences  2.  Analyses:  data are expressed  were t e s t e d  in  were a s s e s s e d w i t h an I n s t r o n  f o r by  b e t w e e n means was  one-way ANOVA.  identified  p<0.05 s i g n i f i c a n c e  as mean +_ SEM.  by  a  level.  121  The  source of a d i f f e r e n c e  multiple  range  test  at the  Results In V i t r o  CPP  After  Purification:  incubation  ultracentrifugation, whey  fraction  casein  exchange  steps,  radiolabel original  for  indicate  f r a c t i o n was  a  crude  protein  the  S  the  in  the  casein-  and  a s i n g l e peak The  ion-  containing recovery  of t h a t p r e s e n t the  in  Upon f u r t h e r 4.1)  in  of the  column f r a c t i o n s  and  phosphorus content,  casein  phosphopeptide  fortification  source feed  with  significantly  did  not  a  FER  ratio  the (CPP)  CPP  had  a  soy  positive  e f f e c t on  (FER;  than C fed animals.  Table  influence Feed  i n f l u e n c e animal growth  Taken t o g e t h e r ,  s o u r c e and  characteristics. 122  4.2).  (p<0.05) l o w e r  d i f f e r e n t b e t w e e n d i e t a r y g r o u p s , and  dietary protein  both  Moreover, d i e t a r y  protein diet.  i n f l u e n c e growth parameters. that  significant  efficiency  p a r a m e t e r s when added t o t h e not  had  diet exhibited a significantly  body w e i g h t and  indicate  present  and  obtained.  body w e i g h t and  Animals fed final  that  (Figure  From m o n i t o r i n g  absorbance  Ca  present  digest.  100%  4 5  67.88% of  obtained.  was  the  Study:  Dietary final  peptide  4.2),  was  purification  digestion mixture.  collected  In V i v o  radioactivity  after  the  ge1-fi1tration (Figure  was  digestion,  remained i n  by  with  78.48% b e i n g  tryptic  chromatography and  preparation  remaining  radioactivity  phosphate  results  the  Following  purification  MPC  21.52% of r a d i o a c t i v i t y  with  pellet.  associated  of the  CPP  on  these  intake  was  therefore these  fortification  data can  CO  c © Q  "5 o +*  a O  6  7  8  9  10  11  12  13  14  15  16  Fraction Number 280 nm  Fig.  4.1  -B-  215 n m  Gel f i l t r a t i o n p u r i f i c a t i o n of casein phosphopeptide (CPP). Phosphorus and * C a r a d i o a c t i v i t y c o n t e n t of f r a c t i o n s ( a ) , and p e p t i d e a b s o r b a n c e o f f r a c t i o n s ( b ) . 5  123  1  1  I  1  I  I  5  I  I  1  1  1  1  1  1  1  10  1  1  1  15  1  1  1  20  1  1  1  1  1  25  Fraction Number 280 nm  Fig.  4.2  ~ ~ 215 nm s  Ion-exchange p u r i f i c a t i o n of c a s e i n phosphopeptide (CPP). Phosphorus and Ca radioactivity content of f r a c t i o n s ( a ) , and p e p t i d e a b s o r b a n c e o f f r a c t i o n s ( b ) . 4 5  124  TABLE 4.2 D i e t e f f i c i e n c y  Diet  2  I n i t i a l body wt . 3 (8)  of r a t s  fed experimental  F i n a l body wt .* (8)  Dry M a t t e r I n t a k e (8)  diets  Feed  1  Efficiency Ratio  c  76 . 7 +_ 2 .0»  259.3 +.8.4"  867 + 23»  0 .211 +. 0 .004*  s  78 .7 + 2 .2*  225 .2 + 5.2°  861 +  0. 170 + 0 .004"  C-CPP  75 . 5 +_ 2 .3*  248 .8 +_ 4 . 4« b  865 +_ 17*  0 .200 i 0 .004  a b  S-CPP  77 .0 ± 2 .2*  236.3 +_ 5.3»*  848 + 11*  0 . 188 +. 0 .006  b  1 2  3 4  D a t a a r e e x p r e s s e d as mean +. SEM. C = c a s e i n ; S = s o y p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP. 5 weeks o f a g e . 14 weeks o f a g e .  Means s h a r i n g t h e same l e t t e r w i t h i n s i g n i f i c a n t l y d i f f e r e n t a t p<0.05).  125  t h e same c o l u m n a r e n o t  Plasma presented  mineral in  a f f e c t e d by  Table  significantly  l o o p ) and  0.32 dpm/ dietary 4 5  Ca  mg  Ca s p e c i f i c 4 0  Ca)  the  animals  P l a s m a p r o t e i n was n o t  content  source or  (range  not  (range  (range  treatment.  0.13  to  2.48 +.0.22  3.08 +_ 0.35  significantly  the  significantly  CPP f o r t i f i c a t i o n  2.04 +_  The p r e s e n c e both  are  M o r e o v e r , p l a s m a m i n e r a l s were  activity  were  treatments.  animals  t o 4.64 +_  d i f f e r e n t between  o f added d i e t a r y CPP i n c r e a s e d  casein  e f f e c t was s i g n i f i c a n t  and  soy  protein  diets,  (p<0.05) i n o n l y t h e S-CPP f e d  (Figure 4.3).  Femoral acute  4 5  Ca  4 0  experimental  (p<0.05) a f f e c t e d by d i e t a r y  a b s o r p t i o n from  albeit  of  t o 5.06 +_ 0.22 g / d L ) .  Intestinal mg/  4.3.  dietary protein  4.78 +_ 0.32 not  profiles  d e p o s i t i o n of  deposition  activities  of  of  the  4 5  4 5  Ca  femora  Ca  i s presented  to  femora,  were  not  i n Table  significantly  a n i m a l s d i d however e x h i b i t  both a  greater  specific  femora  when  animals, i n d i c a t i n g c a l c i u m by t h e s e M i n e r a l Balance The  24  T a b l e s 4.5  the  a more  efficient  affected  by  The s o y p r o t e i n f e d 4 5  Ca  d e p o s i t i o n and  compared t o c a s e i n f e d  translocation  of  absorbed  animals. Study:  hour  t o 4.7.  significantly  of  The  as w e l l as t h e s p e c i f i c  d i e t a r y p r o t e i n s o u r c e o r CPP f o r t i f i c a t i o n .  activity  4.4.  mineral  balance  study data are presented i n  C a l c i u m i n t a k e and  (p<0.05)  different  4.5).  However,  fortification  resulted  in a significantly  fecal  excretion  between d i e t a r y groups of  the  casein  diet  (p<0.05) i n c r e a s e d u r i n a r y 126  were n o t (Table  w i t h CPP calcium  TABLE 4.3 Effect of diet on plasma minerals of animals fed experimental diets . 1  Diet*  Ca  Ca * +  Mg  Na  K  P  (mg/dL)  1 d  c  9.4 + 0.2  5.1 + 0.1  1.9 + 0.1  389 + 16  15.5 + 1.9  7.1 + 0.8  s  9.2 +. 0.4  4.9 + 0.1  1.9 + 0.1  424 + 12  19.2 + 4.6  7.1 + 0.6  C-CPP 9.0 + 0.2  4.8 + 0.1  2.0 + 0.1  388 + 26  19.8 + 1.8  7.4 + 0.4  S-CPP 8.6 + 0.2  4.6 + 0.1  1.9 + 0.1  359 + 22  15.1 + 2.5  7.2 + 0.8  Data are expressed as mean + SEM. C = casein; S = soy protein isolate; C-CPP = C + 3% CPP; S-CPP = S + 3%CPP.  Ca  +2  = [(6 Ca - P/3)/(P + 6)j; where Ca , Ca = mg/dL; P = protein g/dL. +2  821  45Calcium a b s o r b e d (% dose)  Tl TO  CO  fl "B It A T) O. O w O O 0> cn ca  w  a H>  ro  6)  3  ro  H -  Cu S CO  i-»  o cr w  CO  '  O fl> + 1 3 CO H3 O O rt  ro co  H -  O 3 O  i-h CO  I O  .  0)  to  O  rt g  *  to  HH-»  fD W Cl) 3 w H» O -  toO O  co  O  CO + (->• TO CO 3 3 H- O i-n h3  O  to .—. 3  O  TABLE 4.4  E f f e c t o f p r o t e i n s o u r c e and CPP f o r t i f i c a t i o n calcium d e p o s i t i o n to the femur .  45  Diet  on  1  C a Deposited (% d o s e / b o n e )  Bone Ca (% d o s e / g A s h )  C  0.148 i 0.046  0.63 +. 0.20  1.70 + 0.60  S  0.274 +_ 0.080  1.36 +_ 0.41  3.40 +_ 1.00  C-CPP  0.157 + 0.041  0.66 ± 0.17  1.60 + 0.40  S-CPP  0.264 + 0.068  1 .22 + 0.32  3.40 + 0.80  1 2  2  4 5  4 5  D a t a a r e e x p r e s s e d as mean +. SEM. C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; S-CPP = S + 3% CPP.  129  Ca S.A. dose/g Ca) 45  4 0  C-CPP = C + 3% CPP;  TABLE 4.5  E f f e c t of p r o t e i n source and CPP f o r t i f i c a t i o n on 24 h r . c a l c i u m balance . 1  .  ?  Diet  Ca Intake (mg/day)  Urine Ca (mg/day)  Fecal Ca (mg/day)  C  68.75 + 1.25  d  1.114 + 0.138  b  S  66.25 + 5.54  a  0.939 + 0.042  b  C-CPP  73.12 + 5.24  a  2.016 + 0.231  a  S-CPP  72.50 + 2.70  a  0.969 + 0.200  b  36.03 + 3.54 39.88 + 1.46 41.51 + 3.83 49.83 + 4.10  Ca Apparent Absorption (%)  Ca Balance (mg)  a  33.46 + 2.51  a  41.11 + 1.47  a  28.60 + 3.11  a  40.00 + 2.12  a  38.39 + 2.91  a  43.15+ 0.94  a  29.26 + 3.08  a  a  32.40 + 5.79  1  Data a r e expressed as mean + SEM. !: C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP. i Ca Balance (mg) = Ca Intake (mg/day) - U r i n a r y Ca (mg/day) - Fecal Ca (mg/day). Ca Apparent A b s o r p t i o n (%) = {[Ca Intake (mg/day) - Fecal Ca (mg /day)] /Ca Intake (mg/day)) x 100. 1  Means s h a r i n g the same l e t t e r w i t h i n a column are not s i g n i f i c a n t l y at p<0.05.  different  e l i m i n a t i o n by t h i s CPP  fed animals.  group  o f a n i m a l s when compared t o C, S and S-  Apparent  a b s o r p t i o n o f c a l c i u m was i n c r e a s e d i n  t h e C-CPP f e d a n i m a l s o n l y , b u t n o t s i g n i f i c a n t l y was n o t d i f f e r e n t that the  between  increase i n  was n o t u t i l i z e d  dietary  c a l c i u m absorbed  between d i e t a r y  diets  when  groups  not  (Table  4.6).  (p<0.05) g r e a t e r i n compared  animals  to  compared  significantly  with casein alone. balance  and  dietary  to  diets.  to  of  the  the casein  the  soy  diet  lower u r i n a r y  C and S d i e t s .  were  protein  actually  data  intestinal  not  suggest  resulted  different  than  that  Mg  between  absorption  o f Mg  fortified  t h e enhanced  w i t h CPP f o r t i f i c a t i o n magnesium  diets.  i n Mg e x c r e t i o n ,  i n t h o s e a n i m a l s f e d t h e CPP these  fortified  e x c r e t i o n o f Mg  (Table 4.6). Apparent  c a l c i u m seen  affected  t h e CPP  (p<0.05) g r e a t e r i n c a s e i n  fed  absorption  be d e c r e a s e d  Taken t o g e t h e r ,  adversely  fed  Despite these d i f f e r e n c e s  apparent  bioavailability  of  (p<0.05) d i f f e r e n t  animals f e d  those  (p<0.05)  t r e a t m e n t groups  appeared  indicating  F e c a l e x c r e t i o n o f Mg was  counterparts  H o w e v e r , CPP f o r t i f i c a t i o n in a  groups,  by t h e C-CPP f e d a n i m a l s  significantly  U r i n a r y Mg c l e a r a n c e was s i g n i f i c a n t l y fed  Ca b a l a n c e  b u t r a t h e r , was e x c r e t e d .  Magnesium i n t a k e was  significantly  treatment  so.  absorption  may have in  these  animals. Fortification  of  d i e t s w i t h CPP r e s u l t e d  (p<0.05) h i g h e r i n t a k e o f p h o s p h o r u s diets  were  reflected  not  balanced  i n the f e c a l  by  significantly  these animals  f o r phosphorus  and u r i n a r y p h o s p h o r u s 131  in a  s i n c e the  (Table 4.7). This i s excretion  data  TABLE 4.6  E f f e c t o f p r o t e i n source and CPP f o r t i f i c a t i o n on 24 h r . magnesium b a l a n c e . 1  o Diet  Mg Intake (mg/day)  Urine Mg (mg/day)  Fecal Mg (mg/day)  c  6.88 + 0.12  a  1.91 + 0.03  a  1.49 + 0.10  s  6.62 + 0.55  a  0.58 + 0.06  c  2.29 + 0.03  c- -CPP  7.31 + 0.52  a  1.45 + 0.08  b  s- -CPP  7.25 + 0.27  a  0.63 + 0.03  c  2.92 + 0.18 3.12 + 0.23  Mg Apparent Absorption 4 (%)  Mg Balance (mg)  c  3.18 + 0 . 3 1  a  71.16 + 7.08  a  b  3.81 + 0.27  a  67.76 + 2.52  a  a  3.65 + 0.17  a  62.53 + 3.21  a  3.06 + 0.24  a  56.69 + 3.93  a  a  Data are expressed as mean + SEM. ^ C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP. ^ Mg Balance (mg) = Mg Intake (mg/day) - U r i n a r y Mg (mg/day) - Fecal Mg (mg/day). Mg Apparent A b s o r p t i o n (%) = {[Mg Intake (mg/day) - F e c a l Mg (mg /day)] /Mg Intake (mg/day)} x 100. 1  Means s h a r i n g t h e same l e t t e r w i t h i n a column a r e not s i g n i f i c a n t l y at p<0.05.  different  TABLE 4.7  E f f e c t o f p r o t e i n source and CPP f o r t i f i c a t i o n on 24 h r . phosphorus balance . 1  Urine P (mg/day)  P Balance * (mg)  Diet^  P Intake (mg/day)  Fecal P (mg/day)  C  79.48 + 1.44  s  84.38 + 5.48  C-CPP  99.89 + 7.16  a  32.58 + 2.13  a  37.80 + 3.78  S-CPP  100.63 + 3.75  a  27.60 + 0.92  b  39.71 + 2.69  c  23.61 + 2.18  c  22.18 + 1.31  21.74 + 1.05  b  ab  17.82 + 1.36  P Apparent Absorption (%)  1  b  b  a  a  34.12 + 3.54 44.38 + 4.39 29.51 + 3.79 34.82 + 3.01  a  68.40 + 3.00  a  a  72.52 + 2.24  a  a  61.67 + 4.47  a  62.62 + 4.83  a  a  , Data a r e expressed as mean + SEM. i C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP. . P Balance (mg) = P Intake (mg/day) - U r i n a r y P (mg/day) - F e c a l P (mg/day). P Apparent A b s o r p t i o n (%) = {[P Intake (mg/day) - F e c a l P (mg /day)] /P Intake (mg/day)} x 100. q  Means s h a r i n g t h e same l e t t e r w i t h i n a column are not s i g n i f i c a n t l y at p<0.05.  different  which  are both s i g n i f i c a n t l y  fed animals data  when compared  complement  absorption  which  between d i e t a r y data  indicate  fortified  of  treatments that  g r e a t e r i n C-CPP  and S  fed counterparts.  phosphorus no  balance  significant  (Table  and S-CPP  4.7).  and  (p<0.05)  These  apparent  differences  Taken t o g e t h e r , t h e s e  t h e i n c r e a s e i n d i e t a r y p h o s p h o r u s o f t h e CPP  did  not  result  in  a  greater  o f p h o s p h o r u s by a n i m a l s f e d t h e s e  a b s o r p t i o n or  diets.  Mineralization: Femur  mineralization  Femur a s h w e i g h t a f f e c t e d by  and  diets.  affected  and bone  Bone P h y s i c a l  The f e m o r a l Ca/P  by  protein effect  content  energy  was  The  significantly also  animals f e d the  than soy  a g a i n , not  protein fed  significant.  Parameters:  source  on  n o r CPP  femur p h y s i c a l  Bone d r y w e i g h t s and l e n g t h s were groups.  (p<0.05)  than those f e d  not  although  with  mineralization  Femur Mg c o n t e n t was  treatment,  and B i o m e c h a n i c a l  (p<0.05)  r a t i o was  fortification.  dietary  Neither dietary  failure  femur  4.8.  (p<0.05)  Dietary f o r t i f i c a t i o n affect  a n i m a l s ; t h e d i f f e r e n c e h o w e v e r , was  dietary  Table  significantly  calcium content  c a s e i n d i e t s had a h i g h e r femur Mg  4.9).  in  Soy p r o t e i n f e d a n i m a l s had a s i g n i f i c a n t l y  by d i e t a r y CPP  significant  summarized were  significantly  l o w e r femur a s h w e i g h t  affected  are  d i e t a r y p r o t e i n . source.  