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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 BIOAVAILABILITY: IMPACT ON UTILIZATION AS INDEXED BY BONE MINERAL COMPOSITION AND BIOMECHANICS. By YVONNE VERONICA YUAN B.Sc., 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 i n THE FACULTY OF GRADUATE STUDIES (Department of Food S c i e n c e ) We accept t h i s t h e s i s as conforming to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September 1990 (c) Yvonne V e r o n i c a Yuan, 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 be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Fnnrl ^ripnrp The University of British Columbia Vancouver, Canada Date September 19, 1990 DE-6 (2/88) A b s t r a c t C a l c i u m b i o a v a i l a b i l i t y was i n v e s t i g a t e d u s i n g i s o t o p i c i n t e s t i n a l a b s o r p t i o n , and b a l a n c e s t u d y t e c h n i q u e s w i t h bone m i n e r a l i z a t i o n and biomechanics as e ndpoint d e t e r m i n a n t s of c a l c i u m u t i l i z a t i o n . In experiment 1, l a c t o s e enhancement of 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 was c o n f i r m e d , and i t was suggested t h a t a c r i t i c a l l u m i n a l c o n c e n t r a t i o n of l a c t o s e was n e c e s s a r y f o r i t s a c t i o n . 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 a n i m a l s f e d the 50% l a c t o s e c o n t a i n i n g d i e t , bone m i n e r a l i z a t i o n was not d i f f e r e n t from c o n t r o l s ; and f u r t h e r , a d e c r ease i n bone s t r e n g t h of these a n i m a l s was found to be secondary to n u t r i e n t m a l a b s o r p t i o n . In t h i s s t u d y , t h e r e was no e vidence to i n d i c a t e a d i f f e r e n c e i n the b i o a v a i l a b i l i t y of c a l c i u m from m i l k ( c o l l o i d a l ) or y o g u r t ( i o n i z e d ) s o u r c e s . In experiment 2, the a b s o r p t i o n of c a l c i u m from the i l e u m was s i g n i f i c a n t l y enhanced in_ normal W i s t a r r a t s f e d m i l k p r o t e i n d i e t s c o n t a i n i n g c a s e i n as compared to whey and soy p r o t e i n d i e t s . The i n c r e a s e i n absorbed c a l c i u m was shown to have l i t t l e p h y s i o l o g i c a l s i g n i f i c a n c e i n bone m i n e r a l i z a t i o n and biomech-a n i c s when a n i m a l s were f e d a d i e t adequate i n d i e t a r y c a l c i u m . In experiment 3, 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 was s i m i l a r between g e n e t i c a l l y 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 n ormotensive c o n t r o l W i s t a r - K y o t o (WKY) r a t s , s u g g e s t i n g t h a t d i f f e r e n c e s i n c a l c i u m metabolism between these two s t r a i n s was not due to d i f f e r e n c e s i n i l e a l c a l c i u m t r a n s p o r t . The e f f e c t of c a s e i n and soy p r o t e i n d i e t s c o n t a i n i n g h i g h ( 2 . 0 % ) , adequate (0.5%) and low (0.05%) l e v e l s of c a l c i u m , r e s p e c t i v e l y on c a l c i u m b i o a v a i l a b i l i t y and subsequent u t i l i z a t i o n was d etermined i n SHR and WKY a n i m a l s . I l e a l c a l c i u m a b s o r p t i o n was g r e a t e r i n c a s e i n f e d a n i m a l s than those f e d soy a t the adequate and low l e v e l s of c a l c i u m . Femur c a l c i f i c a t i o n was enhanced by c a s e i n d i e t s at the h i g h and medium l e v e l s of d i e t a r y c a l c i u m o n l y . Femur biomech-a n i c s were not i n f l u e n c e d by d i e t a r y p r o t e i n s o u r c e , but were however, a d v e r s e l y a f f e c t e d by the low d i e t a r y c a l c i u m l e v e l . In experiment 4, the e f f e c t of d i e t a r y f o r t i f i c a t i o n w i t h c a s e i n phosphopeptides (CPP) was i n v e s t i g a t e d i n c a s e i n and soy f e d SHR a n i m a l s . CPP added to c a s e i n and soy p r o t e i n d i e t s appeared to r e s u l t i n a g r e a t e r i l e a l a b s o r p t i o n of c a l c i u m . T h i s i n c r e a s e i n c a l c i u m b i o a v a i l a b i l i t y from the c a s e i n d i e t had l i t t l e e f f e c t however, on bone 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 s t r e n g t h , due to the e x c r e t i o n of excess absorbed c a l c i u m . In experiment 5, severe t h e r m a l p r o c e s s i n g of d i e t a r y p r o t e i n s was shown to reduce i n v i t r o d i g e s t i b i l i t y . Animals f e d heat denatu r e d c a s e i n and soy d i e t s e x h i b i t e d reduced 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 , c a l c i u m b a l a n c e as w e l l as bone 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 . These e f f e c t s were i n f l u e n c e d by the n u t r i e n t m a l a b s o r p t i o n e x p e r i e n c e d by these a n i m a l s . In experiment 6, a low (6%) p r o t e i n , low phosphorus d i e t r e s u l t e d i n d e c r e a s e d food i n t a k e and animal growth. However, i l e a l c a l c i u m a b s o r p t i o n (% dose) was s i m i l a r between 6% and 20% p r o t e i n f e d a n i m a l s . A low l e v e l of d i e t a r y p r o t e i n i n f l u e n c e d c a l c i u m b a l a n c e and u t i l i z a t i o n f o r bone 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 s t r e n g t h . These r e s u l t s i n d i c a t e t h a t 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 may not n e c e s s a r i l y equate w i t h those o b t a i n e d from a c a l c i u m b a l a n c e s t u d y . N o t w i t h s t a n d 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 from the i l e u m was shown to be enhanced i n anim a l s f e d c a s e i n , which was l i k e l y due to the p r o d u c t i o n of b i o a c t i v e p e p t i d e s (CPP) t h a t are i n v o l v e d i n s e q u e s t e r i n g c a l c i u m and r e t a i n i n g i t i n a s o l u b l e form. By r e d u c i n g p r o t e i n d i g e s t i b i l i t y w i t h heat d e n a t u r a t i o n , t h i s e f f e c t was l o s t . Reducing the p r o t e i n c o n t e n t i n the d i e t however, d i d not reduce the enhancement of c a l c i u m b i o a v a i l -a b i l i t y observed i n c a s e i n f e d a n i m a l s . F i n a l l y , bone 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 parameters were shown to be s e n s i t i v e i n d i c a t o r s of c a l c i u m u t i l i z a t i o n from d i e t s t h a t v a r i e d i n c a l c i u m c o n t e n t or b i o a v a i l a b i l i t y of t h i s m i n e r a l . Table of Contents Page T i t l e page i A b s t r a c t i i Table of Contents v L i s t of T a b l e s v i i i L i s t of F i g u r e s x i i Acknowledgements x i v I n t r o d u c t i o n 1 L i t e r a t u r e Review . 4 1. 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 4 a. P h y s i o l o g y and B i o p h y s i c s 4 b. Animal Models f o r S t u d y i n g 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 9 2. F a c t o r s I n f l u e n c 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 15 a. F i b r e and P h y t a t e 15 b. O x a l a t e s 17 c. Fat 17 d. P r o t e i n and Phosphorus 17 e. L a c t o s e 22 f . M a i l l a r d Browning R e a c t i o n P r o d u c t s 24 g. M i n e r a l I n t e r a c t i o n s 25 h. E n d o c r i n e F a c t o r s 27 i . Impact of 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 . 30 j . P h y s i o l o g i c a l S t a t u s 32 3. D a i r y Foods 35 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 35 b. C a s e i n M i c e l l e s 36 c. C a l c i u m E x c h a n g e a b i l i t y 37 d. C a s e i n Phosphopeptides 38 e. Soy P r o t e i n 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 and A b s o r p t i o n 42 a- JjL V i t r o S o l u b i l i t y 43 b. Balance S t u d i e s 43 c. N u t r i t i o n a l S t a t u s 45 d. F u n c t i o n a l T e s t s 45 e. Model Systems 45 v 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 46 a. Treatment of O s t e o p o r o s i s 48 b. Anatomy of the Femur 50 c. Bone Biomechanics 51 Experiment 1: The e f f e c t of 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 femur biomechanics i n r a t s 54 I n t r o d u c t i o n 54 M a t e r i a l s and Methods 54 R e s u l t s 61 D i s c u s s i o n 69 Experiment 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 biomechanics i n r a t s f e d s e l e c t e d d i e t a r y p r o t e i n s 74 I n t r o d u c t i o n 74 M a t e r i a l s and Methods 74 R e s u l t s 79 D i s c u s s i o n 86 Experiment 3: C a l c i u m b i o a v a i l a b i l i t y i n c a s e i n and soy f e d r a t s 91 I n t r o d u c t i o n 91 M a t e r i a l s and Methods 91 R e s u l t s 95 D i s c u s s i o n 110 Experiment 4: E f f e c t of c a s e i n phosphopeptide 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 biomechanics i n c a s e i n and soy f e d r a t s . 