parameters.  the c a s e i n  data  calcification  CPP h o w e v e r , d i d n o t  not  to C  indicate  diets  utilization Bone  those  (p<0.05)  biomechanical  significantly  had a  parameters  (Table  s i m i l a r between a l l f o u r parameter,  (p<0.05)  134  fortification  femur  bending  lower i n soy p r o t e i n  TABLE 4.8  E f f e c t o f p r o t e i n s o u r c e a n d CPP f o r t i f i c a t i o n on femur m i n e r a l i z a t i o n . 1  Diet  2  Ash wt. (g)  Ca (mg/bone)  Ca/P ratio  Mg (mg/bone)  c  0 .237  +  0 .006*  94 .60  +  3 .85"  2 .00  +  0 .01  a  1 . 84  +_  0 .06  s  0 .206  +  0 .006b  78 .75  +  3 .21<>  1 . 97  +  0 .05  a  1 . 62  +_  0 .07  C-CPP  0 .238  +_  0 .002*  98 .75  +  1 .88*  2 .02  +_  0 .02"  1 .88  +  0 .04  S-CPP  0 .218  +_  0 .004b  80 .35  +_  6 .83b  1 .99  +_  0 • 05  1 .59  +  0 . 10  1 2  D a t a a r e e x p r e s s e d as mean +_ SEM. C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; S-CPP = S + 3% CPP.  Superscripts different.  with  C-CPP  a  a  a  a  = C + 3% CPP;  d i f f e r e n t alphabets are s i g n i f i c a n t l y  135  a  (p<0.05)  TABLE 4.9  E f f e c t o f p r o t e i n source and CPP f o r t i f i c a t i o n on femur p h y s i c a l and biomechanical p a r a m e t e r s . 1  _ Diet  Dry wt.  Length  (g)  (mm)  Bending F a i l u r e Energy (x 1 0  c  0.387 + 0.010  a  30.61 + 0.47  a  s  0.360 + 0.009  a  30.49 + 0.21  a  c- -CPP  0.393 + 0 . 0 U  a  30.95 + 0.50  a  s- -CPP  0.360 + 0.012  3  30.02 + 0.65  a  0 1  - 2  J)  9.24 + 0.93 6.12 + 0.60  Maximum Bending S t r e s s (N/mm ) 2  a  65.82 + 1 . 4 5  b  8.37 + 0 . 5 2 6.78 + 0 . 8 0  68.94 + 3 . 0 6  ab  74.61 + 4.80  ab  62.23 + 2.08  ab  ab  a  b  Data a r e expressed as mean + SEM. C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; C-CPP = C + 3% CPP; S-CPP = S + 3% CPP.  Means s h a r i n g t h e same l e t t e r w i t h i n a column are not s i g n i f i c a n t l y d i f f e r e n t a t p<0.05.  fed  animals  fortification casein  fed  compared had l i t t l e animals.  femur maximum b e n d i n g CPP  fortified diets,  to  those  effect  fed  casein.  Dietary  on femur b i o m e c h a n i c s  in  CPP  s o y and  D i e t a r y p r o t e i n source d i d not i n f l u e n c e stress. compared  A  similar  result  to n o n - f o r t i f i e d  137  was  diets.  observed i n  Discuss ion The calcium  importance binding  of  activity  dephosphorylated  casein  experiments conducted aration  was  associated  peptides.  Baumy  of calcium  phosphoseryl  of 2  Previous  also  bioactive  of  a n i m a l s when ( N a i t o et. a i - , 1983). profile  4 5  activity Ca  after  digestion  recently  reported  that p r e f e r e n t i a l  on CPP o c c u p i e d  by c l u s t e r s o f  pK v a l u e ,  and c h a r g e  T h e s e w o r k e r s were a b l e t o phosphoseryl  clusters  occurs  residues.  have i n d i c a t e d t h e p o t e n t i a l p h y s i o l o g i c a l CPP  in  demonstrating,  contents  o f CPP  of casein  1974; M e i s e l f o r calcium  calcium  in  the  compared t o c o u n t e r p a r t s 1972; N a i t o  P r o t e i n composition of peptides  s e t of  CPP p r e p -  binding of  with  casein  demonstrated  of  and S u z u k i ,  soluble  crude  to  (1989)  groupings.  the i n t e s t i n a l  the a f f i n i t y  proportion  bound  coworkers  at s i t e s  studies  the enzymatic d i g e s t , which  to s i n g l e phosphoseryl  ejt aj,. , 1 9 7 2 ; N a i t o but  in  strongly  saturation  studies  of the  presence i n  that  which  occurs  that Ca *  to binding  value  is  data  these  a  recovery  r e s i d u e s , due t o t h e f a v o u r a b l e  distribution  prior  to  The  to the  In the f i r s t  study,  phosphate.  and  saturation kinetics  et. a j . . , 1 9 8 3 ) .  which possessed calcium  equal  calcium  phosphate m o i e t i e s  established i n  i n the present  suggested that  establish  (Sato  with organic was  organic  o f CPP was  obtained,  purification,  binding  the  and  Suzuki,  138  their (Naito  and F r i s t e r ,  1989),  i o n s , to increase the  digesta  fed other 1974;  released  only  fed animals  and d i g e s t i b i l i t y  and amino a c i d s  not  of  casein fed  dietary  Lee e t  proteins  a_l. , 1980;  can i n f l u e n c e the  during  the d i g e s t i v e  process.  A reduced  can be a t t r i b u t e d released  weight  the  proteins  thus n u t r i t i o n a l  digestibility  acids  and  FER  be e x p l a i n e d  high  to  and  these animals.  1984).  relatively  N a r a s i n g a Rao,  ratio  amino The  may  lower  1984).  decreased fed  digestibility soy  A d d i t i o n o f CPP body w e i g h t  be a t t r i b u t e d  of  acids  structured, globulin  i n increased f i n a l  This response  the p l a n t  the  nonessential  weight, highly  resulted  and  value,  e x h i b i t e d h e r e i n by soy p r o t e i n  by t h e  molecular  (Raghunath  q u a l i t y of  and  R a g h u n a t h and N a r a s i n g a Rao,  soy p r o t e i n d i e t s in  amino  gain  a n i m a l s , may of  to decreased  essential  ( M a u r o n , 1973; body  protein quality,  t o an  p r o t e i n d i e t when s u p p l e m e n t e d  and  to FER  improved  with animal  protein derived peptides. The  enhanced i l e a l  protein diets  a b s o r p t i o n of  supplemented  w i t h CPP  * Ca 5  extends  in  animals  fed  the f i n d i n g s of  soy Sato  e t a l . ( 1 9 8 6 ) t h a t CPP  can a c t t o e n h a n c e c a l c i u m b i o a v a i l a b i l i t y  from  lumen.  the  intestinal  d i e t a r y CPP  would  enzymes i n  the d i g e s t i v e  after  presence rats  be d e t e r m i n e d  intraluminal  s t u d i e s by  Lee  and  after  Furthermore,  CPP  Ca  chyme.  coworkers  in  to  hr  by p r e v i o u s  were a b l e t o d e t e c t t h e digesta  not  at  of 13.5  susceptibility  activity  (Reeves  be o f s i g n i f i c a n c e  and in  (Van d e r Meer, 1 9 8 8 ) . 139  proteolytic  o b t a i n e d 1.0  exhibited  w h i c h may  human s t u d i e s  the but  supplemental  i s supported  ( 1 9 8 0 ) , who  meal-feeding, have  of  This r e s u l t ,  administration,  phosphatase  M e l l a n d e r , 1963) model and  4 5  effectiveness  by i t s r e s i s t a n c e  of macrophosphopeptides  early  alkaline  The  to  casein fed o r 25.5  hr.  intestinal  Latour,  1958;  both r a t animal P r e v i o u s work o f  Mellander  (1947),  demonstrated the r e s i s t a n c e of a high  weight  peptide  residue  further  a t t a c k from i n t e s t i n a l  The t r a n s l o c a t i o n inversely  related  decreased  uptake of  animals,  respectively  proteolytic  Ca  by  for  only  femora  likely  due  the  balance  and  mechanisms  excretion,  rather  Ca  than  diet  fecal  calcium  resulted  excreted  in  of  and  studies  of  by  other.  (Greger  4 5  homeostatic  of  the greater  Alternatively,  Ca  with  magnesium  mineral  occur, but 4 0  Ca  on t h e  i s associated thus,  can  impair  are  homeo-  calcium  towards  (Allen,  1982).  It is  study,  animals  f e d the  of c a l c i u m i n the u r i n e ;  was  not  often  different.  considered  This this  of  e t a l . . 1 9 8 1 ) ; and c o n v e r s e l y ,  reported  that  magnesium o r o t h e r t h a t magnesium  from supplements (Greger 140  together i n  s i n c e one i s o f t e n a f f e c t e d  c o w o r k e r s have  absorption  the a b s o r p t i o n o f c a l c i u m  with  balance.  bioavailability,  G r e g e r and  a 1  excess  levels  however,  C-CPP f e d  calcium  repletion  utilization  The  an e n h a n c e d a p p a r e n t a b s o r p t i o n o f c a l c i u m f r o m  Calcium  calcium  and  deposition to  direct  elevated  f e m o r a was data.  bone c a l c i f i c a t i o n ,  also  d i e t , b u t an u n c h a n g e d c a l c i u m  the  the  C  animals.  bone  Calcium  excretion,  of to  noteworthy t h e r e f o r e , that i n the present C-CPP  to  utilization  exchange  adequate  will  enzymes.  absorption  the  4 5  casein d i g e s t i o n , to  radiolabel  the eventual  s u r f a c e o f t h e bone m a t r i x .  static  i n vitro  c a l c i u m a b s o r b e d by t h e s e  reflects  calcium  an  intestinal  was  h r p e r i o d was t o o s h o r t rather,  4 5  the  Ca  mechanisms c o n t r o l l i n g amount o f  of  to 4 5  from  molecular  excess  minerals  can i m p a i r  e t a l . . 1987).  In the p r e s e n t s t u d y , exhibited  animals  increased  apparent  fecal  absorption  of  calcium b i o a v a i l a b i l i t y on t h e i n t e s t i n a l u r i n a r y Mg  fed  Mg  Mg,  the  excretion, suggesting  a b s o r p t i o n o f Mg.  calcium The  the r e l a t i v e l y  to casein  Mg  phorus  balance  and  balance apparent  u r i n a r y P and f e c a l  supplemented  enhanced  diets.  et  significantly  were  and  experiment  3.  accompanies  Mg i n  soy f e d  was l i k e l y  Mg  by  these  due t o animals  Similarly,  were  paralleled  physical  calcium  These  b a l a n c e and  not  by  phos-  a f f e c t e d by i n t a k e s of  both i n c r e a s e d  the Bone  failure  t h e CPP  may  (Horowitz  e t a 1. .  the present experiment, the  observed  141  not  bone r e m o d e l l i n g i n e l d e r l y  energy  biomechanical  of  bioavailability  metabolism In  were  e x t e n d t h o s e o f o t h e r s who  high calcium inhibit  trend  parameters  bioavailability  results  a l . . 1988).  lower bending  confirms  c o n s e r v a t i o n of  The i n c r e a s e d p h o s p h o r u s  s u b j e c t s , b u t does n o t a f f e c t bone Sinha  of  absorption  have r e p o r t e d t h a t a d i e t w i t h calcium  effect  P excretion.  mineralization the  increase i n  i n turn  was n o t d i f f e r e n t .  C-CPP and S-CPP f e d a n i m a l s  by  i n a lower  the decreased  excretion of  excretion  d i e t a r y CPP s u p p l e m e n t a t i o n .  Bone  which  fed counterparts  greater fecal  overall  animals  the  Accordingly,  decreased u r i n a r y  since  1988;  that  diets  a b s o r p t i o n ( M o r r i s and O ' D e l l , 1963; G r e g e r e t  a n i m a l s , compared  improve  resulting  e l i m i n a t i o n by C-CPP a n i m a l s r e f l e c t s  a l . . 1981).  affected  supplemented  i n t h e s e a n i m a l s had an i n h i b i t o r y  Mg, due t o an i n c r e a s e d Mg r e q u i r e m e n t increased  CPP  o f femora  in  from soy f e d  t h e soy f e d group i n  parameters  were  similarly  unaffected data of  by d i e t a r y CPP  supplementation.  i n d i c a t e t h a t h o m e o s t a t i c mechanisms  increased  absorbed calcium  bone d e p o s i t i o n  i n calcium  needed t o d e t e r m i n e i f t h i s and c a l c i u m  Taken t o g e t h e r , control  the metabolism  i n e x c r e t i o n , r a t h e r than  replete animals.  Further  142  increased  studies are  i s the case i n o s t e o p o r o t i c  deficient individuals.  these  subjects,  Experiment  5  E f f e c t o f p r o t e i n h e a t d e n a t u r a t i o n on c a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n r a t s f e d c a s e i n and s o y p r o t e i n s . Introduction Protein quality ability  from  the  proteins w i l l peptides  and  t u r n , may  be  The the  amino a c i d s  value  c a s e i n and  procedure of  heat d e n a t u r a t i o n .  A reduction in  and  study,  p r o t e i n sources and  peptides.  The  o b j e c t i v e s of t h i s  determine  mineralization  taneously K y o t o (WKY)  by  and  hypertensive  biomechanical (SHR)  r a t s were a g a i n  and  used.  143  to  the  of c a s e i n  e x p e r i m e n t were  w i t h the  e f f e c t s on  during to the  r e s u l t e d i n extreme  to e l i m i n a t e  production  dairy  heat treatment  p a n c r e a t i c enzymes and  these  amino a c i d s  performed according  c a l c i u m phosphate a s s o c i a t e d  subsequently bone  production  thus,  explained  in particular  a severe  performed  casein  of  be  consumption or  Schneeman ( 1 9 7 9 )  T h i s was and  and  treated before  of  esterified  availability  proteins,  digestion  p o s s i b l e CPP  potentially bioactive  calcium absorption.  In the p r e s e n t  Percival  of  lumen, which i n  Moreover,  soy  digestibility  h e a t t r e a t e d p r o t e i n s can  p r o t e i n s , are u s u a l l y heat pasteurization.  and  bioavail-  intestinal  digestibility  1972).  of  w i t h i n the  of  of c a l c i u m  composition  release  affect paracellular  by a d e c r e a s e d  both  a determinant  diet.  influence  the n u t r i t i o n a l  (Mauron,  may  tryptic phospho-  to c o n t r o l f o r eliminate  the  m i c e l l e and  to  calcium absorption strength.  The  and  spon-  normotensive c o n t r o l Wistar-  M a t e r i a l s and Methods In V i t r o An  Digestibility in  followed  vitro  by  Study:  two-step  pancreatin  proteolysis  enzymatic  soy  protein  were  S o l u t i o n s were with  pepsin  suspended  adjusted  (porcine  shaking  water  samples  were  was  et a l .  ( 1986).  incubated  once  I I , Sigma).  a l . . 1983).  The  obtained  by l i n e a r  used t o  compare t h e  native  mucosa  Following  with  regression  slope  initial  c a s e i n and h e a t d e n a t u r e d  and  at  incubated 37°C  in a  digestion,  were removed a t  (porcine frequent  w i t h TNBS (Kwan  o f p r o t e i n d i g e s t i o n was  a n a l y s i s (Maga  relative  casein  pancreatin  2 0 % TCA and r e a c t e d  initial  a  Proteins,  HC1 and  a 30 m i n u t e p e p s i n  more  in  water, r e s p e c t i v e l y .  