117 I n t r o d u c t i o n 117 M a t e r i a l s and Methods 117 R e s u l t s 122 D i s c u s s i o n 138 Experiment 5: E f f e c t of p r o t e i n heat 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 biomechanics i n r a t s f e d c a s e i n and soy p r o t e i n s 143 I n t r o d u c t i o n 143 M a t e r i a l s and Methods 144 R e s u l t s 147 D i s c u s s i o n 169 v i Page Experiment 6: C a l c i u m b a l a n c e and femur biomechanics 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 176 I n t r o d u c t i o n 176 M a t e r i a l s and Methods 176 R e s u l t s 179 D i s c u s s i o n 193 C o n c l u s i o n s 199 R e f e r e n c e s 204 Appendix 223 v i i L i s t of T a b l e s Table Page 1.1 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 f e d to r a t s . . 56 1.2 D i e t 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 . 62 1.3 Blood c h e m i s t r 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 . 63 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 4 5 c a l c i u m i n r a t s f e d e x p e r i m e n t a l d i e t s 64 1.5 Femur p h y s i c a l dimensions and b i o m e c h a n i c a l parameters i n r a t s f e d e x p e r i m e n t a l d i e t s . . . . 68 2.1 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 f e d to ani m a l s 76 2.2 D i e t 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 . 80 2.3 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 of 4 5 c a l c i u m i n r a t s f e d e x p e r i m e n t a l d i e t s . . . . 81 2.4 Femur m i n e r a l c o m p o s i t i o n i n r a t s f e d e x p e r i m e n t a l d i e t s 83 2.5 Femur p h y s i c a l dimensions and b i o m e c h a n i c a l parameters i n r a t s f e d e x p e r i m e n t a l 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 i n r a t s f e d e x p e r i m e n t a l d i e t s 85 3.1 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 f e d to a n i m a l s 93 3.2 Body weight g a i n and food i n t a k e of e x p e r i m e n t a l a n i m a l s 96 3.3 E f f e c t of d i e t a r y p r o t e i n source on plasma 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 9 7 3.4 E f f e c t of d i e t a r y p r o t e i n 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 of c a l c i u m 99 3.5 Femur d e p o s i t i o n of 4 5 c a l c i u m i n r a t s f e d e x p e r i m e n t a l d i e t s 102 3.6 Femur m i n e r a l c o m p o s i t i o n 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 103 v i i i Table Page 3.7 Femur p h y s i c a l parameters 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 105 3.8 Femur b i o m e c h a n i c a l parameters 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 106 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 f e d e x p e r i m e n t a l d i e t s 108 3.10 T i b i a b i o m e c h a n i c a l parameters 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 r 109 4.1 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 f e d to a n i m a l s 120 4.2 D i e t 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 . 125 4.3 E f f e c t of d i e t on plasma m i n e r a l s of a n i m a l s f e d e x p e r i m e n t a l d i e t s 127 4.4 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 * 5 c a l c i u m d e p o s i t i o n to the femur 129 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 b a l a n c e 130 4.6 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 . magnesium b a l a n c e 132 4.7 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 . phosphorus b a l a n c e 133 4.8 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 femur m i n e r a l i z a t i o n 135 4.9 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 femur p h y s i c a l and b i o m e c h a n i c a l parameters . 136 5.1 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 f e d to a n i m a l s 145 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 on d i e t e f f i c i e n c y 149 5.3 E f f e c t of 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 151 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 on 4 5 c a l c i u m d e p o s i t i o n to the femur 154 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 on 24 hour c a l c i u m e x c r e t i o n 155 i x 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 on 24 hour c a l c i u m b a l a n c e 157 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 on 24 hour magnesium e x c r e t i o n 158 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 on 24 hour magnesium b a l a n c e 159 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 on 24 hour phosphorus e x c r e t i o n 161 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 on 24 hour phosphorus b a l a n c e 162 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 on femur p h y s i c a l parameters 164 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 on femur m i n e r a l i z a t i o n 166 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 on femur b i o m e c h a n i c a l parameters 167 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 f e d to a n i m a l s 178 6.2 E f f e c t of p r o t e i n l e v e l on d i e t e f f i c i e n c y . . . 180 6.3 E f f e c t of p r o t e i n l e v e l on plasma m i n e r a l s . . . 181 6.4 E f f e c t of 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 185 6.5 E f f e c t of p r o t e i n l e v e l on 24 h r . magnesium ba l a n c e 186 6.6 E f f e c t of p r o t e i n l e v e l on 24 h r . phosphorus b a l a n c e 188 6.7 E f f e c t of p r o t e i n l e v e l on femur m i n e r a l i z a t i o n . 191 6.8 E f f e c t of p r o t e i n l e v e l on femur p h y s i c a l and b i o m e c h a n i c a l parameters 192 x Appendix Table Page 1. I n t e r a c t i o n between a n i m a l s t r a i n and c a l c i u m i n t a k e i n femur u t i l i z a t i o n 225 2. I n t e r a c t i o n between p r o t e i n source and c a l c i u m l e v e l i n femur u t i l i z a t i o n 226 3. I n t e r a c t i o n between a n i m a l s t r a i n and c a l c i u m l e v e l i n t i b i a magnesium c o n t e n t 227 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 and p r o t e i n heat t r e a t m e n t i n magnesium b a l a n c e 228 x i L i s t of F i g u r e s F i g u r e Page 1. Summary of c a l c i u m metabolism 5 1.1 L a c t o s e c o n t e n t of i l e a l l o o p of r a t s 67 3.1 E f f e c t of d i e t a r y p r o t e i n source 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 from the i l e a l l o op of SHR and WKY r a t s f e d c a s e i n and soy d i e t s 98 3.2 Femur d e p o s i t i o n of 4 5 C a i n SHR and WKY r a t s f e d c a s e i n and soy d i e t s 101 4.1 Gel f i l t r a t i o n p u r i f i c a t i o n of c a s e i n phosphopeptide (CPP) 123 4.2 Ion exchange p u r i f i c a t i o n of c a s e i n phosphopeptide (CPP) 124 4.3 I n t e s t i n a l a b s o r p t i o n of 4 5 C a from i l e a l l o o p i n r a t s f e d c a s e i n , soy 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 of n a t i v e and heat d e n a t u r e d c a s e i n and soy 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 of 4 5 C a from i l e a l l o o p of SHR and WKY r a t s f e d n a t i v e and heat dena t u r e d c a s e i n and soy 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 of 4 5 C a from i l e a l l o op of r a t s f e d 20% and 6% c a s e i n and soy 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 of absorbed 4 5 C a to the femora of r a t s f e d 20% and 6% c a s e i n and soy p r o t e i n d i e t s 184 X I 1 Appendix F i g u r e Page 1. T i m e - f o r c e d e f o r m a t i o n curve of I n s t r o n 3 - p o i n t bending of bone 224 x i i i Acknowledgements I would l i k e t o exp r e s s my g r a t i t u d e t o the many i n d i v i d u a l s who a s s i s t e d me d u r i n g the course of t h i s s t u d y . In p a r t i c u l a r , I would l i k e to acknowledge the Department of Food S c i e n c e f o r the use of the animal 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 expr e s s my s i n c e r e g r a t i t u d e t o my t h e s i s a d v i s o r , Dr. David D. K i t t s , Department of Food S c i e n c e , f o r h i s d e d i c a t e d p a r t i c i p a t i o n i n the animal s u r g e r i e s as w e l l as i n the comple-t i o n of t h i s t h e s i s . I am a l s o 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 the course of t h i s s t u d y . Thanks are a l s o g i v e n to the members of my t h e s i s committee, Dr. L e s l i e H a r t , of the Department of Animal S c i e n c e , and Drs. Timothy Durance, W i l l i a m Powrie and John Van d e r s t o e p , of the Department of Food S c i e n c e f o r t h e i r s u g g e s t i o n s and h e l p d u r i