1:10,000)  Aliquots  intervals, deproteinized with et  deionized  pepsin,  used  and h e a t d e n a t u r e d  pH 1.9 w i t h d i l u t e  stomach  bath.  p a n c r e a s ; Grade  to  in  using  hydrolysis  m o d i f i c a t i o n o f t h e method o f J a c q u e s namely c a s e i n , soy p r o t e i n i s o l a t e ,  method  et a1..  1973) and  p r o t e o l y s i s r a t e s between  proteins.  A n i m a l s and D i e t s : Four-week normotensive  old  male  Wistar  Kyoto  PQ) were e a c h d i v i d e d animals per soy  group).  protein isolate  spontaneously (WKY)  into four Dietary  (S)  (ICN  hypertensive  rats (Charles experimental  dietary  treatments included Biochemicals,  h e a t d e n a t u r e d C (DC) and S (DS) d i e t s ,  144  River,  (SHR) and Montreal, g r o u p s (6  20% c a s e i n ( C ) ,  Cleveland,  OH)  r e s p e c t i v e l y (Table  and  5.1).  TABLE 5.1 C o m p o s i t i o n  of experimental  Casein D i e t a r y Component ( g / 1 0 0 g)  Casein* Soy p r o t e i n  isolate*  20%  20 .0 —  d i e t s f e d to  animals.  Soy  Diets H.D.  20.0 —  20%  Diets H .D.  20 .0  20 .0  0.3 15.0 48.77 7.0 5.0  0 .3 15 .0 48 .77 7 .0 5 .0  0.3 15.0 48 .77 7.0 5.0  0.3 15.0 48 .77 7.0 5.0  Ca f r e e m i n e r a l m i x t u r e * Vitamin mixture* Choline b i t a r t r a t e *  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3. 5 1 .0 0 .2  Calcium  1 .17  1. 17  1 . 17  1 .17  D.L. m e t h i o n i n e * Cornstarch* Sucrose Fibre* Vegetable o i l  carbonate**  H.D. = h e a t d e n a t u r e d diet. * I C N B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. * U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , * * BDH C h e m i c a l s , T o r o n t o , ON.  145  OH.  Thermal d e n a t u r a t i o n t o t h e method o f proteins  at  P e r c i v a l and  121°C,  forincorporation calcium  of d i e t a r y p r o t e i n s  level  2  atm  (0.5%).  Schneeman for  into diets.  24  hr,  24  described  hour  described study  followed  autoclaving by  contained  cooling  an a d e q u a t e  was  F e e d i n g and m e a l - f e e d i n g  i n experiment performed,  3. as  previously  i n e x p e r i m e n t 4, when a n i m a l s were 10 weeks o f a g e .  Intestinal described  balance  by  according  a n i m a l s f e d t h e C and S p r o t e i n  d i e t s were t h e same as i n e x p e r i m e n t 4.  A  (1979),  A l l diets  The SHR  t r a i n i n g were as p r e v i o u s l y  was p e r f o r m e d  calcium  absorption  was  measured  as  previously  i n e x p e r i m e n t 1 when a n i m a l s were 14 weeks o f a g e .  Analyses: Analyses feces as  intestinal  lumen,  blood  plasma, feed,  and femur m i n e r a l i z a t i o n and b i o m e c h a n i c s  previously described  Statistical All  i n experiment  source  urine,  were c a r r i e d o u t  4.  Analyses!  d a t a a r e e x p r e s s e d as mean +_ SEM.  were t e s t e d the  of  f o r by one-way of  the  ANOVA.  difference  r a n g e t e s t a t a p<0.05 l e v e l  Where was  Treatment  d i f f e r e n c e s d i d occur,  identified  of s i g n i f i c a n c e .  146  differences  using  a multiple  Results In  Vitro  Digestibility  The  initial  c a s e i n , soy protein the  products  ultimately 1978).  time  protein  i s shown of  protein  isolate,  pepsin-pancreatin and h e a t  digestion,  inhibit  No  denatured  attempt  which  the process  However, t h e  protein  course  i n F i g u r e 5.1.  this disadvantage. soy  Study:  was  of  c a s e i n and s o y made t o remove  a r e known t o a c c u m u l a t e  of enzymatic  use of  digestion  the i n i t i a l  and  digestion  (Robbins,  s l o p e method  overcomes  A s l o w e r r a t e o f p r o t e o l y s i s was o b s e r v e d f o r  compared  to  sources r e s u l t e d  casein.  Heat  d e n a t u r a t i o n of both  i n a marked d e c r e a s e  in their  respective  digestibilities . In  Vivo  Study:  At  the  initiation  significantly fed  the  strains  of  the  experiment,  (p<0.05) g r e a t e r body w e i g h t  same d i e t remained  (Table 5.2).  significant  than  WKY  a n i m a l s had a  SHR c o u n t e r p a r t s  This difference  between a n i m a l  a t the c o n c l u s i o n of the experiment.  Dietary protein  s o u r c e and h e a t d e n a t u r a t i o n o f d i e t a r y  had  (p<0.05)  significant  istics. lower  Animals final  f e d the S d i e t  body  respectively,  effects  weight  when  heat d e n a t u r a t i o n of d i e t a r y b o t h body  weight  gain  i n t a k e of  these  animals.  experienced  animal  growth c h a r a c t e r -  had b o t h a s i g n i f i c a n t l y  and  compared  on  proteins  feed to  efficiency  ratio  C fed counterparts.  p r o t e i n s had  (p<0.05)  an a d v e r s e  (FER),  Moreover, effect  and FER as i n f l u e n c e d by t h e r e d u c e d  s i g n s of d i a r r h e a ,  The  DC  and  indicating 147  DS  fed  animals  on  feed also  nutrient malabsorption  c Fig.  5.1  DC  S  Dietary Proteins  DS  P e p s i n - p a n c r e a t i c d i g e s t i o n e s t i m a t e s of c a s e i n ( C ) , soy protein isolate ( S ) , heat d e n a t u r e d C ( D C ) , and h e a t d e n a t u r e d S (DS) c a l c u l a t e d from th£ i n i t i a l reaction r a t e s (0 - 10 minutes). R e s p e c t i v e s l o p e s were: C = 35.5 x 10-3; = u,5 10-3; c = 5.5 x 1 0 " 3 ; DS = 7.5 x 1 0 " ; r e g r e s s i o n range = 0.996>_ rjC0.944. S  3  x  D  TABLE 5.2  Diet  1  2  E f f e c t o f heat t r e a t i n g p r o t e i n on d i e t e f f i c i e n c y .  I n i t i a l body w t . (g) SHR WKY  3  F i n a l body w t . (g) SHR WKY  Dry M a t t e r Intake (g) SHR WKY  4  c  77 + 2ax  97 +  ay  259 + 8  a x  5  298 + gay  s  79 + 2&x.  97 +  2  ay  225 + 5  bx  267 + 2*>y 861 +  1 4  ax  821 + 1 3  DC  77 +  3  ax  98 +  4  ay  179 + 3  213 + c y  733 +  1 7  bx  787  DS  78 +  3  ax  96 +  3  ay  120 + 4  155 + d y  652 + 36  CX  dx  7  3  867  + 23  ax  cx  865 + 26  + 20  720 + 2 1  Feed SHR  0.211 + 0.004  ax  0.234 +  abx  0.170 + 0.004  bx  0.208 +  bcx  0.140 + 0.004  cx  0.146 +  0.065 + 0.005  dx  0.082 +  ax  cy  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S. 5 weeks o f age. 14 weeks o f age.  a,b,c,d i g i f i t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. » ' = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. =  x  Efficiency Ratio WKY  y  S  n  c a n  during  the  exhibited  experimental period.  significantly  body w e i g h t s diets.  (p<0.05)  decreased  and FER, r e s p e c t i v e l y  source or  Plasma  groups.  l e v e l s were  heat d e n a t u r a t i o n  g/dL).  minerals  calcium  (range  affected  mg/dL),  by d i e t a r y  influence  the  Intestinal  4 5  Ca  animals  fed  4 0  Ca  the  fed  casein  4 5  Ca  the  activities  protein 4 5  fed Ca  or  specific  (range  ionized  9.0  plasma  were f o u n d t o be  ileal  the  loop  animals  absorption  same  and s o y p r o t e i n  treatments d i d not  ileal  loop  also  dietary  protein  protein  diets  (Table 5.3).  not  affected  source.  by  However,  exhibited  higher  a c t i v i t i e s when compared t o t h o s e  and s o y d i e t s  (Table was  diet  exhibited  a  5.3). Absorption affected  of the  diets resulted  150  proteins  significantly  animals.  Both  of  by d i e t a r y in  ( T a b l e 5.3; F i g u r e  than those f e d c a s e i n ;  common t o b o t h SHR and WKY casein  between d i e t a r y  calculated  were  s o u r c e as w e l l as h e a t d e n a t u r a t i o n fed  protein  t o 6.03 +_ 0.30  plasma c a l c i u m  and d i e t a r y  of  denatured  ligated  animals  +_ 0.40  5.2 +_ 0.4 mg/dL)  content  differences  loop  the  the  animals.  by d i e t a r y  different  the t o t a l  i n animal s t r a i n  intestinal native  not  nor  final  treatment.  specific  animal s t r a i n  not a f f e c t e d  were  4.6 +. 0.1 t o  Differences  ileal  intakes,  t h a n c o u n t e r p a r t s f e d C and S  ( r a n g e 4.70  Furthermore, neither  +_ 0.3 t o 9.9 +_ 0.3  WKY  feed  T h e s e r e s u l t s were s i m i l a r f o r b o t h SHR and WKY  Plasma p r o t e i n  from  A n i m a l s f e d t h e DC and DS d i e t s  this the  4 5  Ca  protein SHR and  5 . 2 ) . Soy  (p<0.05) l o w e r observation heat  was  denatured  i n decreased absorption  of  TABLE 5.3  Diet  E f f e c t o f heat t r e a t i n g p r o t e i n on c a l c i u m a b s o r p t i o n from i l e a l l o o p .  2  SHR  40Ca i n l o o p (mg/loop)  4 5  Ca Specific Activity (dpm/mg ^ C a ) SHR WKY  4 5  n  u  WKY  Ca  SHR  Absorbed (% dose)  3  WKY  C  2.25 + 0 . 2 2  a x  2.36 + 0 . 2 0  ax  4.30 + 0 . 7 0  ax  3.59 + 0 . 4 0  b x  43.2 + 2 . 0  a x  38.5 + 3 . 7  a x  S  2.16 + 0 . 2 0  a x  3.04 + 0 . 2 0  ax  4.60 + 0 . 3 0  ax  3.05 + 0 . 3 7  b x  29.2 + 3 . 4  b x  30.4 + 2 . 5  b x  DC  2.26 + 0 . 4 1  a x  2.28 + 0 . 1 8  ax  5.08 + 0 . 6 5  ax  5.03 + 0 . 6 5  ax  20.4 + 2 . 6  b x  24.7 + 8 . 8  b x  DS  2.02 + 0 . 3 9  a x  1.59 + 0 . 1 5  ax  5.39 + 0 . 4 9  ax  5.56 + 0 . 4 4  ax  22.6 + 4 . 8  b x  18.4 + 3 . 4  b x  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . z. C_= c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat d e n a t u r e d C; DS = h e a t d e n a t u r e d S. ^ C a a b s o r p t i o n (% dose) = [ 1 - Ca(dpm) a t 1 hr/dose Ca(dpm) a d m i n i s t e r e d ] x 100. 1  6  a x  s  ' '  b y  qt5  = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n c o l u m n s . = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows.  Fig.  5.2  I n t e s t i n a l a b s o r p t i o n of * C a from i l e a l l o o p of spontaneously hypertensive (SHR) and control WistarK y o t o (WKY) rats fed n a t i v e and heat denatured casein (C) and soy p r o t e i n i s o l a t e (S) d i e t s , r e s p e c t i v e l y . * denotes significant (p<0.05) difference from casein treatments. 5  4 5  Ca  i n a n i m a l s f e d t h e s e d i e t s compared t o n a t i v e  protein  fed  animals.  Deposition  of  4 5  Ca  t o t h e femur i s summarized  T h e r e were no s i g n i f i c a n t the  diet.  effect  on  4 5  Animal Ca  fed animals. the  strain  SHR  i n SHR and WKY  differences  Soy f e d WKY  bone,  were  the  than t h a t  results  and s p e c i f i c  denatured  l e s s (p<0.05)  Heat d e n a t u r a t i o n the acute  these  soy p r o t e i n  (p<0.05) f e m u r s p e c i f i c  appeared to a l s o i n f l u e n c e  radioactivity  were shown t o have an  animals deposited  counterparts.  but  5.4.  animals f e d the  d e p o s i t i o n t o t h e bone i n o n l y n a t i v e  femora, r e s u l t i n g i n lower  than  i n Table  d i f f e r e n c e s i n r a d i o l a b e l d e p o s i t i o n to  f e m u r due t o d i e t a r y t r e a t m e n t  same  c a s e i n and s o y  not s i g n i f i c a n t .  activity  Ca  4 5  proteins  Ca  to the  The amount o f  of the femora of  animals fed  c a s e i n and s o y p r o t e i n d i e t s a p p e a r e d t o be  of c o u n t e r p a r t s  fed native  to  activities  of d i e t a r y  d e p o s i t i o n of  4 5  p r o t e i n sources,  higher  respect-  ively. Mineral  Balance  The  24  Study:  hour  mineral  T a b l e s 5.5 t o 5.10. denatured  diets  i n calcium  fed the  same d i e t .  data,  in  excreted  asmuch  (Table  fed  resulted  as  in  fed  the  (p<0.05) l e s s  native  a  protein  e l i m i n a t i o n was i n f l u e n c e d by 153  are presented  in  animals f e d the  significant o f SHR  Ca e x c r e t i o n  animals  data  i n t a k e of  5.5) i n t a k e ,  Fecal  significantly  counterparts  study  The r e d u c e d f e e d  protein  decrease  balance  and WKY  (p<0.05) animals  complements t h e i n t a k e denatured  calcium diets.  in  protein diets the feces  Urinary  than  calcium  TABLE 5.4  E f f e c t o f heat t r e a t i n g p r o t e i n d e p o s i t i o n t o the f e m u r .  on  4 5  calcium  1  Diet  4 3  2  C a Deposited (%dose/bone)  SHR  4 5  WKY  C  0 . 148 +_ 0 .046* X  0 . 101  s  0. 274  +_  0 .080°  DC  0 . 250  +_  0 .090* X  DS  0 . 180 + 0 .069* X  X  0 .039"  Ca Specific (% d o s e / g SHR  4 0  Activity Ca) WKY  1 . 7 + 0 . 6* X  1. 1  +_  0 .4* X  0 .098 + 0 . 019* y  3 .4  1 .0*  X  1 .2  +_  0 .2* y  0 . 174  0 .053**  4 .0 + 1 .6*  X  2.8  +_  0 . 9* X  0 .111 + 0 .018**  3 .2 + 0 .8*  X  2 . 1 + 0 .4* X  +_  1 D a t a a r e e x p r e s s e d a s mean +_ SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r K y o t o r a t s . C = c a s e i n ; S = s o y p r o t e i n i s o l a t e ; DC = h e a t d e n a t u r e d C; DS = h e a t d e n a t u r e d S.  2  *>  = s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n columns. * • y - s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n rows . b  154  TABLE 5.5  E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour c a l c i u m e x c r e t i o n .  Diet^  Ca Intake SHR  WKY  SHR  U r i n e Ca (mg/ day)  c  68.75 + 1.25  ax  71.25 + 4.62  ax  1.11 + 0.14  s  66.25 + 5.54  ax  68.12 + 3.12  ax  0.94 + 0.04  DC 46.88 + 4.13  bx  50.00 + 2.04  bx  1.93 + 0.11  DS 45.00 + 2.28  bx  41.88 + 2.13  bx  0.67 + 0.08  F e c a l Ca WKY  SHR  2.03 + 0.50  ay  38.95 + 3.84  ax  49.09 + 5.97  1.78 + 0.35  ay  39.88 + 1.46  ax  41.47 + 6 . 4 9  ax  2.13 + 0.21  ax  19.83 + 2.07  bx  22.62 + 2.61  cx  1.63 + 0.38  ay  24.34 + 3.08  bx  27.74 + 3 . 5 9  bx  bcx  ax  abx  cx  bcx  D a t a a r e e x p r e s s e d as mean + SEM; SHR = Spontaneously h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n ' i s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S.  1 d  a,b,c _ i g i f i t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. ' = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. s  X,  WKY  V  n  c a n  both animal  strain differences  animals excreted s i g n i f i c a n t l y in  the  urine  than  protein  fed animals  casein  fed  animals  tended  both  respectively.  