n g the p r e p a r a t i o n of t h i s t h e s i s . I would l i k e t o acknowledge my i n d e b t e d n e s s to Dr. T a k a s h i Nagasawa and Mr. Y o s h i o Moriyama f o r t h e i r a s s i s t a n c e w i t h the animal experiments and l a b o r a t o r y work. I would a l s o l i k e to thank Dr. B e r n i c e M i l l s and the D a i r y Bureau of Canada f o r t h e i r support and f u n d i n g of t h i s p r o j e c t . F i n a l l y , I would l i k e to thank my f a m i l y , and e s p e c i a l l y my mother, Mrs. Janet M. Yuan, f o r t h e i r s u p port and u n d e r s t a n d i n g d u r i n g the course of t h i s t h e s i s . x i v I n t r o d u c t i o n The importance of c a l c i u m as a major component i n h a r d , as w e l l as s o f t t i s s u e s , and i n the e t i o l o g y of c a r d i o v a s c u l a r and bone d i s e a s e p r e v e n t i o n has been w e l l e s t a b l i s h e d (McCarron and M o r r i s , 1985; Marcus, 1987). In the N o r t h American d i e t , d a i r y p r o d u c t s p l a y a v i t a l r o l e , p r o v i d i n g as much as 75% of d i e t a r y c a l c i u m . In a d d i t i o n to the importance of the l e v e l of c a l c i u m i n the d i e t , i s i t s b i o a v a i l a b i l i t y from the food system. Numerous i n v e s t i g a t o r s , a d e q u a t e l y reviewed by Bronner et a l . . 1987, Greger et a1.. 1988, and Heaney et a1.. 1989, have shown t h a t c a l c i u m must be p r e s e n t i n a s o l u b l e and i o n i z e d form w i t h i n the s m a l l i n t e s t i n a l lumen t o f a c i l i t a t e o p t i m a l a b s o r p t i o n . A v a r i e t y of d i e t a r y c o n s t i t u e n t s p r e s e n t s i m u l t a n e o u s l y i n the i n t r a l u m i n a l c o n t e n t s have the p o t e n t i a l to b i n d i o n i z e d c a l c i u m and i m p a i r a b s o r p t i o n , and t h e r e f o r e b i o a v a i l a b i l i t y . In g e n e r a l , t h e r e i s a p a u c i t y of i n f o r m a t i o n c o n c e r n i n g the r o l e of s p e c i f i c d a i r y p r o d u c t components i n i n f l u e n c 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 and u t i l i z a t i o n f o r bone d e p o s i t i o n , as indexed by bone 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 parameters. Such i n f o r m a t i o n i s of c o n s i d e r a b l e i n t e r e s t both a c a d e m i c a l l y , as w e l l as p r a c t i c a l l y , to Food S c i e n t i s t s and N u t r i t i o n i s t s because of the i n c r e a s e d knowledge to be g a i n e d c o n c e r n i n g the r o l e of d i e t i n g e n e r a l , and the c h e m i s t r y of d a i r y p r o d u c t s i n p a r -t i c u l a r , i n c a l c i u m b i o a v a i l a b i l i t y . S p e c i f i c a l l y , the c h e m i s t r y of d a i r y p r o t e i n c o n s t i t u e n t s and the v a r i o u s forms of a v a i l a b l e c a l c i u m ( c o l l o i d a l and i o n i z e d ) as they p e r t a i n to r e l a t i v e p r o t e i n d i g e s t i b i l i t y , a l t e r a t i o n w i t h t h e r m a l p r o c e s s i n g and q u a n t i t y consumed; as w e l l as the presence of c o - n u t r i e n t s such as l a c t o s e or l a c t o s e f e r m e n t a t i o n p r o d u c t s i n p o t e n t i a l l y i n f l u e n c i n g c a l c i u m a b s o r p t i o n , r e q u i r e f u r t h e r s t u d y . For example, the p r o d u c t i o n of c a s e i n phosphopeptides (CPP) produced on t r y p t i c d i g e s t i o n of c a s e i n , may s e q u e s t e r c a l c i u m and t h e r e b y , p r e v e n t the p r e c i p i t a t i o n of i n s o l u b l e c a l c i u m complexes by o t h e r d i e t a r y c o n s t i t u e n t s . F u r t h e r s t u d i e s were r e q u i r e d to as s e s s the s i g n i f i c a n c e of CPP on c a l c i u m b a l a n c e and u t i l i z a t i o n f o r bone m i n e r a l i z a t i o n . The experiments r e p o r t e d i n t h i s t h e s i s were conducted to determine the s i g n i f i c a n c e of the a f o r e m e n t i o n e d f a c t o r s a s s o c i a t e d w i t h d a i r y p r o d u c t s or p r o c e s s e s t h e r e o f , on 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 as indexed by bone 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 p r o p e r t i e s . In. v i t r o s t u d i e s conducted to determine c a l c i u m b i n d i n g to c a s e i n d e r i v e d b i o a c t i v e p e p t i d e s , and the r e l a t i v e d i g e s t i b i l i t y of t h e r m a l l y t r e a t e d p r o t e i n s were a l s o i n v e s t i g a t e d . In s p e c i f i c e x p e r i m e n t s , the 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 (SHR) was chosen as the e x p e r i m e n t a l a n i m a l model f o r s e v e r a l r e a s o n s . F i r s t l y , c a l c i u m a b s o r p t i o n i n these a n i m a l s has been suggested as a f a c t o r i n the d i s t u r b e d c a l c i u m homeostasis c h a r a c t e r i s t i c t o these a n i m a l s . S e c o n d l y , the SHR has been r e p o r t e d to be prone to o s t e o p o r o s i s , and t h e r e f o r e p o t e n t i a l l y more s e n s i t i v e to the s u b t l e changes a s s o c i a t e d w i t h d i e t a r y m o d i f i c a t i o n s i n l e v e l of c a l c i u m i n t a k e and b i o a v a i l a b i l i t y . 2 F i n a l l y , v a r i o u s e x p e r i m e n t a l e n d p o i n t measurements used to a s s e s s c a l c i u m b i o a v a i l a b i l i t y have been r e p o r t e d and c r i t i q u e d ( G r e g e r , 1988; Pak and A v i o l i , 1988). I t i s apparent t h a t each method has i t s advantages and p o t e n t i a l d i s a d v a n t a g e s i n 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 . In the p r e s e n t s t u d y , a p h y s i c a l method was used to a s s e s s the b i o -m e c h a n i c a l parameters of bone t h a t would complement the c a l c i u m i s o t o p i c and b a l a n c e s t u d i e s used to q u a n t i t a t e bone u t i l i z a t i o n of c a l c i u m i n a n i m a l s f e d d i f f e r e n t d i e t a r y p r o t e i n s or m o d i f i c a t i o n s of d a i r y p r o d u c t s . 3 L i t e r a t u r e Review I n t r o d u c t i o n : C a l c i u m c o n t r i b u t e s t o 1.5 to 2.0% of body w e i g h t , or 39% of the m i n e r a l c o n t e n t of the human body. W i t h i n the body, c a l c i u m i s a major component of the hard t i s s u e s (99% of body c a l c i u m i s p r e s e n t i n the bones and t e e t h ) , w i t h the remainder d i s p e r s e d i n the b l o o d , e x t r a c e l l u l a r f l u i d s and w i t h i n the c e l l s of the s o f t t i s s u e s . The r o l e s of c a l c i u m i n the body i n c l u d e bone s t r e n g t h , b l o o d c l o t t i n g a c t i v i t y , c e l l membrane t r a n s p o r t , nerve t r a n s m i s s i o n and r e g u l a t i o n of c a r d i a c f u n c t i o n . Thus, c a l c i u m i s c o n s i d e r e d to be an e s s e n t i a l m a c r o n u t r i e n t of the human d i e t (U.S. 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. 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 l a . P h y s i o l o g y and B i o p h y s i c s : D i e t a r y c a l c i u m e n t e r s the i n t e s t i n a l lumen from the g a s t r i c chyme by m e c h a n i c a l and enzymatic a c t i v i t y . C a l c i u m i s absorbed from the lumen by c r o s s i n g the i n t e s t i n a l mucosa and c e l l j u n c t i o n s t o the s e r o s a and f i n a l l y e n t e r i n g the lymph and b l o o d ( F i g u r e 1 ) . T r a n s e p i t h e l i a l c a l c i u m a b s o r p t i o n from the s m a l l i n t e s t i n e i s the sum of two independent p r o c e s s e s ( Z o r n i t z e r and Bronner, 1971; Behar and K e r s t e i n , 1976; Pansu .et. a_l_. , 1983b). The f i r s t i s an a c t i v e , and t h e r e f o r e s a t u r a b l e v i t a m i n D dependent p r o c e s s which o c c u r s i n the p r o x i m a l i n t e s t i n e , the duodenum and upper jejunum (Pansu et a 1. . 1983b). The second i s a p a s s i v e , n o n s a t u r a b l e c o n c e n t r a t i o n dependent p r o c e s s which o c c u r s a l o n g the e n t i r e l e n g t h of the s m a l l i n t e s t i n e , but which 4 D i e t F i g . 1 Summary of 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 a c t i v e c a l c i u m t r a n s p o r t ; (2) endogenous c a l c i u m s e c r e t i o n ; (3) p a s s i v e p a r a c e l l u l a r c a l c i u m 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 r e a b s o r b e d 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 mediated bone r e s o r p t i o n ; s u b j e c t to i n h i b i t i o n by CT. 5 i s e s p e c i a l l y predominant i n the d i s t a l jejunum and i l e u m where the a c t i v e p r o c e s s i s not of s i g n i f i c a n c e ( Z o r n i t z e r and Bronner, 1971; Behar and K e r s t e i n , 1976). The d u a l component n a t u r e of 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 was e l u c i d a t e d from i n . v i t r o as w e l l as i n v i v o a b s o r p t i o n k i n e t i c s t u d i e s . U s i n g k i n e t i c a b s o r p t i o n d a t a , Bronner et a 1. (1986) were a b l e to c a l c u l a t e t h a t the e f f l u x of c a l c i u m from duodenal loop s t u d i e s was much g r e a t e r than had been p r e d i c t e d f o r a s a t u r a b l e p r o c e s s a l o n e . T h i s l e d to the h y p o t h e s i s of a d i f f u s i o n a l component i n the 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 p r o c e s s . I t i s noteworthy t h a t Wasserman and T a y l o r (1969) had p r e v i o u s l y h y p o t h e s i z e d t h a t duodenal c a l c i u m a b s o r p t i o n had both a s a t u r a b l e and a n o n s a t u r a b l e component. The k i n e t i c s of 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 lumen were e x p r e s s e d u s i n g the f o l l o w i n g e q u a t i o n ; Jms = J . . . TCal + D [Ca] [Ca] + K*. where Jms - f l u x of c a l c i u m from mucosa to s e r o s a ; Jmax - maximum f l u x of the a c t i v e s a t u r a b l e p r o c e s s ; [Ca] = l u m i n a l c a l c i u m c o n c e n t r a t i o n ; Kt - t r a n s p o r t c o n s t a n t (which i s comparable to the M i c h a e l i s - M e n t e n c o n s t a n t ) ; and D = p a r a c e l l u l a r d i f f u s i o n c o e f f i c i e n t ( A r n o l d et a1.. 1975). T h i s e q u a t i o n d e s c r i b e s the b i p h a s i c curve of the r a t e of 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 as a f u n c t i o n of the l u m i n a l c a l c i u m c o n c e n t r a t i o n . At r e l a t i v e l y low c a l c i u m c o n c e n t r a t i o n s , 0 to 2 mM, the a c t i v e t r a n s p o r t component i s dominant, as i n d i c a t e d by the h y p e r b o l i c n a t u r e of the curve (Wasserman et. al_. , 1961; K r a w i t t and S c h e d l , 1968) u n t i l 6 s a t u r a t i o n i s a c h i e v e d at c o n c e n t r a t i o n s of 2 to 5 mM (Papworth and P a t r i c k , 1970). Above these c o n c e n t r a t i o n s , the curve becomes l i n e a r , w i t h the t r a n s i t i o n o c c u r r i n g at a p p r o x i m a t e l y 10 mM, i n d i c a t i n g a dominance of the n o n s a t u r a b l e d i f f u s i o n p r o c e s s . The above e q u a t i o n i s a l s o a p p l i c a b l e to the c h a r a c t e r i s t i c k i n e t i c s of s p e c i f i c segments of the s m a l l i n t e s t i n e ( S c h a c h t e r and Rosen, 1959; K r a w i t t and S c h e d l , 1968). Moreover, the a b s o r p t i o n of n u t r i e n t s i s a f u n c t i o n of not o n l y the r a t e of a b s o r p t i o n from a p a r t i c u l a r segment of the i n t e s t i n e , but a l s o the r e s i d e n c e time of the s u b s t r a t e i n t h a t segment (Cramer and Copp, 1959). Thus, w h i l e the e f f i c i e n c y of c a l c i u m a b s o r p t i o n i s much g r e a t e r from the duodenum and jejunum ( a c t i v e t r a n s p o r t ) than from the i l e u m ( p a s s i v e d i f f u s i o n ) , the amounts of c a l c i u m absorbed are q u i t e d i f f e r e n t . In s t u d i e s w i t h r a d i o l a b e 1 1 e d 4 5 C a l c i u m , Marcus and Lengemann (1962) were a b l e t o q u a n t i f y the 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 d i f f e r e n t r e g i o n s of the g a s t r o i n t e s t i n a l t r a c t . Animals were a b l e to absorb 0, 15, 23 and 62% d i e t a r y c a l c i u m from the stomach, duodenum, jejunum and i l e u m , r e s p e c t i v e l y w i t h a l i q u i d dose and 0, 8, 4 and 88% w i t h a s o l i d dose. Moreover, Cramer (1965) r e p o r t e d e s t i m a t e s of the i n t r a l u m i n a l c a l c i u m c o n c e n t r a t i o n s i n the dog to be 2.4, 2.0 and 8.1 mM i n the duodenum, d i s t a l jejunum and d i s t a l i l e u m , r e s p e c t i v e l y . Thus, the r e l a t i v e l y g r e a t e r l u m i n a l c a l c i u m c o n c e n t r a t i o n , c o u p l e d w i t h the l o n g e r r e s i d e n c e time i n the i l e u m (due to i t s g r e a t e r l e n g t h ) , more than compensates f o r the 7 s l o w e r a b s o r p t i o n r a t e of n o n s a t u r a b l e c a l c i u m t r a n s p o r t i n r a t s (16% l u m i n a l c a l c i u m c o n t e n t t r a n s l o c a t e d to body f l u i d s per hour; Z o r n i t z e r and Bronner, 1971; Pansu et a l . , 1983b). The a c t i v e t r a n s p o r t of c a l c i u m i s t r a n s c e l l u l a r , o c c u r r i n g i n t h r e e s t a g e s . The f i r s t s t a g e , i n v o l v e s the e n t r y of c a l c i u m i n t o i n t e s t i n a l e p i t h e l i a l c e l l s v i a brush b o r d e r membranes on the l u m i n a l s i d e , down a c o n c e n t r a t i o n g r a d i e n t (Rasmussen et  a l . . 1979 ; M i l l e r and Bronner, 1981). The next s t e p r e q u i r e s d i f f u s i o n a c r o s s the c y t o p l a s m a g a i n s t a c o n c e n t r a t i o n g r a d i e n t a l l o w i n g the c a l c i u m to p o s s i b l y i n t e r a c t w i t h a v a r i e t y of c a l c i u m - b i n d i n g m o l e c u l e s , u s u a l l y p r o t e i n s (Ca b i n d i n g p r o t e i n ; CaBP), i n the c y t o p l a s m or p a r t of o r g a n e l l e s ( K r e t s i n g e r et a1. . 1982; Feher, 1983). The f i n a l s t a g e i s the e x t r u s i o n of the c a l c i u m at the b a s o l a t e r a l p o l e of the c e l l i n t o e x t r a c e l l u l a r f l u i d on the s e r o s a l s i d e a g a i n s t an e l e c t r o c h e m i c a l g r a d i e n t ( S c h i f f l and Binswager, 1980). As a r e s u l t , the t r a n s c e l l u l a r p r o c e s s of c a l c i u m a b s o r p t i o n i s s u b j e c t to b oth p h y s i o l o g i c a l as w e l l as n u t r i t i o n a l r e g u l a t i o n . The c y t o s o l i c CaBP i s v i t a m i n D dependent; w i t h CaBP a c t i n g as an i n t r a c e l l u l a r m e d i a t o r f a c i l i t a t i n g c a l c i u m t r a n s p o r t a c r o s s the c e l l , and p r o d u c i n g a c a l c i u m g r a d i e n t between the c e l l p o l e s ( K r e t s i n g e r .et. a 1 . . 1982). There i s a c l o s e l i n e a r r e l a t i o n s h i p between the v e l o c i t y of t r a n s m u r a l c a l c i u m t r a n s p o r t and the i n t e s t i n a l CaBP co n t e n t (Pansu e_t aj_. , 1981, 1983b; Roche e_t aj_. , 1986). Another component of a c t i v e c a l c i u m t r a n s p o r t i s age dependency. For example, i n the newborn r a t , c a l c i u m i s not a c t i v e l y t r a n s p o r t e d 8 i n the i n t e s t i n e (Pansu et a l . . 1983a; D o s t a l and Toverud, 1984). T h i s o b s e r v a t i o n was s u p p o r t e d by the absence of d e t e c t a b l e l e v e l s of CaBP (Ueng e_t a 1 . . 1979). A c t i v e t r a n s p o r t cannot be induced i n the newborn w i t h v i t a m i n D t h e r a p y , due to the low number of r e c e p t o r s i t e s f o r t h i s v i t a m i n s t e r o i d hormone (Ueng et a l . , 1979; H a l l o r a n and D e l u c a , 1980, 1981). However, by 30 days of age, a c t i v e t r a n s p o r t and CaBP can be seen to peak i n the r a t and t h e r e a f t e r d e c l i n e to a minimum at 3 months of age (Ueng et a1. . 1979). Human i n f a n t s show a s i m i l a r development p a t t e r n . Thus, they are not a b l e to adapt to a low c a l c i u m i n t a k e as are a d u l t s ( Y o u n o s z a i , 1981). The p a s s i v e t r a n s p o r t p r o c e s s i s a p a r a c e l l u l a r one i n which the c a l c i u m moves between the mucosal c e l l s . T h i s f l u x i s independent of age, v i t a m i n D e n d o c r i n e system and c a l c i u m i n t a k e ( Z o r n i t z e r and Bronner, 1971; N e l l a n s and Kimberg, 1978; Pansu et  a l . . 