the  Animals  a l o w e r Ca b a l a n c e M o r e o v e r , Ca  fed  and  fed  significantly  f e d the denatured  urine  was  protein  casein  than  diets,  exhibited  (Table 5.6).  (p<0.05) r e d u c e d  protein diets.  g r e a t e r (p<0.05)  Soy  similar for  soy p r o t e i n d i e t  counterparts  a b s o r p t i o n was  i n the  denatured  SHR  of calcium  t h e same d i e t .  observation  f e d the native  b a l a n c e was  calcium apparent  This  source.  lower l e v e l s  t o e x c r e t e l e s s Ca  native  than  animals  (p<0.05)  counterparts  counterparts.  fed  SHR and WKY  WKY  and d i e t a r y p r o t e i n  i n both  Conversely, i n these  same  animals . M a g n e s i u m i n t a k e was a l s o SHR and  WKY  animals  significantly  f e d the denatured  counterparts fed native protein  diets  (p<0.05)  reduced i n  protein diets  compared t o  (Table  data taken together w i t h the s i g n i f i c a n t l y Mg e x c r e t i o n o b s e r v e d (p<0.05) e n h a n c e d fed animals by  both  respectively  Soy p r o t e i n  lower  urinary  Mg  Moreover, animals significantly  protein  Mg e l i m i n a t i o n  animals  excretion  (p<0.05)  fecal  was  protein  influenced  s o u r c e and p r o t e i n h e a t d e n a t u r a t i o n , fed  f e d animals e x h i b i t e d  f e d the  intake  a significantly  a b s o r p t i o n o f Mg by d e n a t u r e d  i n SHR and WKY  5.7).  mineral  apparent  The  (p<0.05) r e d u c e d  i n these animals, i n d i c a t e d  (Table 5.8). U r i n a r y  dietary  5.7).  than  same  counterparts  urinary  proteins  fed  (Table  (p<0.05) casein.  experienced a  elimination  than c o u n t e r p a r t s f e d the n a t i v e p r o t e i n s . 156  diet  a significantly  heat denatured  greater  the  of  T h u s , Mg  this  TABLE 5.6  E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour c a l c i u m b a l a n c e . 1  Diet' SHR  Ca Intake (mg/ day)  WKY  SHR  Ca Balance" (mg)  C  68.75 + 1 . 2 5  ax  71.25 + 4 . 6 2  ax  33.46 + 2 . 5 1  S  66.25 + 5 . 5 4  ax  68.12 + 3 . 1 2  ax  28.60 + 3 . 1 1  DC 46.88 + 4 . 1 3  bx  50.00 + 2 . 0 4  bx  23.11 + 3 . 2 2  DS 45.00 + 2 . 2 8  bx  41.88 + 2 . 1 3  bx  19.92 + 1 . 5 5  a x  Ca A p p a r e n t A b s o r p t i o n  3  SHR  WKY  33.42 + 2 . 4 7  a x  WKY  41.11 + 1 . 4 7  ax  38.84 + 5 . 5 0  bx  27.15 + 3 . 0 7  a b x  40.00 + 2 . 1 2  a x  40.75 + 1 . 7 9  bx  bx  24.92 + 1 . 9 3  b c x  54.14 + 5 . 3 3  a x  57.60 + 3 . 5 6  ax  bx  18.54 + 1 . 0 2  53.96 + 4 . 5 8  a x  54.86 + 3 . 9 5  ax  a b x  cx  * Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . z. C = c a s e i n ; S = soy p r o t e i n I s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S. Ca Balance (mg) = Ca I n t a k e (mg/day) - U r i n a r y Ca (mg/day) - F e c a l Ca (mg/day). Ca Apparent A b s o r p t i o n (%) = {[Ca Intake (mg/day) - F e c a l Ca (mg / d a y ) ] / C a I n t a k e (mg/day)}x 100. 4  a,b,c _ i g i f i t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. S  x  y  n  c a n  TABLE 5.7  E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour magnesium e x c r e t i o n .  Diet^  Mg Intake SHR  1 d  a x  WKY  SHR  U r i n e Mg (mg/ day)  F e c a l Mg WKY  SHR  WKY  c  6.88 + 0 . 1 2  ax  7.12 + 0 . 4 6  ax  1.91 + 0 . 0 3  b x  1.18 + 0 . 0 8  by  1.49 + 0 . 1 0  b x  2.07 + 0 . 0 8  ay  s  6.62 + 0 . 5 5  ax  6.81 + 0 . 3 1  a x  0.58 + 0 . 0 6  d x  0.66 + 0 . 0 1  c x  2.29 + 0 . 0 3  ax  2.87 + 0 . 2 5  ax  DC 4.69 + 0 . 4 1  b x  5.00 + 0 . 2 0  bx  2.67 + 0 . 1 8  ax  2.94 + 0 . 1 9  ax  0.48 + 0 . 0 5  c x  0.64 + 0 . 0 5  bx  DS 4.50 + 0 . 2 3  bx  4.19 + 0 . 3 0  bx  1.35 + 0 . 1 2  cx  2.76 + 0 . 1 8  ay  0.66 + 0 . 0 5  c x  0.89 + 0 . 0 6  bx  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat denatured C; DS = h e a t d e n a t u r e d S. » b » c _ s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. v  TABLE 5.8  Diet* SHR  E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour magnesium b a l a n c e .  Mg Intake (mg/ day)  WKY  SHR  Mg Balance" (mg)  c  6.88 + 0 . 1 2  ax  7.12 + 0 . 4 6  ax  3.18 + 0 . 3 1  S  6.62 + 0 . 5 5  ax  6.81 + 0 . 3 1  a x  3.81 + 0 . 2 7  DC 4.69 + 0 . 4 1  bx  5.00 + 0 . 2 0  bx  DS 4.50 + 0 . 2 3  bx  4.19 + 0 . 3 0  bx  Mg Apparent A b s o r p t i o n (%) SHR WKY  3  WKY  4.24 + 0 . 1 9  a x  71.16 + 7 . 0 8  bx  66.41 + 4 . 4 4  ax  3.60 + 0 . 1 3  b x  67.76 + 2 . 5 2  bx  72.99 + 5 . 5 0  1.51 + 0 . 2 4  cx  1.37 + 0 . 1 1  d x  89.38 + 0 . 8 4  ax  86.07 + 1 . 4 3  2.45 + 0 . 1 9  b x  1.92 + 0 . 0 5  c x  87.43 + 1 . 6 7  ax  80.78 + 3 . 6 9  a b x  bx  a b x  ax  a b x  D a t a a r e e x p r e s s e d as mean + SEM; SHR = Spontaneously h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat denatured C; DS = heat d e n a t u r e d S. Mg B a l a n c e (mg) = Mg Intake (mg/day) - U r i n a r y Mg (mg/day) - F e c a l Mg (mg/day). Mg A p p a r e n t A b s o r p t i o n (%) = {[Mg Intake (mg/day) - F e c a l Mg (mg /day)]/Mg I n t a k e (mg/day)}x 100. b , c y  = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n columns. = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows.  balance fed  was s i g n i f i c a n t l y  the  denatured  comparison to a result,  casein  and  soy  treatment  was f o u n d t o e x i s t  interaction  significantly  diets  between  f o r Mg  (p<0.05)  respectively in  5.9).  (p<0.05) r e d u c e d o n l y protein diets,  (Table  animal  reduced  Fecal i n WKY  compared t o  the feed in  animals  of  P was  f e d denatured  counterparts  fed  of d i e t a r y protein urine  than  counterparts  P i n SHR and  higher  WKY  and s o y  significantly  significantly  dietary  treatment  i n f l u e n c e d by  SHR than  source  T h i s o b s e r v a t i o n was  f e d the  excreted  P in  similar  of d i e t a r y p r o t e i n s  same d i e t .  significant  For  significantly P balance  counterparts  was s i g n i f i c a n t l y  less  Soy  (p<0.05) r e d u c e d u r i n a r y e x c r e t i o n o f  counterparts. in  (p<0.05)  Heat d e n a t u r a t i o n  animals  animals  d i f f e r e n c e was n o t balance  or  significantly  fed casein.  animals.  WKY  the u r i n e than was  excreted  in a significantly  treatments,  denatured  p r o t e i n as w e l l as by p r o t e i n h e a t d e n a t u r a t i o n .  i n SHR and WKY resulted  differences,  U r i n a r y e l i m i n a t i o n o f P was  f e d animals  4)  the n a t i v e p r o t e i n  by  5.10).  and  c a s e i n and s o y  affected  strain  the  fed native casein  excretion animals  fed  A p p a r e n t a b s o r p t i o n o f P h o w e v e r , was n o t  (Table  strain  As  i n t a k e d a t a , and were  sources.  animal  5.8).  balance.  compared t o c o u n t e r p a r t s  (Table  animals  ( F ( 3 , 2 4 ) = 5 . 8 5 , p<0.004; A p p e n d i x T a b l e  Phosphorus i n t a k e s p a r a l l e l e d  protein diets  diets,  n a t i v e c a s e i n and s o y f e d a n i m a l s  a significant  p r o t e i n heat  (p<0.05) r e d u c e d i n SHR and WKY  fed  (Table  (p<0.05) more P i n i n soy  casein,  5.10).  (p<0.05) r e d u c e d i n SHR 160  a l l dietary  fed animals although  the  Furthermore, P and WKY  animals  TABLE 5.9 E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour phosphorus  Diet^  P Intake SHR  WKY  SHR  Urine P (mg/ day)  c  79.5 + 1.4  ax  82.4 + 5 . 3  ax  21.74 + 1.05  s  84.4 + 5.5  ax  84.2 + 3.9  ax  17.82 + 1.36  bx  57.8 + 2.4  bx  15.21  + 2.07  bx  51.8 + 2.6  bx  16.21  + 0.95  DC 55.6 + 3.8 DS 56.4 + 2.3  excretion . 1  Fecal P WKY  SHR  31.45 + 3.17  ay  23.61  22.14 + 0.81  bx  bx  29.65 + 1.75  bx  22.52 + 1.09  ax  abx  WKY  + 2.18  ax  30.02 + 2.41  ay  22.18 + 1.31  ax  27.54 + 2.07  ax  ay  23.31  + 2.19  ax  13.24 + 1.61  by  by  17.09 + 1.22  ax  15.28 + 2.01  bx  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . ^ C - c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S. 1  a,b,c _ i g i f i t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. . » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. S  x  y  n  c a n  TABLE 5.10  Diet' SHR  E f f e c t o f heat t r e a t i n g p r o t e i n on 24 hour phosphorus b a l a n c e . 1  P Intake (mg/ day) WKY  SHR  P Balance (mg)  c  79.5 + 1 . 4  ax  82.4 + 5 . 3  a x  34.12 + 3 . 5 4  s  84.4 + 5 . 5  a x  84.2 + 3 . 9  a x  44.38 + 4 . 3 9  DC 55.6 + 3 . 8  b x  57.8 + 2 . 4  b x  DS 56.4 + 2 . 3  b x  51.8 + 2 . 6  b x  P Apparent A b s o r p t i o n (%) SHR WKY  0  WKY  29.57 + 3 . 3 5  ax  68.40 + 3 . 0 0  ax  68.29 + 3 . 4 0  ax  a x  37.79 + 3 . 4 4  ax  72.52 + 2 . 2 4  ax  70.68 + 4 . 7 6  ax  26.75 + 3 . 2 5  b x  16.70 + 2 . 2 2  bx  72.49 + 3 . 4 8  ax  77.18 + 2 . 4 6  a x  22.97 + 1 . 8 7  bx  18.95 + 1 . 8 6  bx  69.75 + 1 . 4 4  ax  69.78 + 5 . 1 6  ax  a b x  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . ^ C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat denatured C; DS = h e a t d e n a t u r e d S. P B a l a n c e (mg) = P Intake (mg/day) - U r i n a r y P (mg/day) - F e c a l P (mg/day). P Apparent A b s o r p t i o n (%) = {[P Intake (mg/day) - F e c a l P (mg / d a y ) ] / P I n t a k e (mg/day)}x 100. 1  q  a x  » b » c _ s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n c o l u m n s . ' = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows. y  fed  the  denatured  protein  diets  compared t o t h o s e f e d n a t i v e  proteins . Taken t o g e t h e r , t h e f e e d indicate  that  intake  and  malabsorption  associated  dietary proteins.  The  a  likely  data  with  of  the n u t r i e n t  the e x t e n s i v e heat  p r o c e s s i n g of  longterm  result  deficient  a b s o r p t i o n of these a n i m a l s , r e s u l t e d  mineral balances of  of these minerals  While  calcium  and  mineral  elimination,  The  reduced  i n enhanced apparent magnesium,  source of d i e t a r y p r o t e i n  influence urinary  i n t a k e and  mineral balances  proteins.  animals r e s u l t e d for  mineral  i n reduced  when c o m p a r e d t o c o u n t e r p a r t s f e d n a t i v e  phosphorus.  balance  the m i n e r a l a b s o r p t i o n of animals f e d the denatured  p r o t e i n d i e t s was d e c r e a s e d ,  absorption  mineral  but  not  c o u l d be o b s e r v e d t o  these  effects  d i d not  disturb mineral balance. Bone P h y s i c a l  Parameters!  Femur a s h  weight  and d r y w e i g h t  were r e d u c e d  soy p r o t e i n  compared t o c o u n t e r p a r t s f e d c a s e i n ,  WKY  (Table 5.11).  animals  diets  exhibited  weights protein.  and  significantly  dry  weights  (p<0.05)  native proteins. WKY  femur  not  femur  to  fed native  those  significantly  d i e t a r y p r o t e i n s had  lengths  affected  163  ash  by  However,  significantly  compared t o c o u n t e r p a r t s f e d  The r e d u c t i o n i n o v e r a l l  animals f e d the denatured  protein  decreased  or source of d i e t a r y p r o t e i n .  f e d the heat denatured decreased  comparison  was  f o r b o t h SHR and  f e d the heat denatured  (p<0.05)  in  Femur bone l e n g t h  animal s t r a i n d i f f e r e n c e s animals  Animals  i n animals fed  bone  size  in  SHR and  TABLE 5.11  E f f e c t o f heat t r e a t i n g p r o t e i n on femur p h y s i c a l  Diet*  Ash Wt. (g) SHR  1 i  parameters . 1  Dry Wt. (g) WKY  SHR  Length (mm) WKY  SHR  c  0.237 + 0 . 0 0 6  ax  0.228 + 0 . 0 0 6  ax  0.387 + 0 . 0 1 0  ax  0.358 + 0 . 0 1 2  ax  30.6 + 0 . 5  a x  31.2 + 0 . 6  a x  s  0.206 + 0 . 0 0 6  bx  0.215 + 0 . 0 0 5  ax  0.360 + 0 . 0 0 9  bx  0.347 + 0 . 0 1 6  ax  30.5 + 0 . 2  a x  31.2 + 0 . 4  ax  DC 0.176 + 0 . 0 0 4  cx  0.167 + 0 . 0 0 7  bx  0.323 + 0 . 0 0 6  cx  0.292 + 0 . 0 1 3  bx  29.2 + 0 . 2  b x  29.0 + 0 . 5  b x  DS 0.121 + 0 . 0 0 3  dx  0.145 + 0 . 0 1 2  bx  0.235 + 0 . 0 0 7  dx  0.263 + 0 . 0 2 2  bx  25.6 + 0 . 4  C X  27.9 + 1 . 0  b v  D a t a a r e e x p r e s s e d as mean + SEM; SHR = Spontaneously h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S.  a,b,c,d _ i g i f i t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between treatment means i n rows. S  x  WKY  v  n  c a n  protein diets istics  was a  f e e d i n t a k e and FER  T h i s i s o b s e r v e d when f e m u r a s h w e i g h t  a per cent of f i n a l 0.006 %  Bone  of r e t a r d e d animal growth c h a r a c t e r -  as i n f l u e n c e d by t h e r e d u c e d  animals.  0.001  result  Final  body w e i g h t  body  weight  % F i n a l body w e i g h t  ( r a n g e 0.076 +.  f o r WKY;  and 0.091  i s expressed  0.001  as  0.093 +_  t o 0.101  +  f o r SHR).  Mineralization:  calcification  was  lower i n  counterparts i n  Moreover, icantly those  animals  SHR  native  animals  native proteins, b o t h SHR  and WKY  procedure energy  were b o t h  denatured  t h e work  reduced  when compared t o  significantly  parameters  in  SHR  h o w e v e r , was  femur  magnesium  (p<0.05)  reduced  as compared t o t h o s e f e d  required  t h e femur maximum b e n d i n g  from  5.13.  and  WKY  those  the Femur  was  common t o  to break  3-point  bending  bending  failure  animals f e d the denatured fed  the  only s i g n i f i c a n t  c a s e i n and s o y d i e t s .  energy  signif-  Parameters:  p r o t e i n d i e t s when compared t o  the denatured  same d i e t .  p r o t e i n d i e t s had  This observation  are presented i n Table  The d i f f e r e n c e  Bone  animals.  biomechanical  was  the  Similarly,  proteins  respectively.  Bone B i o m e c h a n i c a l Femur  fed  calcium contents sources.  5.12.  