1983b). R e p o r t s suggested t h a t a m o d i f i c a t i o n of c e l l u l a r j u n c t i o n s would a l l o w movement of c a l c i u m from the lumen mucosa to the s e r o s a ( C a s s i d y and T i d b a l l , 1967). I n t e s t i n a l mucosa c e l l s have t h r e e r e g i o n s from mucosa to s e r o s a , namely t i g h t and gap j u n c t i o n s ; an i n t e r m e d i a t e j u n c t i o n ; and l a s t l y a w i d e r b a s o l a t e r a l r e g i o n (Guyton, 1977). The t i g h t and gap j u n c t i o n s both appear to be the r a t e - l i m i t i n g f a c t o r s c o n t r o l l i n g the non-s a t u r a b l e c a l c i u m f l u x ( B r o n n e r , 1987). l b . Animal Models f o r S t u d y i n g 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 : An abnormal c a l c i u m m e tabolism and p o t e n t i a l l y d i s t u r b e d 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 have f r e q u e n t l y been r e p o r t e d i n 9 the 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 (SHR) compared to i t s g e n e t i c a l l y r e l a t e d normotensive W i s t a r - K y o t o (WKY) c o u n t e r p a r t . H y p e r t e n s i v e b l o o d p r e s s u r e i n c r e a s e s b e g i n t o appear by about 10 weeks of age i n the SHR; these changes are co u p l e d w i t h a l t e r -a t i o n s i n the c a l c i u m metabolism of the SHR when compared to age-matched WKY a n i m a l s . Research i n t o the r e g u l a t i o n of c a l c i u m homeostasis i n the SHR has f o c u s s e d on 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 ; serum t o t a l and i o n i z e d c a l c i u m l e v e l s , " c i r c u l a t i n g p a r a t h y r o i d hormone and c a l c i t r i o l e n d o c r i n e f a c t o r s ; i n t e s t i n a l b rush b o r d e r membrane f l u i d i t y ; as w e l l as bone histomorphometry. 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 by SHR an i m a l s has been v a r i o u s l y r e p o r t e d to be l o w e r , not s i g n i f i c a n t l y d i f f e r e n t from, as w e l l as g r e a t e r t h a n , i n age-matched WKY c o n t r o l s (Toraason and W r i g h t , 1981; Lau et al_. , 1984; G a f t e r et_ a_l. , 1986). These s t u d i e s have used a v a r i e t y of a b s o r p t i o n s t u d y m e t h o d o l o g i e s i n c l u d i n g , b a l a n c e s t u d i e s , i n v i t r o U s s i n g chambers and i n s i t u i n t e s t i n a l p e r f u s i o n t e c h n i q u e s i n measuring duodenal c a l c i u m t r a n s p o r t i n mature SHR a n i m a l s . U s i n g an i n s i t u l i g a t e d i l e a l l o o p t e c h n i q u e , K i t t s et a l . (1989) were unable to d e t e c t a d i f f e r e n c e 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 c a l c i u m from the d i s t a l s m a l l i n t e s t i n e i n SHR an i m a l s compared to WKY c o u n t e r p a r t s . To i n v e s t i g a t e whether a d i s t u r b e d c a l c i u m metabolism c o n t r i b u t e d t o the development of g e n e t i c h y p e r t e n s i o n , Lau et  a1 . (1986) examined c a l c i u m b a l a n c e i n p r e h y p e r t e n s i v e SHR a n i m a l s . A g r e a t e r p o s i t i v e c a l c i u m b a l a n c e i n SHR a n i m a l s , compared to WKY, as ev i d e n c e d by decrea s e d f e c a l and u r i n a r y 10 c a l c i u m was s u g g e s t i v e of an abnormal r e n a l c a l c i u m r e t e n t i o n . The f i n d i n g s of Lau and coworkers (1986) h e l p e d to e s t a b l i s h t h a t changes i n the c a l c i u m metabolism of SHR an i m a l s are not caused by an e l e v a t i o n i n b l o o d p r e s s u r e . Plasma l e v e l s of t o t a l c a l c i u m have been r e p o r t e d to be s i m i l a r between mature SHR and WKY c o u n t e r p a r t s (McCarron et a 1. . 1981; Merke ejt a_l_. , 1989). However, plasma i o n i z e d c a l c i u m c o n c e n t r a t i o n has been found to be decreas e d i n mature SHR i n comparison to age-matched WKY c o n t r o l s , s u g g e s t i n g an i r r e g u l a r i t y i n e x t r a c e l l u l a r c a l c i u m b i n d i n g a c t i v i t y (McCarron et a1. , 1981) . S u p p o r t i n g e v i d e n c e f o r t h i s h y p o t h e s i s can be found i n e a r l i e r work which r e p o r t e d an i n c r e a s e i n v a s c u l a r smooth muscle c e l l p e r m e a b i l i t y t o c a l c i u m , w i t h d e c r e a s e d i n t r a c e l l u l a r o r g a n e l l e uptake of c a l c i u m i n SHR an i m a l s g i v i n g r i s e t o an a l t e r e d d i s t r i b u t i o n of c a l c i u m between e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments ( A o k i et a 1 . . 1976 ," Sc h e d l et a l . . 1988). I t i s noteworthy t h a t a d i e t h i g h i n c a l c i u m (4%) can r e s t o r e SHR i o n i z e d c a l c i u m l e v e l s t o t h a t of WKY c o n t r o l s (McCarron et a l . . 1981). C e l l u l a r p e r m e a b i l i t y t o c a l c i u m may be a l t e r e d i n the SHR animal model, l e a d i n g to changes i n c a l c i u m t r a n s p o r t . The l i p i d f l u i d i t y of i n t e s t i n a l b r ush border membrane c e l l s 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 SHR an i m a l s when compared to age-matched WKY c o n t r o l s (Lau et a l . . 1986). T h i s change i n membrane f l u i d i t y and c a l c i u m p e r m e a b i l i t y was due to an a l t e r e d l i p i d p r o f i l e i n the membrane c o m p o s i t i o n , namely a 11 decrease i n the amount of s a t u r a t e d l i p i d i n f a v o u r of u n s a t u r a t e s . The r o l e of e n d o c r i n e f a c t o r s namely, p a r a t h y r o i d hormone (PTH) and c a l c i t r i o l , i n the a l t e r e d c a l c i u m homeostasis of SHR a n i m a l s has not been c l e a r l y e l u c i d a t e d as y e t . C i r c u l a t i n g l e v e l s of immunoreactive PTH have been r e p o r t e d to be s i g n i f -i c a n t l y i n c r e a s e d i n SHR a n i m a l s over t h a t of WKY c o u n t e r p a r t s (McCarron e_t_ a_L- , 1981). The i n c r e a s e d s e c r e t i o n of PTH may have been the r e s u l t of p a r a t h y r o i d g l a n d h y p e r p l a s i a as observed i n 11 week o l d SHR a n i m a l s by Merke and coworkers (1989), as opposed to h y p e r p a r a t h y r o i d i s m . The e l e v a t i o n i n c i r c u l a t i n g l e v e l s of PTH d i d not appear to a f f e c t the h y p e r c a l c i u r i a of mature SHR a n i m a l s , which may be i n d i c a t i v e of an i m p a i r e d end-organ response t o PTH by these a n i m a l s . A l t e r n a t i v e l y , i f serum l e v e l s of PTH were not e l e v a t e d , the d i f f e r e n c e i n u r i n a r y c a l c i u m l e v e l s between SHR and WKY a n i m a l s may have been prominent at an e a r l i e r age (McCarron et a1.. 1981). Support f o r t h i s h y p o t h e s i s i s found i n the o b s e r v a t i o n s of Lau and coworkers (1986) who observed a decrease i n u r i n a r y c a l c i u m , and i n c r e a s e d c a l c i u m b a l a n c e of p r e h y p e r t e n s i v e SHR a n i m a l s compared to WKY c o u n t e r -p a r t s . The r o l e of v i t a m i n D and i t s m e t a b o l i t e s has been s i m i l a r l y u n d e f i n e d ; t h e r e are r e p o r t s of d e c r e a s e d , s i m i l a r , as w e l l as i n c r e a s e d serum c a l c i t r i o l l e v e l s i n SHR compared t o WKY a n i m a l s ( S c h e d l et_ al_. , 1984; Lau e t aj_. , 1986 ; Lucas et_ al_. , 1986). A d m i n i s t r a t i o n of v i t a m i n D3 and 2 5 - h y d r o x y v i t a m i n D3 had no 12 e f f e c t on duodenal c a l c i u m t r a n s p o r t i n SHR and WKY a n i m a l s (Toraason and W r i g h t , 1981). However, c a l c i t r i o l , the a c t i v e m e t a b o l i t e of v i t a m i n D, d i d i n c r e a s e duodenal c a l c i u m a b s o r p t i o n i n both mature SHR and WKY, a l b e i t the i n c r e a s e was not s i g n i f -i c a n t i n the SHR a n i m a l s . The d e c r e a s e d e f f e c t of c a l c i t r i o l i n SHR a n i m a l s may i n d i c a t e a d i s t u r b e d c a l c i u m h o m e o s t a t i c mechanism, or t h a t i n t e s t i n a l a c t i v e t r a n s p o r t of c a l c i u m i s a l r e a d y o p e r a t i n g at a near-maximal l e v e l (Toraason and W r i g h t , 1981). S i m i l a r l y , G a f t e r et a l . (1986) observed t h a t duodenal t i s s u e from c a l c i t r i o l t r e a t e d a n i m a l s had a g r e a t e r , but not s i g n i f i c a n t l y so, net f l u x of c a l c i u m , u s i n g an i n v i t r o U s s i n g chamber. P r e h y p e r t e n s i v e (3.5 weeks o l d ) SHR a n i m a l s e x h i b i t e d an i n c r e a s e i n serum c a l c i t r i o l l e v e l s compared to age-matched WKY c o n t r o l s (Lau e_t_ a_l_. , 1986). A c o n f l i c t i n g r e p o r t from Merke and coworkers (1989) found serum c a l c i t r i o l t o be d e c r e a s e d i n SHR a n i m a l s at 5 and 11 weeks, but not at 24 weeks of age. F u r t h e r , these workers r e p o r t e d t h a t SHR had g r e a t e r numbers of i n t e s t i n a l c a l c i t r i o l r e c e p t o r s . Much of the c o n t r o v e r s y i n the r e p o r t s of i n t e s t i n a l c a l c i u m t r a n s p o r t and plasma c a l c i t r i o l l e v e l s may be due to d i f f e r e n c e s i n the age of the SHR a n i m a l s used as w e l l as d i f f e r e n t t e c h -n i q u e s of measuring i n t e s t i n a l c a l c i u m f l u x . As w e l l , S c h e d l and coworkers (1988) i n v e s t i g a t e d the p o t e n t i a l c o n t r i b u t i o n of r o d e n t breeder v a r i a t i o n to the d i s p a r i t y of r e p o r t e d f i n d i n g s i n the c a l c i u m m e tabolism of SHR and WKY a n i m a l s . From e v e r t e d duodenal sac s t u d i e s as w e l l as measuring plasma l e v e l s of 13 v i t a m i n D m e t a b o l i t e s , they were a b l e to conclude t h a t c a l c i u m t r a n s p o r t i s d e c r e a s e d i n the SHR compared to WKY, i n d e p e n d e n t l y of breeder source and plasma c a l c i t r i o l c o n c e n t r a t i o n . The d i f f e r e n c e s i n c a l c i t r i o l l e v e l s appeared to be s u b j e c t to breeder v a r i a b i l i t y . I n c r e a s e d t i s s u e r e c e p t o r s i t e s f o r c a l c i t r i o l may be a h o m e o s t a t i c response to the d e c r e a s e d c a l c i u m t r a n s p o r t by the SHR. Taken t o g e t h e r , these d a t a suggest t h a t the mature SHR a n i m a l may be i n a h y p o c a l c e m i c s t a t e w i t h r e s p e c t to plasma i o n i z e d c a l c i u m d e s p i t e e l e v a t e d PTH l e v e l s and i n c r e a s e d t i s s u e c a l c i t r i o l r e c e p t o r s , which are unable to a m e l i o r a t e the enhanced r e n a l c a l c i u m l e a k r e p o r t e d i n these a n i m a l s . D e s p i t e numerous r e p o r t s on the abnormal c a l c i u m m e tabolism of SHR a n i m a l models, t h e r e i s a p a u c i t y of i n f o r m a t i o n on the p o t e n t i a l l i n k w i t h bone d i s o r d e r s , s p e c i f i c a l l y o s t e o p o r o s i s . D i s t u r b a n c e s i n c a l c i u m metabolism and p a r t i c u l a r l y PTH l e v e l s may impact upon the bone h e a l t h and q u a l i t y of SHR a n i m a l s i n comparison to WKY c o u n t e r p a r t s . The f i n d i n g s of Izawa and coworkers (1985), of a d e c rease i n femur c o r t e x t h i c k n e s s , bone l e n g t h , per cent c o r t i c a l a r e a , as w e l l as bone ash as a per cent d r y w e i g h t , r e s p e c t i v e l y i n mature SHR a n i m a l s compared to WKY c o u n t e r p a r t s support t h i s h y p o t h e s i s . Moreover, i n c r e a s e d numbers of 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 have been observed i n the s h a f t of the t i b i a i n SHR a n i m a l s , i n c o m b i n a t i o n w i t h a s m a l l d e c rease i n bone m i n e r a l i z a t i o n r a t e (Merke et a l . . 1989). The i n c r e a s e i n o s t e o c l a s t i c a c t i v i t y may be a r e f l e c t i o n of the 14 e l e v a t e d PTH l e v e l s r e p o r t e d i n the SHR. These f i n d i n g s suggest t h a t the SHR a n i m a l model may be at r i s k f o r the development of o s t e o p o r o t i c bone f r a g i l i t y . 2. F a c t o r s I n f l u e n c 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 A b s o r p t i o n of c a l c i u m from the d i e t i s dependent on numerous f a c t o r s i n c l u d i n g the a c t i o n of food c o n s t i t u e n t s ; and the n u t r i -t i o n a l , m e t a b o l i c and p h y s i o l o g i c a l s t a t e s , r e s p e c t i v e l y of the s u b j e c t . The m a j o r i t y of c a l c i u m i n food i s complexed w i t h o t h e r d i e t a r y c o n s t i t u e n t s . The c a l c i u m must be r e l e a s e d i n a s o l u b l e and i o n i z e d form f o r a b s o r p t i o n from the i n t e s t i n e (Pak and A v i o l i , 1988). D a i r y p r o d u c t s are the major d i e t a r y source of c a l c i u m i n the N o r t h American d i e t , p r o v i d i n g upto 75% of t o t a l c a l c i u m i n t a k e (78% i n Canada; Shahani and Kaup, 1989). In c o n t r a s t , v e g e t a b l e s , f r u i t s and g r a i n s s u p p l y j u s t 20% of d i e t a r y c a l c i u m ; w i t h meat, f i s h and p o u l t r y p r o v i d i n g the r e m a i n i n g 5% ( A l l e n , 1982). The l e v e l of d i e t a r y c a l c i u m i s i m p o r t a n t , as are the many f a c t o r s which can i n f l u e n c e the b i o a v a i l a b i l i t y of t h a t c a l c i u m , namely f i b r e components ( p h y t a t e s , u r o n i c a c i d ) , o x a l a t e s , f a t , p r o t e i n , phosphorus, l a c t o s e , M a i l l a r d Browning R e a c t i o n p r o d u c t s and v i t a m i n D. 2a. F i b r e and P h y t a t e : I t has been r e p o r t e d t h a t the a d d i t i o n of r e a s o n a b l e amounts of wholewheat, c e l l u l o s e , f r u i t s and v e g e t a b l e s to a normal d i e t c o n s i s t e n t l y r e s u l t e d i n n e g a t i v e c a l c i u m b a l a n c e s , i n s p i t e of an o t h e r w i s e adequate c a l c i u m i n t a k e (McCance and Widdowson, 1942a; Ke1say et a1. , 1979; A l l e n , 1982). T h i s area of r e s e a r c h remains 15 h i g h l y a c t i v e and c o n t r o v e r s i a l . V a r i o u s components of f i b r e have been i m p l i c a t e d i n the impairment of c a l c i u m a b s o r p t i o n . The s t r u c t u r a l p o l y s a c c h a r i d e c e l l u l o s e i s not c o n s i d e r e d t o be a c o n t r i b u t o r s i n c e pure c e l l u l o s e does not b i n d c a t i o n s i n v i t r o . H e m i c e l l u l o s e , which c o n t a i n s u r o n i c a c i d r e s i d u e s , may b i n d i o n i z e d c a l c i u m at pH 7.4 w i t h i t s charged c a r b o x y l groups. However, over 80% of d i e t a r y u r o n i c a c i d s are fermented by microbes i n the i n t e s t i n e , f r e e i n g c a l c i u m f o r a b s o r p t i o n i n the c o l o n . N o n s t r u c t u r a l p e c t i n s have a l s o been c o n s i d e r e d because of t h e i r u r o n i c a c i d c o n t e n t , however m e t h y l a t i o n of the a c i d r e s i d u e s p r e c l u d e s them from b i n d i n g c a l c i u m . P h y t a t e s b i n d c a l c i u m w i t h t h e i r i o n i z e d phosphate m o i e t i e s making the c a l c i u m u n a v a i l a b l e f o r a b s o r p t i o n . B e r l y n e et a l . (1973) r e p o r t t h a t the consumption of unleavened, wholewheat bread was a s s o c i a t e d w i t h the development of o s t e o m a l a c i a i n Bedouin p e o p l e . Reports have argued t h a t foods c o n t a i n i n g p h y t a t e s i n v a r i a b l y a l s o c o n t a i n f i b r e and t h a t i t i s the f i b r e per se which i s i n h i b i t o r y to c a l c i u m a b s o r p t i o n (McCance and Widdowson, 1942b; R e i n h o l d et  a l . . 1973; Greger, 1988). S u p p o r t i n g e v i d e n c e f o r a s m a l l e r r o l e of p h y t a t e s i n d e c r e a 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 i s found i n human s t u d i e s which i n d i c a t e t h a t p h y t a t e s can be d i g e s t e d t o some e x t e n t i n the lower i n t e s t i n e (McCance and Widdowson, 1942a; R e i n h o l d et a l . . 1973). D i g e s t i o n of the p h y t a t e s would r e l e a s e the c a l c i u m f o r a b s o r p t i o n from the c o l o n . Thus, w h i l e the r o l e of f i b r e c o n s t i t u e n t s i n the i n h i b i t i o n of c a l c i u m a b s o r p t i o n i s 16 s i g n i f i c a n t , c o n s i d e r a b l e c o n t r o v e r s y s t i l l e x i s t s as to the r e l a t i v e e f f e c t s of i n d i v i d u a l f i b r e components. 2b. O x a l a t e s : O x a l a t e s are found i n l a r g e amounts i n green l e a f y v e g e t a b l e s . Other s o u r c e s of d i e t a r y o x a l a t e s i n c l u d e t e a and c h o c o l a t e cocoa powder. O x a l a t e s are capable of b i n d i n g a l l of the c a l c i u m p r e s e n t i n l e a f y v e g e t a b l e s making i t p o o r l y absorbed (Kikunaga e_t. a l . . 1988; Doane ejt_ a l . . 1989). Moreover, o x a l a t e s may reduce the a v a i l a b i l i t y of c a l c i u m from o t h e r d i e t a r y source s consumed i n the same meal (Weaver e_t a l . . 1987 ; P o n e r o s - S c h n e i e r and Erdman, 1989) The o x a l i c a c i d c o n t e n t of c h o c o l a t e m i l k has been found to have l i t t l e e f f e c t on c a l c i u m a b s o r p t i o n from the d i e t i f the d i e t a r y c a l c i u m i n t a k e i s adequate (Roberge, 1986; Recker ejt. a_l . , 1988). 2c . F a t : Under normal c i r c u m s t a n c e s , the amount of d i e t a r y f a t i s not a f a c t o r i n c a l c i u m a b s o r p t i o n ( S t e g g e r d a and M i t c h e l l , 1951; Kaup et a l . . 1990). However, i n c o n d i t i o n s marked by f a t m a l a b s o r p t i o n , 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 can be reduced due to the f o r m a t i o n of i n s o l u b l e c a l c i u m soaps i n the lumen. The c a l c i u m i s p r e c i p i t a t e d by f a t t y a c i d s w i t h the a v a i l a b i l i t y of c a l c i u m d e c r e a s i n g as f a t t y a c i d c h a i n l e n g t h i n c r e a s e s , and as the degree of s a t u r a t i o n i n c r e a s e s ( A l l e n , 1982; Greger, 1988). T r i g l y c e r i d e s have not been observed to a f f e c t c a l c i u m a b s o r p t i o n . 