f e d animals than c a s e i n  animals  denatured  bone  protein  f e d the  WKY  the  (p<0.05) r e d u c e d fed  soy p r o t e i n  and  fed  c o n t e n t and Ca/P r a t i o in  to  + 0.001  Bone m i n e r a l i z a t i o n d a t a a r e s u m m a r i z e d i n T a b l e  fed  of these  native i n SHR  animals fed  Despite these d i f f e r e n c e s i n the femora of these a n i m a l s ,  s t r e s s was n o t s i g n i f i c a n t l y 165  proteins.  affected  TABLE 5.12  Diet  E f f e c t o f heat t r e a t i n g p r o t e i n on femur m i n e r a l i z a t i o n . 1  2  SHR  1  Ca (mg/bone)  WKY  SHR  Mg (mg/bone)  WKY  SHR  WKY  C  94.60 + 3 . 8 5  a x  92.20 + 2 . 9 1  ax  1.84 + 0 . 0 6  ax  1.92 + 0 . 0 6  ax  2.00 + 0 . 0 1  a x  2.04 + 0 . 0 2  S  78.75 + 3 . 2 1  b x  82.35 + 4 . 9 3  ax  1.62 + 0 . 0 7  ax  1.78 + 0 . 0 7  ax  1.97 + 0 . 0 5  a x  1.88 + 0 . 0 7  DC  64.60 + 4 . 0 3  cx  63.45 + 2 . 6 3  bx  1.32 + 0 . 0 6  bx  1.42 + 0 . 0 5  bx  1.77 + 0 . 1 4  ax  1.83 + 0 . 0 2  DS  51.20 + 4 . 2 6  dx  53.55 + 4 . 7 1  bx  1.14 + 0 . 1 3  bx  1.16 + 0 . 1 0  cx  1.82 + 0 . 0 4  ax  1.91 + 0 . 0 4  ax  a b x  bx  a b x  Data a r e e x p r e s s e d as mean + SEM; SHR = S p o n t a n e o u s l y h y p e r t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DC = heat d e n a t u r e d C; DS = heat d e n a t u r e d S.  a,b,c,d _ s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n columns. » = s i g n i f i c a n t (p<0.05) d i f f e r e n c e between t r e a t m e n t means i n rows.  x  Ca/P Ratio  y  E f f e c t o f h e a t t r e a t i n g p r o t e i n on biomechanical parameters .  TABLE 5.13  femur  1  Diet  1 2  2  Bending F a i l u r e Energy (x 10-2 J ) SHR WKY  +  X  68 . 94 + 3 .06  +  0 .41°  X  65 .82  +  1 • 45  5 .89  +  0 . 55  a  X  73 .22  +  2 .30  4 .24  +  0 . 57  a  X  66 .46  +  2.ll  9 .24  0 . 92 * 6 .65  s  6 . 12 + 0 .60»>*  6 .51  DC  5 . 72 +_ 1 .21«>*  DS  3 .09  a  0 .33°*  2  0 .85"  c  +  Maximum B e n d i n g S t r e s s (N/mm ) SHR WKY  D a t a a r e e x p r e s s e d as mean +_ SEM. C = c a s e i n ; S = soy p r o t e i n i s o l a t e ; DS = h e a t d e n a t u r e d S.  a  a  a  X  75 .31  +  4 . 95  a  '  X  78 .77  +  1 . 89  a  y  X  75 . 15 + 3 . 45 *  a  X  a  71 .09  +  1 . 71«y  DC = h e a t d e n a t u r e d C;  a , b , c , d = s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n c o l u m n s . *•y = s i g n i f i c a n t (p<0.05) d i f f e r e n c e b e t w e e n t r e a t m e n t means i n rows.  167  by d i e t a r y  treatment.  significantly  (p<0.05)  •SHR c o u n t e r p a r t s needed t o  The  greater  f e d t h e same  break the  in diet  femora of  may be due t o t h e g r e a t e r c o m p a r i s o n t o SHR  femur  final  maximum WKY  counterparts.  168  animals  indicating  these body  bending  animals. weight of  stress  was  when compared t o a  greater  This these  force  observation animals i n  Discussion Percival  and  h e a t damaged  Schneeman  casein diet  (1979)  experienced  i n t a k e and d i a r r h e a , s i m i l a r r e d u c e d body w e i g h t g a i n diets  may  protein  be  (Ikegami  This  ejt. a_l_. ,  by  result  1975 ;  that  weight  t o the r e s u l t s  and PER  explained  sources.  reported  animals  loss,  reduced  feed  reported  herein.  The  of animals  f e d the  the reduced n u t r i t i v e i s supported  Percival  value  by t h e work  and Schneeman,  secretion i n rats fed poorly digestible protein  Studies  have of  pancreatic  protein quality Percival  and  ibility,  in  protective  (ikegami  casein  a  al. .  in  a  be  1966;  have  pancreatic sources.  composition  and  i n f l u e n c e d by d i e t a r y Ikegami  et  a 1 . . 1975;  Thus, reduced p r o t e i n d i g e s t vitro  intestinal  on i n t e s t i n a l  reduced  enzyme  can  1979).  greater  effect  residue,  enzymes  suppression  study  of  h e r e i n , may and  have  therefore, a  from a u t o d i g e s t i o n . further  pancreatic  This output  e_t_ a_l_. , 1 9 7 5 ) . product  of  pancreatic  phosphopeptides  casein. sequester the  (Rebound e t Schneeman,  result  A  protein  as d e m o n s t r a t e d by t h e i n  resulted  would  pancreatic  others  1979) who  exocrine  that  and  DS  of the  of  increased  shown  contents,  DC and  reported  turnover  intestinal  fed a  These calcium  enzymatic  (CPP),  peptides  proteolysis,  r e l e a s e d by t r y p t i c  have  been  extensively  ions w i t h i n the i n t e s t i n a l  intraluminal soluble  calcium  and S u z u k i ,  1974; Lee  observations  were e x t e n d e d by t h e f i n d i n g s 169  e_t a l . . ,  d i g e s t i o n of reported  to  m i l i e u , to increase  concentration  1972; N a i t o  are the  (Naito  et a l . .  1980; 1 9 8 3 ) .  These  of experiments  3 and  4,  which  showed e n h a n c e d p a r a c e l l u l a r c a l c i u m  f e d c a s e i n and have  also  lysine,  shown  enhance  (Wasserman  ejb.  absorption of  can  protein  dairy The  CPP  containing diets, that  amino  calcium a_l.. ,  be  proteins, could  a b s e n c e o f an intestinal  calcium  is  further  evidence  of  damaged p r o t e i n s  the  Schneeman ( 1 9 7 9 )  heat  substantial  casein  diets  residues  bind m i c e l l a r c o l l o i d a l  of  the  in  on  calcium  intestinal calcium  Weeks and processing  of  reduced i l e a l  or  amino a c i d the  dietary calcium  lumen.  i n denatured  would  release  similarly  phosphate.  The  casein  proteins  at  Dephosphorylation  of  capacity for calcium  to  1983),  which i n  p o t e n t i a l of  u n c e r t a i n , given  calcium of  4 5  Ca  adverse  absorption. in  170  the  normally  the  turn  would  peptides  subsequent e f f e c t that t h i s would is  CPP  been shown t o r e s u l t i n  calcium  et a1..  of  decreased.  Wright, 1934), which  reduced b i n d i n g  diet.  digestion  and  solubilizing  absorption  the  CPP  animals  of t h i s  that the  fed  (Howat  a  on  be has  ( 1 9 8 5 ) w h i c h showed no milk  casein  thus,  of  bioavailability.  poor d i g e s t i b i l i t y  milk  L-  profiles  digestibility  the  bioavailability King  and  small i n t e s t i n e  of  casein phosphopeptides (Sato decrease the  peptide  i s impaired;  treatment  resulted  workers  since p a r a c e l l u l a r calcium  have s u g g e s t e d  dephosphorylation  phosphoseryl  Other  L-arginine  the  affecting  absorption  of heat  caseins  the  influence  and  Further,  Thus,  factors  Percival  denatured  from  in rats  o b s e r v e d e n h a n c i n g e f f e c t of p o s t - d i g e s t i o n  on  from  s u c h as  absorption  a f f e c t e d by  digestion;  respectively.  acids,  1956).  absorption  rats fed  the  in  have  f i n d i n g s of  e f f e c t s of  thermal  A l t e r n a t i v e l y , the the  heat  denatured  protein diets time.  may be e x p l a i n e d by a d e c r e a s e d  An i n c r e a s e i n t h e t r a n s i t  also result  i n the  seen e a r l i e r  i n experiment  Despite the heat  observed  proteins  animals  maintained  h e r e i n , plasma  system,  which  calcium  and  w i t h the  Ca  relatively  reducing  from  o n l y on t h e s u r f a c e  of  levels  observed  p r o t e i n s are l i k e l y content, resulting former  important necessarily  to  bone.  loop.  that  animals to  a predictive  I n an a c u t e that  loss  bone  (Greger,  mechanism  may  Ca  4 5  a  index of c a l c i u m  C o n s i d e r i n g the  4 5  4 5  the  similar  denatured  Ca  Ca  in  plasma  and n a t i v e bone  activities  such Ca  4 5  w o u l d be d e p o s i t e d  decrease  study  r e l a t e d to  s t u d y t o measure  fed  femoral  171  endocrine  mobilizing  was i n v e r s e l y  i n the g r e a t e r femoral  emphasize  l e v e l s are  calcium  Therefore,  attributable  group of a n i m a l s .  calcium  t o the femora i n animals  ileal  expected  in  t h e 10 week  c a l c i u m by  Ca  a t t r i b u t e d to  over  t h i s homeostatic 4 5  animals fed  parathyroid  s h o r t time p r o t o c o l used i n t h i s i t is  of  be  diets  urinary  of  the  femoral d e p o s i t i o n ,  calcium  may  the  p r o t e i n d i e t s , which  absorption  would  p r o t e i n and m i n e r a l  plasma  by  t r a n s l o c a t i o n of  fed the denatured 4 5  constant  A possible indication  be s e e n  the  example,  regulates extracellular  reserves  1988).  result  the r e s p e c t i v e  For  relatively  malabsorption  This  to  experimental period.  contents  s i g n s o f n u t r i e n t m a l a b s o r p t i o n , as  nutrient  l e v e l s were n o t a f f e c t e d . a d a p t a t i o n by  r a t e of l u m i n a l  transit  1.  the observed  denatured  intestinal  utilization.  Ca  of the  as t h i s ,  activity  4 0  i t is is  not  Alternatively, extracellular calcium not  calcium  excretion  observed  calcium  calcium  balance  reduced  is  fed  s t u d y , as  as  tubular  calcium  denatured  w e l l as  deficiency  reabsorption  of  excretion.  and  calcium  absorption  and  magnesium i n  The  increased animals fed  excretion  in  consequently, between  indicates urinary  that  the  excretion  denatured  extracellular  have i n d i c a t e d  altered calcium  calcium  (Aoki  S c h e d l .et. aj_. ,  1988).  absorption  was  not  confirming  previous  In  different  exhibited  respectively.  positive effect  metabolism,  therefore,  decreased  tubular  increased  urinary  magnesium b a l a n c e  and  influenced  that  intestinal  occurred  present  b e t w e e n SHR  r e s u l t s from 172  our  for  soy  diets.  by  the  As  inter-  treatment.  the  SHR  and  animal  model  distribution  ejt JLL- , 1976 ; M c C a r r o n .et. a_L. , the  can  negative  diets  also  casein  p r o t e i n heat  b o t h an  and  a decreased  a c t i o n b e t w e e n a n i m a l s t r a i n and  exhibits  fecal  denatured  (Zemel, 1985);  also  studies  not  was  i t i s noteworthy  protein  result  and  the  Thus,  a r e s u l t , magnesium b a l a n c e was  Previous  urinary  effect  animals fed  a direct  reabsorption  similarity  data,  this  phosphorus balances,  would  calcium  reduced  u r i n a r y , but  in  al_. , 1979).  heat  calcium  deficiency,  known t h a t a p h o s p h o r u s d e f i c i e n c y  urinary  the  by  However,  have shown t h a t p h o s p h o r u s has  phosphorus  data  present  ( R a d e r et_  calcium,  renal  calcium  It  increased  animals  calcium  homeostasis i s regulated  the  diets. in  on  of  (Agus e t a l . . 1 9 8 1 ) .  in  result  Studies  conditions  l o s s e s , were a c t u a l l y i n c r e a s e d  protein  that  under  study, and  WKY  laboratory  ileal  of  1981;  calcium  counterparts, (Kitts  e_t_ al_. ,  1989). SHR  The r e l a t i v e  animals  results  of  metabolism decreased t o WKY  was  amount o f  g r e a t e r than  experiment of  3.  Similarly,  overall  calcium  have  been  respectively  in  overall  suggest  study  absorption, in situ  detected  to  strains.  animals  is  urinary  et  the  was n o t  t h e young  i n young  1988).  SHR  to  was  1988). in  The  SHR  greater  i n d i c a t i v e o f an  c o n t r o l s (McCarron et  Taken  together,  the  SHR.  The r e s u l t s  technique,  hr balance paracellular  to  either  play  altered  SHR  calcium  metabolism.  method i n b o t h calcium  significant,  173  before  paracellular  are  data  partic-  early,  d i e t a r y p r o t e i n source  appear  these  occur  differences in  loop  a  and u n c h a n g e d ,  c a l c i u m m e t a b o l i s m , and  in  with  ileal  t h e 24  Therefore,  increased  calcium handling,  associated  al. .  a g e - m a t c h e d WKY  hypertension  ligated  both  et  balance  e x c r e t i o n of calcium balance  a1. .  (Lau  balance  employing longer  calcium  be  i n d i c a t e that  using  altered  i n older  extracellular  present  an  c a l c i u m e x c r e t i o n and c a l c i u m b a l a n c e  abnormalities  of  for  to the  Alter-  compared  development  similar  1986).  reported  that  SHR  calcium  workers  retention  a l . . 1 9 8 1 ; J o n e s ejt. a l . . ,  the  in  positive  balance  natively, urinary  ularly  calcium  decreased  more  abnormal r e n a l c a l c i u m  SHR  evidence  ( L a u e t a 1. . 1986 ; J o n e s  positive  counterparts,  Further  other  s t u d i e s , have r e p o r t e d a  animals  t r a n s l o c a t e d t o the femora of  c a l c i u m e x c r e t i o n a t 10 weeks o f a g e , compared  c o u n t e r p a r t s ; however,  an  Ca  i n WKY  extracellular  urinary  affected.  with  4 5  the  of the calcium  m e a s u r e d by  not n e c e s s a r i l y t h e SHR  absorption  or s p e c i f i c  and  WKY  does n o t  role  i n the  In t h e p r e s e n t soy  diets  exhibited  parameters. considered al . .  to  be  1983).  bone  and  browning  protein  digestion  or presence  length  reaction  observed  cortical  1969;  products  and  made  e_t  this  study.  Decreased  hypercalcemic calcium  in  P  deficiency  response  levels  in  the observations  i n P d e f i c i e n c y may have n o r m a l i z e d  plasma  the  denatured  fed  animals,  Alternatively,  which plasma  have been r e p o r t e d t o be i n c r e a s e d due t o e n h a n c e d  1975).  the  study,  present  by m a l a b s o r p t i o n  P  i n animals  respectively,  resulted  deficiency  femur  bending  fed  ejt. a l . .  i n C a , Mg, and P  the denatured  i n decreased  failure 174  (Castillo  deficiencies  magnesium c o n t e n t s and l o w Ca/P r a t i o s . biomechanical  a1. .  protein  hypocalcemic.  in  soy d i e t s ,  et  development of This  mobilization  caused  deficient  d e p o s i t i o n of c a l c i u m to the  (Rader  bone m i n e r a l In  mineralization  1979).  o t h e r w i s e w o u l d have been calcium levels  the  contributed to  i n phosphorus  s k e l e t o n has been shown t o c o n t r i b u t e t o t h e a c u t e hypercalcemia  Thus,  c a s e i n and s o y  bone  supports  to the  e f f e c t on  1984).  Also,  which  (Garn e t al.. , 1983;  their  the denatured  and s i z e .  ( B r u i n et a l . . 1975),  in  and  Sjodin,  herein with  bone g r o w t h  thickness are  has been a t t r i b u t e d  been r e p o r t e d t o be a c u t e l y i n h i b i t e d  animals  Crosby  of M a i l l a r d Reaction products, l i k e l y  the decreased  c a s e i n and  of p r o t e i n m a l n u t r i t i o n  Berridge,  (Oste  the denatured  and  Protein malabsorption  of  malabsorption  fed  bone m i n e r a l i z a t i o n and p h y s i c a l  symptomatic  Adams  presence  has  decreased  Decreased  1964;  Parfitt,  study, animals  c a s e i n and  bone c a l c i u m and  Therefore, a decrease i n energy,  but  n o t maximum  bending  s t r e s s , was  c a u s e d by r e d u c e d bone g r o w t h  components, secondary fed  the  heat  to n u t r i e n t  denatured  biomechanical parameters mineral  proteins  malabsorption. exhibited  as i n f l u e n c e d  deficiencies.  175  and bone m a t r i x Thus, a n i m a l s  decreased  femur  by r e d u c e d body g r o w t h  and  Experiment  6  C a l c i u m b a l a n c e and femur b i o m e c h a n i c s i n r a t s f e d c a s e i n and soy d i e t s v a r y i n g i n p r o t e i n l e v e l . Introduction In a d d i t i o n to dietary protein intake  shown  levels to  Linkswiler  et  affect  Adams and  Berridge,  porosis.  be  production well  as  of  dietary  subsequent  using  protein  (Allen  et  calcium  structure  of  al_. ,  et  and  intake,  while  peptides  utilization  ileal  limit  their to  1964; 1983),  prone to  osteo-  were t o  calcium  mineral  biomechanical strength. prone  Thus,  Parfitt,  l e v e l , w h i c h may  for  1979 ;  s i z e (Garn et a1..  objectives  on  al..  and  been r e p o r t e d  to i n d i v i d u a l s  protein  been  1981).  a_l. , 1983;  of  protein  excretion  h o m e o s t a s i s may  C r o s b y et.  osteoporosis  quality  level  calcium  balance  f o r concern  of b i o a c t i v e  performed  urinary  p r e s e n t s t u d y , the  m i n e r a l i z a t i o n and  (SHR)  1969;  cause  In the  effect  bone  and  p h o s p h o r u s u n c h a n g e d , has  Schuette  with  the  H o w e v e r , p r o t e i n d e f i c i e n c y has  adversely  w h i c h may  1981;  concerned  intake.  and  calcium  aj_. ,  source  Increasing  increased  negative  individuals protein  calcium in  of  bioavailability,  considered.  of  result  eventually,  importance  to c a l c i u m  must a l s o be  leaving  the  the  determine  influence  the  absorption  as  b a l a n c e and  These  spontaneously  studies  bone were  hypertensive  rats.  Materials  and  A n i m a l s and  Methods Diets:  Four-week o l d (Charles  male  spontaneously  R i v e r , M o n t r e a l , PQ)  hypertensive  were d i v i d e d 176  i n t o four  (SHR)  rats  experimental  dietary  groups  (6  20%  casein,  included  animals  C l e v e l a n d , OH) and  6%  respectively  (Table  calcium level  (0.5%).  protein  isolate  soy  per  protein  casein 6.1).  group).  Dietary  isolate  and  soy  A l l diets  treatments  (ICN B i o c h e m i c a l s ,  protein  isolate  contained  an  diets, adequate  The a n i m a l s f e d t h e 2 0 % c a s e i n and 2 0 % s o y  d i e t s were  t h e same  as i n e x p e r i m e n t s  4 and 5.  F e e d i n g and m e a l - f e e d i n g t r a i n i n g were as p r e v i o u s l y d e s c r i b e d i n experiment A  3.  24  described  hour  i n experiment  Intestinal described  balance  calcium  i n experiment  study  was  performed,  as  previously  4, when a n i m a l s were 10 weeks o f a g e . absorption  was  measured  as p r e v i o u s l y  1 when a n i m a l s were 14 weeks o f a g e .  Analyses: Analyses  of  intestinal  lumen,  blood plasma,  f e c e s and femur m i n e r a l i z a t i o n and b i o m e c h a n i c s p r e v i o u s l y d e s c r i b e d i n experiment Statistical All  the range  source  were p e r f o r m e d  as  4.  Analyses:  d a t a a r e e x p r e s s e d as mean +_ SEM.  were t e s t e d  feed, urine,  f o r by one-way ANOVA. of  the  difference  t e s t a t a p<0.05 l e v e l  Where  Treatment  differences d i d occur,  was i d e n t i f i e d  of s i g n i f i c a n c e .  177  differences  using a multiple  TABLE 6.1  Composition  of experimental  Casein D i e t a r y Component ( g / 1 0 0 g)  Casein* Soy p r o t e i n  isolate*  D.L. m e t h i o n i n e * Cornstarch* Sucrose Fibre* Vegetable o i l  20%  20.0 —  0.3 15.0 48 .77 7.0 5.0  diets  fed to animals.  Soy D i e t s  Diets 6%  6.0 —  0.3 15.0 60 .0 7.0 5.0  20%  6%  20.0  6.0  0.3 15.0 48 .77 7.0 5.0  0.3 15.0 60 .0 7.0 5.0  Ca f r e e m i n e r a l m i x t u r e * Vitamin mixture* Choline b i t a r t r a t e *  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  3.5 1.0 0.2  Calcium  1 . 17  1 . 17  1 . 17  1 . 17  carbonate**  * I C N B i o c h e m i c a l s , I n c . , C l e v e l a n d , OH. * U n i t e d S t a t e s B i o c h e m i c a l Co., C l e v e l a n d , * * BDH C h e m i c a l s , T o r o n t o , ON.  178  OH.  Results Both to  t h e s o u r c e and l e v e l  have  an  effect  ratios  (FER; T a b l e  lower  (p<0.05)  animals. casein  final  6.2).  final  body  animals.  intake  weight  was a l s o  observed  were n o t  protein  intake  h o w e v e r , due t o a Animals fed  (p<0.05) d e c r e a s e d  and FER when  l e v e l s were  4.70 +.  calcium  decreased  was  compared  to  increased protein  to  final  compared t o c o u n t e r -  level  m g / l o o p ) and dpm/  4.91 +_  phosphorus  in fed  45  40  Ca  There  content  4 0  Ca)  were  i n the d i e t .  influenced  protein  level  as  similar  to  that  4 5  Ca  by  f e d animals absorbed  20%  protein  no  total protein  (Table 6.3).  effect  (p<0.05)  of d i e t a r y  2.16 +_  0.20  t o 2.46 +_ 0.31 to  4.64 +.  b o t h n o t a f f e c t e d by d i e t a r y  protein  ( r a n g e 3.39 +_ 0.44  Absorption  dietary  the  Plasma  Mg, Na o r K ( T a b l e 6 . 3 ) .  of  protein  a b s o r p t i o n from  from  g/dL).  treat-  a n i m a l s f e d t h e 6%  was  (range  Ca s p e c i f i c a c t i v i t y  mg  0.16  by d i e t a r y  l e v e l s were s i g n i f i c a n t l y  o r s o u r c e , on p l a s m a  source or l e v e l was  not a f f e c t e d  (p<0.05)  i n these animals.  Intestinal  protein  0.32  counterparts  C o n v e r s e l y , plasma  loop  6% s o y v e r s u s  o f t h e s o y d i e t s compared t o c a s e i n .  ments ( r a n g e  0.32  i n the  f e d 20% p r o t e i n d i e t s . Plasma  diets  exhibited a  and FER t h a n 2 0 % c a s e i n f e d  These r e s u l t s  body w e i g h t , d r y m a t t e r  found  and f e e d e f f i c i e n c y  f e d 20% soy p r o t e i n  t h e 6% p r o t e i n d i e t s had a s i g n i f i c a n t l y  parts  i n t h e d i e t , were  body w e i g h t s  Animals  A s i m i l a r trend  fed  reduced  on  of p r o t e i n  diets  significantly  Ca  from the i l e a l  source  t h e 20%  6% p r o t e i n  179  4 5  moreso, than  protein  diets  was  (Figure  6.1).  Soy  (p<0.05) l e s s  4 5  Ca  TABLE 6.2  Effect  of p r o t e i n  Diet  I n i t i a l body wt. (g)  Final wt.  2  20% p r o t e i n Casein 76.7 +_ 2 . 0 Soy 78.7 +_ 2.2* 6% p r o t e i n Casein 81.6 +_ 1.7* Soy 88.6 +_ 2.9* Data a r e e x p r e s s e d 2 5 weeks o f a g e . 3 14 weeks o f a g e .  1  level  3  a  on d i e t  body (g)  efficiency  Dry M a t t e r Intake (g)  1  Feed  Efficiency Ratio  259.3 + 8.4* 225 .2 + 5 .2«>  867 861  + 23* + 14*  0.211 ± 0.004* 0.170 + 0.004>>  187.0 159.0  659 733  + 14»> + 31b  0.160 + 0.004b 0.097 + 0.004°  + 8.2« + 6.3°  as mean +_ SEM.  Means s h a r i n g t h e same l e t t e r w i t h i n a c o l u m n a r e n o t s i g n i f i c a n t l y d i f f e r e n t p<0.05.  180  TABLE 6.3  Diet  E f f e c t o f p r o t e i n l e v e l on plasma m i n e r a l s .  Ca  Ca^  Mg  Na  K  P  (mg/dL)  20% p r o t e i n Casein 9.4 + 0 . 2 Soy 9.2 + 0.4 7.2 + 0 . 8 8.5 + 0 . 4  a a  b a D  5.1 + 0 . 1 4.9 + 0 . 1 4.2 + 0 . 3 4.8 + 0 . 2  a a  a a  1.9 + 0 . 1 1.9 + 0 . 1 2.4 + 0 . 1 2.7 + 0 . 2  a a  a a  389 + 1 6 424 + 1 2 352 + 6 346 + 1 0  a a  a a  15.5 + 1 . 9 19.2 + 4 . 6 32.2 + 2 . 8 30.8 + 2 . 6  a a  a a  7.1 + 0 . 8 7.1 + 0 . 6  b  12.8 + 1.3 12.5 + 1.4  a  b  a  Data a r e e x p r e s s e d as mean + SEM. C a ^ = [ ( 6 Ca - P/3)/(P + 6 ) ] ; where C a S Ca = mg/dL; P = p r o t e i n g/dL. 1  +  +  Means s h a r i n g t h e same l e t t e r w i t h i n a column a r e not s i g n i f i c a n t l y d i f f e r e n t a t p<0.05.  50  20% C  6% C  20% S  6% S  Experimental Diets Fig-  6.1  I n t e s t i n a l a b s o r p t i o n of * C a from i l e a l loop of r a t s f e d 2 0 % and 6% c a s e i n and s o y p r o t e i n i s o l a t e d i e t s , r e s p e c t i v e l y . * d e n o t e s s i g n i f i c a n t (p<0 05) d i f f e r e n c e . 5  than c a s e i n f e d c o u n t e r p a r t s  at  levels.  a b s o r p t i o n data d i d not correspond  The i n t e s t i n a l  4 5  Ca  both  the  to the subsequent d e p o s i t i o n of r a d i o l a b e l 6.2).  Although  the  amount  f e m o r a was n o t d i f f e r e n t groups, activity  animals  of  4 5  Ca  between t h e  f e d soy  20%  to the  activity  2 0 % and  protein diets  and  6% p r o t e i n  femur  (Figure  measured i n the  6% d i e t a r y  protein  had a g r e a t e r femur  4 5  Ca  t h a n c a s e i n f e d c o u n t e r p a r t s , when e x p r e s s e d as p e r c e n t  d o s e o f 45Ca i n t h e f e m u r . Mineral Balance The  24  Study:  hour  T a b l e s 6.4 t o 6.6. significantly (Table  balance  study data are presented i n  C a l c i u m i n t a k e and  different  6.4).  (p<0.05)  mineral  in  calcium  excretion  animals  fed  the  comparison t o c o u n t e r p a r t s f e d 20% p r o t e i n a b s o r p t i o n of  Ca f r o m  indicating  was s i m i l a r  that a  greater  (Table 6.5).  intake  fed  amounts o f  6%  diets.  proportion  Mg i n  The  diets i n apparent  was  not  excreted  differences,  d i e t a r y treatment of  absorbed  groups  c a l c i u m was  diets. affected  s o u r c e and t h e l e v e l  animals  groups  significantly  protein  by  dietary  However, f e c a l Mg e x c r e t i o n was  dietary protein protein  was  Despite these  among t h e  r e t a i n e d by a n i m a l s f e d 6% p r o t e i n Magnesium  were n o t  t h e 6% p r o t e i n d i e t s was g r e a t e r (p<0.05)  than t h a t from t h e 20% p r o t e i n d i e t s . calcium balance  excretion  b e t w e e n 6% and 2 0 % d i e t a r y p r o t e i n  Urinary  decreased  fecal  i n f l u e n c e d by b o t h  of p r o t e i n  significantly  treatment  i n the d i e t . (p<0.05)  Soy  greater  the f e c e s than c o u n t e r p a r t s f e d c a s e i n a t both  t h e 2 0 % and 6% p r o t e i n  levels,  respectively. 183  This indicated a  20% C  6%C  20% S  Experimental Diets Fig.  6.2  D e p o s i t i o n o f absorbed * C a t o t h e femora o f r a t 20% and 6% casein and s o y p r o t e i n isolate respectively. 5  TABLE 6.4  Diet  E f f e c t o f p r o t e i n l e v e l on 24 h r . c a l c i u m b a l a n c e . 1  Ca Intake (mg/day)  20% p r o t e i n Casein 68.75 + 1.25 Soy 66.25 + 5.54 oo  6% p r o t e i n Casein 61.25 + 4 . 2 7 Soy 76.88 + 2 . 7 7  U r i n e Ca (mg/day)  a a  a a  1.11 + 0.14 0.94 + 0 . 0 4  . Ca B a l a n c e ^ (mg)  F e c a l Ca (mg/day)  a a  0.45 + 0.04? 0.54 + 0.06 b  38.95 + 3.84 39.88 + 1.46  a  31.69 + 0.71 38.30 + 2.28  a  a  a  33.46 + 2 . 5 1 28.60 + 3 . 1 1 33.35 + 3 . 5 2 36.74 + 2 . 0 3  Ca A p p a r e n t Absorption" (%) 3  a a  a a  41.11 + 1.47° 40.00 + 2 . 1 2 b  52.62 + 4.26 . 49.23 + 1 . 1 7 a  ab  D a t a a r e e x p r e s s e d as mean + SEM. ^ Ca B a l a n c e (mg) = Ca Intake (mg/day) - U r i n a r y Ca (mg/day) - F e c a l Ca (mg/day). Ca A p p a r e n t A b s o r p t i o n (%) = {[Ca Intake (mg/day) - F e c a l Ca(mg / d a y ) ] / C a Intake (mg/day)} x 100. 1  6  Means s h a r i n g the same l e t t e r w i t h i n a column a r e not s i g n i f i c a n t l y d i f f e r e n t a t p<0.05.  TABLE 6.5  Diet  co c*  E f f e c t o f p r o t e i n l e v e l on 24 h r . magnesium  Mg I n t a k e (mg/day)  20% p r o t e i n Casein Soy  6.88 + 0 . 1 2 6.62 + 0 . 5 5  6% p r o t e i n Casein Soy  6.12 + 0 . 4 3 7.69 + 0.28  U r i n e Mg (mg/day)  a a  a a  1.91 + 0 . 0 3 0.58 + 0.06 2.08 + 0.18 0.75 + 0.07  balance . 1  F e c a l Mg (mg/day)  a b  a b  1.49 + 0 . 1 0 2.29 + 0 . 0 3  Mg A p p a r e n t Absorption (%)  Mg B a l a n c e ^ (mg)  b a  0.97 + 0.06?; 1.76 + 0 . 1 6 b  3.18 + 0 . 3 1 3.81 + 0 . 2 7 3.82 + 0.28 5.38 + 0.38  13  b b  b a  71.16 + 7 . 0 8 67.76 + 2 . 5 2 83.69 + 2 . 2 0 77.53 + 2 . 8 5  a a  a a  Data a r e e x p r e s s e d as mean + SEM. ^ Mg B a l a n c e (mg) = Mg I n t a k e (mg/day) - U r i n a r y Mg (mg/day) - F e c a l Mg (mg/day). Mg Apparent A b s o r p t i o n (%) = {[Mg Intake (mg/day) - F e c a l Mg (mg /day)]/Mg I n t a k e (mg/day)} x 100. 1  d  Means s h a r i n g t h e same l e t t e r w i t h i n a column a r e n o t s i g n i f i c a n t l y d i f f e r e n t a t p<0.05.  reduced,  but not s i g n i f i c a n t ,  protein  diets.  excreted  l e s s Mg  diets.  Only  higher  animals  fed  protein.  As  casein;  protein  fed  groups.  