2d. P r o t e i n and Phosphorus: 17 P r o t e i n i n t a k e does not a f f e c t c a l c i u m a b s o r p t i o n w i t h i n the range of the normal d i e t . However, w i t h a h i g h p r o t e i n i n t a k e , u r i n a r y c a l c i u m i s i n c r e a s e d i n d e p e n d e n t l y from the l e v e l of d i e t a r y c a l c i u m ( A l l e n e_t_ a_l_. , 1979) . T h i s i n c r e a s e i n c a l c i u r i a i s not a s s o c i a t e d w i t h an i n c r e a s e i n c a l c i u m a b s o r p t i o n , t h e r e f o r e a n e g a t i v e c a l c i u m b a l a n c e r e s u l t s . I n c r e a s i n g the i n t a k e of c a l c i u m has not been shown to a l l e v i a t e t h i s c o n d i t i o n (Zemel, 1988). P r o t e i n induced h y p e r c a l c i u r i a has been a t t r i b u t e d t o a decrease i n f r a c t i o n a l r e n a l t u b u l a r r e a b s o r p t i o n and an i n c r e a s e i n g l o m e r u l a r f i l t r a t i o n of c a l c i u m (Kim and L i n k s w i l e r , 1979; S c h u e t t e et. a l . , 1981; Chu et a l . , 1985). The f i n d i n g s of W h i t i n g and Draper (1981) r e p o r t i n g p r o t e i n s u l f u r amino a c i d c o n t e n t to be r e l a t e d to a c a l c i u r e t i c a c t i o n , f u r t h e r demonstrated t h a t the source of d i e t a r y p r o t e i n , i n a d d i t i o n to the q u a n t i t y f e d , can a d v e r s e l y a f f e c t c a l c i u m b a l a n c e . C a t a b o l -ism of the s u l f u r amino a c i d s to s u l f a t e by h e p a t i c s u l f i t e o x i d a s e (Amy, 1988) l e a d to i n c r e a s e d r e n a l a c i d e x c r e t i o n and changes i n the f i l t r a t i o n and r e a b s o r p t i o n of c a l c i u m by the k i d n e y s ( L i n k s w i l e r et a1.. 1981). The h y p e r c a l c i u r e t i c e f f e c t of p r o t e i n was observed i n s u b j e c t s f e d d i e t s of p u r i f i e d p r o t e i n s . The r e s u l t s of human s t u d i e s w i t h complex d i e t a r y p r o t e i n s such as meat are q u i t e d i f f e r e n t (Spencer e_t_ a_L. , 1978; 1983). These workers were a b l e to show t h a t d e s p i t e a h i g h meat i n t a k e over s e v e r a l months, u r i n a r y c a l c i u m e l i m i n a t i o n was not s i g n i f i c a n t l y a l t e r e d . These o b s e r v a t i o n s were a t t r i b u t e d to the f a c t t h a t r e d meat i n 18 a d d i t i o n to b e i n g h i g h i n p r o t e i n , i s a l s o r i c h i n phosphorus. D i e t a r y phosphorus has been known f o r some time to have a h y p o c a l c i u r i c e f f e c t (Spencer et a l . . 1965). T h i s h y p o c a l c i u r i c a c t i v i t y of phosphorus i s e s p e c i a l l y prominent w i t h a h i g h p r o t e i n h i g h phosphorus d i e t . Numerous animal and human s t u d i e s have r e p o r t e d t h a t the h y p e r c a l c i u r i a i n s u b j e c t s f e d h i g h p r o t e i n a l o n e , i s a m e l i o r a t e d when the phosphorus c o n t e n t of the d i e t i s i n c r e a s e d as w e l l (Hegsted et a 1., 1981; Zemel et a1., 1981; Yuen and Draper, 1983). Thus, Yuen and Draper h y p o t h e s i z e d t h a t phosphorus a f f e c t s c a l c i u m metabolism by p a r a t h y r o i d a c t i v a t i o n and subsequent e f f e c t s on bone c a l c i u m r e s o r p t i o n and i n c r e a s e d f r a c t i o n a l 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 c a l c i u m . C o n t r o v e r s y surrounds the p o s s i b l e e f f e c t t h a t phosphorus may have on o v e r a l l c a l c i u m b a l a n c e and m e tabolism ( G r e g e r , 1988). An improvement i n body c a l c i u m r e t e n t i o n does not a. p r i o r i r e s u l t i n i n c r e a s e d u t i l i z a t i o n of c a l c i u m i n d e p o s i t i o n to bone or o t h e r h o m e o s t a t i c mechanisms. C o n f l i c t i n g r e p o r t s from animal and human s t u d i e s e x i s t as to whether or not excess phosphorus c o n t r i b u t e s to i n c r e a s e d bone r e s o r p t i o n secondary to h y p e r p a r a t h y r o i d i s m (Zemel and L i n k s w i l e r , 1981; Yuen and Draper, 1983). Moreover, Greger and coworkers (1987) r e p o r t e d t h a t i n s t u d i e s on c a l c i u m u t i l i z a t i o n from c a l c i u m phosphate d i b a s i c supplements, a n i m a l s e x h i b i t e d e n l a r g e d k i d n e y s w i t h >20 f o l d i n c r e a s e s i n r e n a l c a l c i u m c o n t e n t w h i l e bone c a l c i u m c o n t e n t s were not d i s t u r b e d . 19 Normal d i e t a r y phosphorus l e v e l s have not been shown to a f f e c t c a l c i u m a b s o r p t i o n a d v e r s e l y . C o n t r o v e r s y i n t h i s area of r e s e a r c h has been confounded by the f a c t t h a t n e i t h e r phosphorus, nor c a l c i u m occur i n i s o l a t i o n i n foods or i n the human body. In g e n e r a l , a h i g h p r o t e i n i n t a k e seems to i n d i c a t e an i n c r e a s e d r e q u i r e m e n t f o r both d i e t a r y phosphorus as w e l l as c a l c i u m (Yuen and Draper, 1983; G r eger, 1988). In c o n t r a s t to r e s e a r c h on the impact of h i g h d i e t a r y l e v e l s of p r o t e i n on c a l c i u m b i o a v a i l a b i l i t y , t h e r e i s a p a u c i t y of i n f o r m a t i o n on the e f f e c t of d e f i c i e n t , or low l e v e l s of p r o t e i n on c a l c i u m b i o a v a i l a b i l i t y and u t i l i z a t i o n . T h i s i s of e s p e c i a l concern g i v e n past r e p o r t s of the 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 d i e t a r y p r o t e i n and phosphorus on c a l c i u m b a l a n c e and r e t e n t i o n . People concerned w i t h c a l c i u m r e t e n t i o n may adopt a d i e t low i n p r o t e i n to m a i n t a i n c a l c i u m h o m e o s t a s i s ; a c r o s s s e c t i o n of the p o p u l a t i o n of concern are the e l d e r l y who may be s u s c e p t i b l e to o s t e o p o r o s i s . A d i e t low i n p r o t e i n i s c o n s e q u e n t l y one t h a t i s a l s o low i n phosphorus; two components which are n e c e s s a r y f o r c o l l a g e n s y n t h e s i s and bone m i n e r a l i z a t i o n as they r e l a t e to bone growth and a r c h i t e c t u r a l m a t r i x components, which c o n t r i b u t e to bone shape, s i z e and t i s s u e c o n t e n t ( F r o s t , 1988). 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 has been r e p o r t e d to have d e l e t e r i o u s e f f e c t s on s k e l e t a l c o r t i c a l bone t h i c k n e s s i n both young a d u l t s and c h i l d r e n (Garn et a l . . 1964; Crosby et a l . . 1983). Moreover, 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 t i o n i n bone l e n g t h i n humans s u f f e r i n g from a p r o t e i n d e f i c i e n c y (Adams 20 and B e r r i d g e , 1969; P a r f i t t , 1983). I t i s noteworthy t h a t a n e g a t i v e p r o t e i n b a l a n c e has been shown to r e s u l t i n a lower body weight g a i n and reduced bone c o l l a g e n c o n t e n t , but not to a f f e c t bone c a l c i u m c o m p o s i t i o n ( B o l l e t , 1973). D i e t a r y phosphorus d e f i c i e n c y can l e a d to d i s t u r b e d bone metabolism and g e n e r a l m a l a i s e ( L o t z et a l . . 