in  casein  fed  animals  f e d the  a  similar  result  a n i m a l s however. of  phosphorus  Urinary  was  in  The  For example,  comparison  a  protein were  result  those  casein  than  not  was  f e d soy  diet excreted  observed  different  animals  fed  the  6%  Furthermore,  less urinary  source  between  and  animals  decreased  fed in  t o t h o s e f e d 20%  signifdiets  f e d the  was  therefore i n f l u -  respective  level  i n the  retained  Also,  more  phosphorus  a n i m a l s f e d 6% p r o t e i n d i e t s i n  protein.  187  dietary  phosphorus than c a s e i n  counterparts. the  soy  apparent  protein  a n i m a l s f e d soy p r o t e i n d i e t s  casein  with  h o w e v e r , was  phosphorus balance  e n c e d by b o t h t h e p r o t e i n  was  than  elimination  diets excreted  counterparts.  was  not  soy p r o t e i n  balance  6%  This  Despite this difference,  phosphorus  (p<0.05) r e d u c e d  phosphorus  protein  since diets  6.6).  animals,  t h o s e f e d 20% p r o t e i n .  diet.  diets  exhibited  c o n t e n t w i t h t h e 6%  (Table  compared t o  fed  soy p r o t e i n d i e t  soy  (p<0.05) l e s s p h o s p h o r u s i n f e c e s t h a n c o u n t e r p a r t s  f e d 20%  absorption  protein t h e 20%  phosphorus i n t a k e ,  mineral  a result,  significantly  6%  counterparts fed t h e 6%  reduced  not b a l a n c e d f o r t h i s moreso  the  from  balance.  in a  apparent  a b s o r p t i o n o f Mg fed  reduction in dietary protein  resulted  icantly  animals  i n feces than  (p<0.05) Mg  The diets  Further,  apparent  TABLE 6.6  E f f e c t o f p r o t e i n l e v e l on 24 h r . phosphorus b a l a n c e  P Intake (mg/day)  Urine P (mg/day)  20% p r o t e i n Casein Soy  79.48 + 1.44 84.38 + 5.48  21.74 + 1.05 17.82 + 1.36  6% p r o t e i n Casein Soy  58.12 + 2.06° 72.88 + 2 . 4 5  Diet  a  13.45 + 1.76 12.52 + 1.01  Fecal P (mg/day)  a a  1  23.61 + 2 . 1 8 22.18 + 1.31  P Balance' (mg)  a a  16.26 + 1.45° 26.76 + 1.79 a  P Apparent Absorption 3  (*)  34.12 + 3.54 ab 44.38 + 4.39 a  68.40 + 3.00 72.52 + 2.24  28.41 + 2.89 33.61 + 2.71 ab  70.23 + 3.56 62.54 + 3 . 5 0  c £  a a  Data a r e e x p r e s s e d as mean + SEM. ^ P B a l a n c e (mg) = P Intake (mg/day) - U r i n a r y P (mg/day) - F e c a l P (mg/day). P A p p a r e n t A b s o r p t i o n {%) = {[P Intake (mg/day) - F e c a l P (mg / d a y ) ] / P Intake (mg/day)} x 100. 1  J  Means s h a r i n g t h e same l e t t e r w i t h i n a column a r e not s i g n i f i c a n t l y d i f f e r e n t a t p<0.05.  Bone M i n e r a l i z a t i o n : Femur  ash  significantly of p r o t e i n had  weight  affected  intake  mineralization  by b o t h  dietary protein  (Table 6.7).  significantly  calcium  and  (p<0.05)  Animals  protein.  Femur Ca/P  femur  s o u r c e and  level  protein  ash  i n comparison  r a t i o was  were  f e d t h e 6%  decreased  and magnesium c o n t e n t s ,  parameters  diets  w e i g h t s , and  t o those  f e d 20%  n o t a f f e c t e d by d i e t a r y  protein  level . Bone P h y s i c a l and B i o m e c h a n i c a l Femur d r y decreased fed  in  weight  animals fed  20% p r o t e i n d i e t s  s i z e observed retarded reduced  and  Parameters:  length  (Table 6.8).  protein  growth intake  ( r a n g e 0.087 +. 0.003 no d i f f e r e n c e  6.8).  characteristics of these animals.  as  influenced  i n bone  source  per cent  of f i n a l  t o 0.092 ±_ 0.002 % F i n a l body w e i g h t )  showed  as w e l l  had l o w e r  were  influenced  as t h e  protein  femur b e n d i n g  fed counterparts. had s i g n i f i c a n t l y  e n e r g i e s compared t o  maximum b e n d i n g  when  weight  by level  A t b o t h t h e 2 0 % and 6% d i e t a r y p r o t e i n  protein diets  by t h e  T h i s was o b s e r v e d  between l e v e l s of d i e t a r y p r o t e i n  those of casein  failure  The o v e r a l l r e d u c t i o n  body  biomechanics  soy p r o t e i n  (p<0.05)  t h e 6% p r o t e i n d i e t s compared t o t h o s e  femur a s h w e i g h t , e x p r e s s e d as a  protein  significantly  i n a n i m a l s f e d t h e 6% p r o t e i n d i e t s was a r e s u l t o f  animal  Bone  were  failure  intake. both  the  i n the d i e t  energy  (p<0.05) d e c r e a s e d  s t r e s s was r e d u c e d 189  protein  (Table  l e v e l s , animals fed  Moreover, animals  20%  dietary  fed  values  than  f e d t h e 6%  femur  bending  animals.  Femur  i n 6% s o y p r o t e i n  fed animals  moreso t h a n 6% c a s e i n f e d a n i m a l s when compared t o t h e r e s p e c t i v e 20% p r o t e i n f e d a n i m a l s .  190  TABLE 6.7  Effect  of p r o t e i n  Diet  Ash wt. (g)  level  on femur  Ca (mg/bone)  mineralization . 1  Ca/P ratio  Mg (mg/bone)  20% p r o t e i n C a s e i n 0.237 +. 0.006* Soy 0.206 +_ 0.006«>  94.60 +_ 3.85* 78 . 75 +. 3.21«>  2.00 ± 0.01* 1.97 +. 0.05*  1.84 +_ 0.06* 1.62 + 0.07*  6% p r o t e i n C a s e i n 0.158 + 0.004° Soy 0.146 +. 0.008"  63.90 +_ 2.22" 58.03 +_ 4.58«  2.10 + 0.05* 2.00 +_ 0.03*  1.33 +_ 0.03» 1.20 +_ 0.09»>  a column  are not s i g n i f -  1  Data a r e expressed  as mean +_ SEM.  Means s h a r i n g t h e same l e t t e r w i t h i n i c a n t l y d i f f e r e n t a t p<0.05.  191  TABLE  6.8  E f f e c t o f p r o t e i n l e v e l on femur biomechanical parameters .  p h y s i c a l and  1  Diet  Dry wt. (g)  Bending F a i l u r e Maximum Energy Bending S t r e s s ( x 10-2 j ) (N/mm )  Length (mm)  2  20% p r o t e i n C a s e i n 0.387 +_ 0.010° 30.61 +_ 0.47 Soy 0.360 +_ 0 . 0 0 9 30.49 +_ 0 . 2 1 a  6% p r o t e i n C a s e i n 0.295 +_ 0 . 0 0 8 Soy 0.266 ±_ 0 . 0 1 7 1  b b  27.82 +. 0 . 3 3 27.07 +_ 0 . 5 8  a a  b b  9.24 + 0.93° 6.12 +_ 0.60  68.94 +_ 3.06 65.82 +_ 1.45  4.68 + 0 . 4 4 3.56 +. 0.34«  63.96 +. 3.70 56.37 +_ 1.38  b  b c  a a  a b  D a t a a r e e x p r e s s e d as mean +_ SEM.  Means s h a r i n g t h e same letter within i c a n t l y d i f f e r e n t a t p<0.05.  192  a column  are not s i g n i f -  Discussion A diet phosphorus, poor  which i s  low i n p r o t e i n  i f the phosphorus  palatability  of  level  synthetic  i s consequently also  i s not compensated diets deficient  r e p o r t e d by o t h e r s ( L o t z e_t_ a i . , 1 9 6 8 ) , l i k e l y reduced feed fed  t h e 6%  calcium content, t h e 6%  diets.  diets,  calcium deficiency, total  1942).  in  it  diets for  i n t a k e of a n i m a l s f e d experimentally induced  phosphorus  Calcium deficiency  that  PTH  absorbed d i e t a r y  i n a release  of  diets.  bone  both  compensated  for  of  excess  by  an  c o n s e r v a t i o n of that urinary  plasma  increased  phosphorus  concentration.  i n animals of  i n these  f e d the  and 6%  phosphate i s  renal  phosphorus  (Agus e_t al_. , 1 9 8 1 ) .  differences  c e l l u l a r ) absorption  with  the f a c t  an  and  phosphate  were r e d u c e d  Normally,  resorption  calcium  coupled  phosphate, r e f l e c t e d  e x c r e t i o n m e d i a t e d by PTH Despite  of  phosphate,  e x c r e t i o n of phosphate  eventually  mediated  l e a d i n g t o a h i g h e r plasma  mobilization  protein  calcium  f o r , by  Thus,  intestine,  of  i s compensated  (Patt  s e c r e t i o n and v i t a m i n D m e t a b o l i s m ( A u r b a c h , 1 9 8 8 ) .  i n t o plasma,  regulation  levels  hormone  suspected  the  i n c r e a s e d plasma  as i n f l u e n c e d by p a r a t h y r o i d  animals, resulted  fecal  the  i n the  is  The  an  to the  metabolism  k i d n e y s , bone and (PTH)  in  i n phosphorus,  as d e m o n s t r a t e d i n t h e s e a n i m a l s , by r e d u c e d  c a l c i u m and  and L u c k h a r d t , alterations  resulted  The  observed i n rats  balancing  the longterm reduced feed  protein  plasma  Despite  for.  contributed  i n t a k e , body w e i g h t g a i n e d and FER, protein  low i n  4 5  in Ca  calcium was 193  not  homeostasis, i l e a l different  (para-  between a n i m a l s  fed  t h e two d i f f e r e n t  levels  of d i e t a r y p r o t e i n .  This  c o n t r a r y to the greater apparent a b s o r p t i o n of calcium fed  the  6% p r o t e i n  absorption along increase protein  in  intestinal  levels  Previous  mediates the the  in  the e n t i r e  fed animals,  calcitriol 1979).  diets  the balance  intestinal  calcium  was l i k e l y  i n chronic workers  tract.  calcium deficiency  due  to  a  time,  or  o f PTH,  an i n t e r a c t i o n  hypothesized  that  severely hypocalcemic therefore  AMP  concentrations,  response  1978).  renal  i n calcium  two c o n d i t i o n s Rader  formation  increase  et a l .  o f cAMP by  1-a-hydroxylase  synthesis.  cells  f o r an e x t e n d e d  between these al. ,  Further,  of t a r g e t  hypocalcemia  decrease  usual  impaired  (cAMP)  activity  These e f f e c t s , i n  in  efficiency  of the  v i t a m i n D-dependent component o f i n t e s t i n a l  absorption 1987).  reduced  calcitriol  the  transcellular  (Bronner,  e_t  r a t s may  impair  t u r n , would i n h i b i t  calcium  (Rader et a1. .  that c y c l i c  depressed  ( H o r i u c h i e t a l _ . , 1977 ; C a r n e s  active,  by t h e 6%  decrease i n c i r c u l a t i n g  have s u g g e s t e d  exposed t o e l e v a t e d l e v e l s  (1979)  reflected  a b s e n c e o f an  absorption efficiency due t o a  was  i n animals  PTH s t i m u l a t i o n o f r e n a l 1 - a - h y d r o x y l a s e .  rats  p e r i o d of  which  The  i n f l u e n c e o f PTH on cAMP was shown t o be  deficient  and  study,  result  reported Thus,  animals  equivalent proportions  as a fed  in  calcium  result the  deficient  of s i m i l a r  6%  protein  of c a l c i u m from the lower  subjects  luminal  calcium  diets  absorbed  small  intestine  compared t o t h e 2 0 % p r o t e i n f e d a n i m a l s . Despite animals  fed  the s i m i l a r t h e 6%  f e e d , and t h e r e f o r e  and 2 0 %  protein diets, 194  calcium,  intakes of  r e s p e c t i v e l y , during  the  24  hr b a l a n c e  study  intake d e f i c i e n c i e s urinary  calcium  indicates calcium a1 . .  a  1981).  from  the  apparent absorption  greater The  tubules  similar  comparison  to  balance  20%  d a t a , w h i c h showed no  transport  in  calcium  absorption was  not  detected  of r e n a l  The  in  protein diets tubular  calcium  increase  due  et  occurs  reabsorption  greater  calcium  of a n i m a l s  to r e n a l  the  increase  fed  6%  intestinal  an  a  other  workers  increased  d e f i c i e n c y (Favus using  calcium  i n p a r a c e l l u l a r calcium  have  herein  conservation.  Alternatively,  may  i n calcium  decrease  p r o t e i n , s u g g e s t s r e t e n t i o n of a  deficiency. colon  longterm  d e f i c i e n c y (Agus  filtrate.  extends  absorption  have i n d i c a t e d t h a t t h e  6%  calcium balance  study  calcium  the  to  p r o p o r t i o n of absorbed c a l c i u m  calcium  which  fed  the  The  of e x t r a c e l l u l a r  glomerular  and  groups.  with a calcium  distal  the  reflect  m e d i a t e d enhancement  conservation  a c t s on  in  6% p r o t e i n  reported  Renal  calcium  the d a t a  e x c r e t i o n of a n i m a l s  reabsorption  protein  the  p o s s i b l e PTH  b e c a u s e PTH of  of  period,  role  in  et a l . . 1980),  ligated  ileal  loop  technique. The  presence  absorption  in  enhancing  effect  (CPP)  was  still  paracellular soluble retained  the  translocation  a  6%  protein  of  p r o t e i n source  the  i n the  feeding of  fed  Thus,  absorption  animals  the by  ability  on  ileal  calcium  indicated  that  lower  absorbed  4 5  (6%)  Ca 195  o f CPP  to  an  enhance  i n c r e a s i n g the p r o p o r t i o n  luminal contents a  effect  casein p o s t - d i g e s t i o n phosphopeptides  apparent.  calcium  calcium by  of  to  (Sato casein the  et  a 1 . . 1983)  protein diet.  femora  was  of was The  however,  inversely specific 20%  related  t o the  activity  soy d i e t  intestinal  o f t h e f e m o r a was  as w e l l t h o s e  is 4 0  likely  Ca  in the  Ca  of t h e bone.  regardless  animals.  This  ability  of  and f u r t h e r  i n acute  was  not  the  fact  diets  paracellular  in  that  limited  only  absorption in  lower  Ca  observed  confirms  that  considered  studies. in this  compared t o s o y f e d  t o i n f l u e n c e the b i o a v a i l (Mellander,  small  1963);  o f magnesium  intestine.  A  e x c r e t i o n observed  thus, from  greater  o f magnesium i n c a s e i n f e d a n i m a l s u r i n a r y Mg  with  u r i n a r y Mg e x c r e t i o n was  than c a l c i u m  the  4 5  significantly  enhance the b i o a v a i l a b i l i t y  the i n c r e a s e  animals.  Since  n o t be  absorption  altered  been o b s e r v e d  other  feasibly  casein  explain  CPP has  of m i n e r a l s  CPP c o u l d  f e d the  r e s u l t was a l s o  i n b o t h 6% and 2 0 % c a s e i n f e d a n i m a l s ,  counterparts.  Ca  secondary to  o n l y an e x c h a n g e o f  1) s t u d y ,  of u t i l i z a t i o n  Magnesium b a l a n c e  higher  of these  d e p o s i t i o n t o f e m u r measurement s h o u l d  study,  animals  4 5  5  the l a c t o s e (experiment 4 5  in  The  f o r * C a d e p o s i t i o n i n t o the femora t o o c c u r , i t  the surface  as a t r u e i n d e x  the  contents  that deposition, r e f l e c t s  on  increased  data.  f e d t h e 6% p r o t e i n d i e t s ,  t h e d e c r e a s e d bone c a l c i u m 1 h r was a l l o w e d  absorption  would  i n these  P o s s i b l e d i f f e r e n c e s i n Mg p a r a c e l l u l a r a b s o r p t i o n , i f to  detection  the  lower  i n the balance  Phosphorus  small  intestine,  was  dietary protein  as w e l l  as t h e  phosphorus  decreased  t h a n 6% s o y , a l i k e l y  have  precluded  study.  balance  was  may  result  influenced  moreso  level  in  both the source  the d i e t .  i n the animals  of the higher 196  by  of  Intake of  f e d 6% c a s e i n ,  phosphorus  content  of  soy  protein  sources  (Zemel,  coupled w i t h decreased caused is  a  relative  reflected  1988).  feed intake  phosphorus  and  diets.  Phosphorus  phosphorus  i n t h e 6%  deficiency  balance  apparent  d i e t a r y protein treatments  protein  bone m e t a b o l i s m mineralization  as  absorption  parameters  animals  fed  t h e 6%  protein  fed animals.  contribute  by  1974).  the p h y s i c a l  and  was  not  phosphorus  It is  mineralization  decreased  of  were low  that  the  Parfitt,  (Bruin  was  The  n o t a d v e r s e l y a f f e c t e d by  deficiencies  et  also  similar  animals fed d i e t s work n e e d e d energy,  of a1..  had  ratio  dietary  the  decreased 197  The  results  other  workers  In the p r e s e n t  treatment  however,  likely  due  to  i n these animals.  indicated  these bones.  to the  of the femora  phosphorus  low i n p r o t e i n ,  coupled with  been r e p o r t e d  l o w e r femur c a l c i u m  i n femur b i o m e c h a n i c a l b e n d i n g  to break  20%  mineralization in  1975).  diets  Ca/P  o f b o t h c a l c i u m and  A decrease  from  compared t o t h e  has  1983);  inhibition  contents.  femora  bone l e n g t h i n s t u d i e s w i t h humans  s t u d y , a n i m a l s f e d t h e 6% p r o t e i n and magnesium  by  therefore,  bone  deficiency  affected  bone  have o b s e r v e d phosphorus  protein  and  Moreover,  acute  phosphorus  synthesis  o b t a i n e d h e r e i n w i t h a r a t animal model. the  This  i s necessary for  noteworthy  Protein deficiency  B e r r i d g e , 1969;  fecal  f e d t h e 6%  collagen  protein diets  to  fed animals  however.  influenced  (Grey,  level,  i n these animals.  of animals  C o n s e r v a t i o n o f b o t h c a l c i u m and  (Adams and  lower p r o t e i n  i n the h y p o p h o s p h a t u r i a , decreased  excretion  to  The  a  failure  energy  reduced  amount o f  reduced  physical  in  amount o f work  size  and  mineral  c o n t e n t of growth  as  these femora, influenced  these animals.  Femur  i n animals f e d the significant  in  by  6%  the r e t a r d e d maximum b e n d i n g  protein  o n l y the  e x p l a i n e d by t h e s l i g h t l y femora  from  t h e 6%  counterparts. that a for  diet  were l i k e l y  6%  diets,  t h e y o u n g and  characteristics  s t r e s s was albeit  also  decreased  T h i s r e s u l t may  be  bone s i z e and m i n e r a l i z a t i o n  of  data  phosphorus  ( S h a h a n i and  198  of  was  these  alike  bone  the d i f f e r e n c e  i n p r o t e i n and  elderly,  of reduced  o f a n i m a l s compared t o 6%  Taken t o g e t h e r , limited  growth  soy f e d a n i m a l s .  reduced  soy g r o u p  a result  support  casein fed the  concept  i s contraindicated Kaup,  1989).  Conclusions Calcium calcium i n  bioavailability  measurements  of  bone  were d e t e r m i n e d .  level  i n the  p r o c e s s i n g on the  relevance  relates  balance  diet,  these  enhance  well  as  the  well  effects  as  the  optimal  concentration  w i t h o n l y the h i g h lactose  on  content,  contribution to  of  i n these  was  that the  Further,  homeostasis  as i t  discussed.  shown  to  be  Lactose  due  u n c h a n g e d bone i n animals  mineral f e d the  has a g r e a t e r  enhanced i n t e s t i n a l The r e l a t i v e l y  transport  low l a c t o s e  m i l k ) w o u l d make o n l y a s m a l l  f i n d i n g s a r e r e l e v a n t t o o s t e o p o r o t i c i n d i v i d u a l s who  199  t o an  achieved  c a l c i u m a b s o r p t i o n f o r humans.  intolerant.  and  compared t o c o n t r o l and 20%  malabsorption  animals.  (5% i n fluid  intestinal  thermal  of c o - n u t r i e n t s which  s t r e n g t h observed  suggests  of d a i r y foods  products,  of d i e t a r y c a l c i u m .  F u r t h e r , the  on bone m e t a b o l i s m t h a n  of c a l c i u m observed  strength  o f l a c t o s e i n t h e lumen w h i c h was  diets.  diets,  endpoint  of c a l c i u m from d a i r y p r o d u c t s ,  transport  b u t r e d u c e d bone  lactose  as  effects  calcium  (50%) l a c t o s e d i e t  containing  intestinal  and b i o a v a i l a b i l i t y .  i s due t o t h e p r e s e n c e  intestinal  of  e x p e r i m e n t s have i n v e s t i g a t e d t h e  enhancement o f c a l c i u m a b s o r p t i o n  content  as  t o bone m e t a b o l i s m and o s t e o p o r o s i s was  milk i n p a r t i c u l a r ,  effect  by i s o t o p i c  studies  calcium absorption of  utilization  o f v a r i o u s d a i r y c o n s t i t u e n t s and  The h i g h b i o a v a i l a b i l i t y  50%  subsequent  m i n e r a l i z a t i o n and b i o m e c h a n i c a l  The p r e c e d i n g  of the chemistry  their  the  c a l c i u m h o m e o s t a s i s as i n d e x e d  absorption methodologies,  role  and  These  are lactose  The  -  importance  intestinal concerning  varying with  calcium the  secondly,  casein proteins  and  a n i m a l model  was  used  the  ileal  casein  calcium  formation  The  e n h a n c i n g e f f e c t of  found to  of  level  and  protein diets.  B o t h SHR  of  intestinal  calcium  in  calcium  absorption.  influenced and  WKY  by  casein  dietary  calcium  rats,  the of  of  of  diet  studies, (SHR)  rat  metabolism and  utiliz-  source,  and  in  found  to  p o t e n t i a l l y due  to  of d a i r y p r o t e i n s was  calcium  and  In s e l e c t e d  bioavailability  sequestering  utilization  processing  hypertensive  i n Wistar  with  fortification  of p r o t e i n i n the  Dietary protein  adequate l e v e l  source  dietary  disturbed  i n calcium  e f f e c t on  bioavailability  high  spontaneously  b i o a c t i v e peptides  c a s e i n p r o t e i n s on  of d i e t a r y  sources;  utilization.  fraction  calcium  have l i t t l e  a n i m a l s f e d an The  and  transport  the  dairy protein  plant protein  level  to  studies  thirdly,  the  component in  (CPP); f o u r t h , thermal  to d i e t a r y treatments.  particular  levels  various  because i t s  changes  protein elucidated  a soy  finally,  prone,  highlight  a t i o n due  be  first, versus  bioavailability  osteoporosis  affect  was  l e v e l s of d i e t a r y c a l c i u m ;  calcium  could  dietary  absorption  casein phosphopeptides  the  in  the  e f f e c t s of  dietary proteins; on  of  calcium  (CPP).  absorption  was  l o n g t e r m bone m i n e r a l i z a t i o n calcium. was  found  calcium  to  from  a n i m a l s f e d the  influence  the  c a s e i n and  soy  a d e q u a t e and  low  c o n t a i n i n g d i e t s e x h i b i t e d enhanced Femur c a l c i u m  p r o t e i n source  adequate d i e t a r y l e v e l s of  200  content  i n SHR  calcium.  appeared  animals fed  to the  Femur b i o m e c h a n i c s  were i n f l u e n c e d  by t h e  dietary protein  source.  To  determine  tryptic  dietary  phosphopeptides  CPP was  but was  sufficiently  sustained,  digestive  influenced effects  indicated  calcium  utilized  great to  was  the  production  of  were  digestion with  or  CPP  and s o y by  m i n e r a l i z a t i o n or The  the  level  o f CPP  potential effect  a f f e c t bone  the  peptides,  showed  that  heat  resulted  i n reduced calcium  bioactive  mineral-  on d i e t a r y  of  the  such  as  absorption,  e x p e r i e n c e d by t h e s e a n i m a l s .  may  be  thus,  the  calcium  heat  bioavail-  In v i t r o  studies  treated  dietary  which i n t u r n would decrease the CPP.  denaturation  secondary  CPP  of d a i r y products,  investigated.  digestibility  further  mineralization  casein  bone  significantly  markedly reduced,  of d i g e s t i v e  In p r e l i m i n a r y  animals.  utilization  that  e f f e c t of  i n casein  excreted.  by t h e t h e r m a l p r o c e s s i n g  and  from  i n absorbed calcium  in  enough,  than  absorption  fortification  increase  was  to  absorption  of a severe heat treatment  ability  proteins  not  Dietary  this  rather,  of soy f e d  calcium  calcium  However,  fortification  The  moreso  peptides  investigated.  of  confirmed.  f e d a n i m a l s was n o t  ization  digestion  (CPP) i n f l u e n c e d  binding  i n enhanced i l e a l  biomechanics,  yield  bioactive  with  was  animals.  casein  of d i e t a r y calcium  d i e t s o b s e r v e d i n e x p e r i m e n t s 2 apd 3, t h e  s t u d i e s , the  resulted  the  of c a s e i n  fortification  in vitro  fed  i f  digestion  from c a s e i n  low l e v e l  to  the The  201  of  In  dietary  calcium nutrient  e f f e c t s of  vivo  studies proteins  b a l a n c e and bone malabsorption m a l a b s o r p t i o n on  bone  biomechanical  lactose  experiment  A final digestion study,  (experiment  variable  peptides,  the  proportion  of  was of  to  i s the  effect  bioavailability The  parameters  the  positive  e f f e c t of  casein  dietary  level.  The  b a l a n c e and  a n i m a l s were  due  factors dietary  In  well,  and  intraluminal  absorption  calcium  are  levels  casein.  This  result  e x t e n d e d t o bone  a soy  6%  of a  the  lower  absorbed,  protein  fed  deficient state  likewise  reduced  Thus,  detrimental  bioavailability  was  from  were  similar  at  calcium  the  calcium  animals.  was  bioavailability  of  calcium  retention  metabolism.  absorption  Dietary  fed  this  of in  dietary  decreased  to  calcium  t o bone m e t a b o l i s m .  high  calcium  soy  In  a r e s u l t of d e c r e a s e d  phosphorus  of  the  casein  on  diets  i n amount o f  i n bone  calcium  presence  and  the  diet.  diets  indicating  calcium  sequester  absorption.  from  biomechanics  at  bioactive  i n the  casein  chronically  i t pertains  of  protein  in  p r o t e i n d i e t s as  lactose  ileal  20%  levels on  the  conclusion,  containing  which  6%  protein  e n h a n c e d by  and  absorbed  a v a i l a b l e f o r use  m e t a b o l i s m , as  of p r o t e i n  reductions  Femur  the  level  bone m i n e r a l i z a t i o n  to  these animals. animals fed  production  protein  in  above.  determined  between  calcium  1)  those observed  the  6%  calcium  two  confirmed  casein and  of d i e t a r y  shown  intake  enhanced i n a n i m a l s was  likely  to  only. fed  influenced  retain  protein diet, at 202  i t in a soluble with  CPP  by  a l b e i t the the  level  the  for  calcium  e f f e c t was of  As  (CPP)  state  enhanced  be  diets  d i g e s t i o n phosphopeptides  fortification  calcification  was  not  fortification  used.  Thermal p r o c e s s i n g of c a s e i n l e d t o a r e d u c t i o n i n p r o t e i n  digestibility  as  w e l l as  absorption.  The l e v e l  enhancing e f f e c t experiments isotopic  tract.  from  a  using  specific  the a b s o r p t i o n The  use  of  on  calcium  both  acute  of  segment calcium  bone  diets  animals  varying i n calcium  on  calcium  were  bioavailability. intestinal  data  indicate  a b s o r p t i o n of that  calcium  the  i n t e s t i n e may n o t  from  the  entire  utilization  fed different  bioavailability.  203  The  of  mineralization  p a r a m e t e r s as i n d i c e s o f c a l c i u m s e n s i t i v e when  effect  of p r o t e i n i n the d i e t d i d not a f f e c t the  casein  herein,  the c a s e i n  c a l c i u m , as w e l l as b a l a n c e  transport reflect  of  l o s s of  intestinal  and  biomechanical  were  found  levels  t o be  of c a l c i u m , or  References Adams, P. and cortical  B e r r i d g e , F.R. 1969. 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I n t e r a c t i o n between a n i m a l s t r a i n i n femur u t i l i z a t i o n .  Source of Variation  Sum o f Squares  Within cases  6719.54  Anima1 strain  1817.36  1817.36  37536.43  18768.22  1613.93  806 .96  Calcium level Animal x Calcium  Degrees o f Freedom  Mean Square  and c a l c i u m  F-value  intake  P-value  110 .16  61  225  16 . 50 170.38 7 . 33  0.000 0.000 0 .001  TABLE 2.  I n t e r a c t i o n between p r o t e i n i n femur u t i l i z a t i o n .  Source of Variation  Sum o f Squares  Within cases  7396 .83  Protein source C a l c ium level Protein x Ca 1c ium  Degrees of Freedom  s o u r c e and c a l c i u m  Mean Square  F-value  level  P-value  121 .26  61  36 .17  36 .17  0.30  0.587  37991.53  18995.77  156.65  0.000  2286.36  1143.18  9.43  0.000  226  TABLE 3.  Source of Variation  I n t e r a c t i o n between a n i m a l s t r a i n i n t i b i a magnesium c o n t e n t .  Sum o f Squares  Degrees of Freedom  Mean Square  and c a l c i u m  level  F-value  P-value  Within cases  4 .77  Animal strain  1 . 17  1 . 17  13.26  0.001  Ca1c ium level  4.36  2 . 18  24.68  0.000  Animal x Ca1c ium  2 .33  1 .16  13 .17  0.000  54  0.09  227  TABLE 4.  I n t e r a c t i o n between a n i m a l s t r a i n t r e a t m e n t i n magnesium b a l a n c e .  Source of Variation  Sum o f Squares  Within cases  4 .00  Animal strain  0.01  P r o t e i n heat treatment  30.98  Animal x P r o t e i n Tmt.  2.92  Degrees of Freedom  Mean Square  24  0 . 17 0.01 10.33 0.97  228  and p r o t e i n  heat  F-value  P-value  0 .08  0 .774  62 .04  0 .000  5.85  0.004  

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