1968). Taken t o g e t h e r , these r e p o r t s suggest t h a t the r e s t r i c t i o n of d i e t a r y p r o t e i n and phosphorus i s c o n t r a i n d i c a t e d as a t h e r a p y to improve c a l c i u m metabolism and r e t e n t i o n i n the young and e l d e r l y a l i k e . The q u a l i t y of d i e t a r y p r o t e i n may be a c o n t r i b u t i n g f a c t o r i n the b i o a v a i l a b i l i t y of c a l c i u m from the d i e t . T h i s concept i s h i g h l i g h t e d by the f a c t t h a t p r o t e i n s , and e s p e c i a l l y those of d a i r y o r i g i n , are seldom consumed i n the n a t i v e form, but r a t h e r , are s u b j e c t e d to t h e r m a l p r o c e s s i n g e i t h e r p r i o r to consumption, or d u r i n g p a s t e u r i z a t i o n . Heat t r e a t m e n t can reduce the n u t r i t i o n a l v a l u e of the m i l k c a s e i n p r o t e i n s and a f f e c t c a l c i u m a b s o r p t i o n . A l t e r a t i o n s to the c a s e i n p o l y p e p t i d e s from t h e r m a l t r e a t m e n t i n c l u d e i n t e r a c t i o n of d e n a t u r e d whey p r o t e i n s ( p -l a c t o g l o b u l i n ) w i t h K - c a s e i n ; c r o s s - l i n k i n g of p r o t e i n s ; d e p h o s p h o r y l a t i o n of p h o s p h o s e r y l r e s i d u e s ; and M a i l l a r d browning r e a c t i o n s (Swaisgood, 1985). P a s t e u r i z a t i o n or s t e r i l i z a t i o n p r o c e s s i n g of f l u i d m i l k r e s u l t s i n the d e n a t u r a t i o n of whey p r o t e i n s . Of p a r t i c u l a r i n t e r e s t i s ( J - l a c t o g l o b u l i n , which forms an a s s o c i a t i o n w i t h K-c a s e i n v i a a t h i o l - d i s u l p h i d e i n t e r c h a n g e . P r o t e i n s can be c r o s s - l i n k e d as a r e s u l t of the r e a c t i o n of d e h y d r o a l a n y l 21 r e s i d u e s (from B - e l i m i n a t i o n of c y s t i n e , p h o s p h o s e r i n e or s e r i n e ) w i t h l y s i n e r e s i d u e s at the e-amino group p o s i t i o n t o form l y s i n o a l a n i n e (Swaisgood, 1985). Heat tr e a t m e n t of m i l k i n the range 110 to 140°C r e s u l t s i n e x t e n s i v e d e p h o s p h o r y l a t i o n of the c a s e i n p o l y p e p t i d e s at p h o s p h o s e r y l r e s i d u e s due to s c i s s i o n of an oxygen-phosphorus bond (Howat and W r i g h t , 1934; B e l e c and Jenness, 1962a,b). M a i l l a r d Browning R e a c t i o n p r o d u c t s can be formed from the c o n d e n s a t i o n of p o l y p e p t i d e amino a c i d r e s i d u e e-amino groups w i t h l a c t o s e t o form m e l a n o i d i n s . The a v a i l a b i l i t y of amino a c i d s such as l y s i n e i s reduced under these c o n d i t i o n s . Moreover, m e l a n o i d i n s may b i n d t o i o n i c c a l c i u m , p o t e n t i a l l y r e d u c i n g m i n e r a l b i o a v a i l a b i l i t y (Rendleman, 1987). Taken t o g e t h e r , these e f f e c t s of t h e r m a l p r o c e s s i n g of m i l k and o t h e r d i e t a r y p r o t e i n s , may reduce t h e i r d i g e s t i b i l i t y and b i o l o g i c a l v a l u e (Mauron, 1973; D e s r o s i e r s ejt. a 1 . . 1987). D e s p i t e the e f f e c t of t h e r m a l t r e a t m e n t on m i l k p r o t e i n q u a l i t y , Weeks and K i n g (1985) d i d not observe any adverse e f f e c t s of heat p r o c e s s i n g on c a l c i u m a b s o r p t i o n from p a s t e u r i z e d m i l k . S u p p o r t i n g e v i d e n c e f o r these o b s e r v a t i o n s i s the work of Ho and Waugh (1965), who r e p o r t e d t h a t m i l k m i c e l l a r c a l c i u m . i s a s s o c i a t e d w i t h not o n l y c a s e i n p h o s p h o s e r y l r e s i d u e s , but a l s o p o s s i b l y w i t h c a r b o x y l groups. Thus, m i l k p r o t e i n s dephosphor-y l a t e d by heat t r e a t m e n t may s t i l l b i n d i o n i z e d c a l c i u m k e e p i n g i t h i g h l y b i o a v a i l a b l e . 2e. L a c t o s e : 22 V a r i o u s hypotheses have been suggested t o e x p l a i n the mechanism of the l a c t o s e e f f e c t on c a l c i u m b i o a v a i l a b i l i t y and a b s o r p t i o n ( K l i n e et. a l . , 1932; S c h a c h t e r e_t al_. , 1961; C h a r l e y and Saltman, 1963). These i n c l u d e d the r e d u c t i o n of i n t e s t i n e l u m i n a l pH and enhancement of c a l c i u m s o l u b i l i t y due to the f e r m e n t a t i o n of l a c t o s e by i n t e s t i n a l m i c r o f l o r a ( K l i n e et a1. . 1932); the f o r m a t i o n of a c a l c i u m - l a c t o s e complex f o r co-a b s o r p t i o n ( C h a r l e y and Saltman, 1963); and the i n h i b i t i o n of i n t e s t i n a l mucosa c e l l u l a r a e r o b i c m e t abolism to d i s t u r b mucosal c e l l p atency and a l l o w leakage of c a l c i u m to the s e r o s a ( S c h a c h t e r e_t_ a_l_. , 1961). These t h e o r i e s have been r e f u t e d i n l i g h t 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 i s not unique to t h i s sugar a l o n e , and f u r t h e r t h a t o p t i m a l l u m i n a l c o n c e n t r a t i o n s of both c a l c i u m and l a c t o s e are r e q u i r e d f o r an e f f e c t (Vaughan and F i l e r , I960; Armbrecht and Wasserman, 1976). The m i l k s u g a r , l a c t o s e , has been r e p o r t e d to enhance the b i o a v a i l a b i l i t y of c a l c i u m and p o s s i b l y o t h e r m i n e r a l s (Lengemann et a1. , 1959; Greger et a 1 ., 1987) . T h i s d i s a c c h a r i d e has been shown to enhance the p a r a c e l l u l a r t r a n s p o r t of c a l c i u m i n both the p r o x i m a l as w e l l as d i s t a l s m a l l i n t e s t i n a l segments (Armbrecht and Wasserman, 1976; Armbrecht, 1987). The e f f e c t was independent of c a l c i u m i n t a k e , age and the v i t a m i n D e n d o c r i n e system (Lengemann et a l . . 1959; Behar and K e r s t e i n , 1976; N e l l a n s and Kimberg, 1978; Armbrecht, 1987). Lengemann and coworkers ( 1 9 5 9 ) , i n i t i a l l y i d e n t i f i e d the i l e u m as the s i t e of l a c t o s e enhanced c a l c i u m a b s o r p t i o n . F u r t h e r , Pansu e t a1. ( 1979) 23 r e p o r t e d t h a t c a l c i u m a b s o r p t i o n was enhanced i n a n i m a l s f e d l a c t o s e , d e s p i t e the f a c t t h a t both the amount of c a l c i u m b i n d i n g p r o t e i n , as w e l l as the a c t i v e t r a n s p o r t of c a l c i u m were reduced i n the p r o x i m a l s m a l l i n t e s t i n e . T h i s demonstrated the p o t e n t i a l importance of the p a s s i v e , 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 p r o c e s s i n the lower i n t e s t i n e , or i l e u m , by l a c t o s e mediated enhancement of c a l c i u m b i o a v a i l a b i l i t y . L a c t o s e mediated enhancement of c a l c i u m a b s o r p t i o n has been r e p o r t e d to be c h a r a c t e r i z e d by a f l u i d s h i f t from the i l e u m as w e l l as an i n c r e a s e d e f f l u x of c a l c i u m from mucosa to s e r o s a (Behar and K e r s t e i n , 1976). The f l u i d s h i f t from the i l e u m i s dependent upon an o s m o t i c a l l y a c t i v e l o a d of l a c t o s e w i t h i n the lumen which would d i s t u r b mucosal c e l l p atency i n c r e a s i n g the space between c e l l s ( B r o n n e r , 1987). S t u d i e s i n a post-weaning rodent a n i m a l model as w e l l as a d u l t humans support t h i s t h e o r y s i n c e both l a c k i n t e s t i n a l l a c t a s e a c t i v i t y and so, do not h y d r o l y z e t h i s d i s a c c h a r i d e , a l l o w i n g i t to r e a c h the d i s t a l s m a l l i n t e s t i n e i n t a c t ( L e i c h t e r and T o l e n s k y , 1975). 2 f . M a i l l a r d Browning R e a c t i o n P r o d u c t s : 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 , are c h a r a c t e r i z e d by the c o n d e n s a t i o n of p r o t e i n amino a c i d r e s i d u e s w i t h r e d u c i n g sugars to form m e l a n o i d i n s . I n v e s t i g a t i o n s have been c a r r i e d out w i t h numerous s u g a r s , namely g l u c o s e , f r u c t o s e , g a l a c t o s e , m a l t o s e , l a c t o s e , rhamnose and p e n t o s e s , w i t h p r i m a r y and secondary amino a c i d s i n the f r e e form or the e-amino group of l y s i n e (Powrie et a1. . 1986). The browning r e a c t i o n p r o d u c t s 24 ( m e l a n o i d i n s ) c o n t r i b u t e to the c o l o u r ( p i g m e n t s ) , aroma and f l a v o u r of cooked f o o d s . S t u d i e s conducted by Homma et a1. (1986) r e p o r t e d a copper c h e l a t i n g p o t e n t i a l of browning components p r e s e n t i n c o f f e e . A d h i k a r i and Tappel (1973) p r o v i d e d f u r t h e r e v i d e n c e f o r the f u n c t i o n a l group of g l u c o s e -g l y c i n e m e l a n o i d i n s t h a t p o ssessed metal c h e l a t i n g p o t e n t i a l . Rendleman (1987) examined the c a l c i u m c h e l a t i n g p o t e n t i a l of m e l a n o i d i n s , which was thought t o be r e l a t e d to the number of a c i d i c donor groups i n the f o o d pigment, i n b oth a model browning r e a c t i o n , as w e l l as browning pigments d e r i v e d from a c o f f e e brew. These s t u d i e s showed no measurable c a l c i u m b i n d i n g a c t i v i t y of m e l a n o i d i n s i n pigments from c o f f e e and t o a s t . Thus, the c a l c i u m b i n d i n g p o t e n t i a l of m e l a n o i d i n s d e r i v e d from food systems and model r e a c t i o n s , r e s p e c t i v e l y may be q u i t e d i f f e r e n t . 2g. M i n e r a l I n t e r a c t i o n s : Because n u t r i e n t s , and t h e r e f o r e m i n e r a l s , do not e x i s t i n i s o l a t i o n i n e i t h e r food or p h y s i o l o g i c a l systems, i n t e r a c t i o n s must be c o n s i d e r e d i n any d i s c u s s i o n of n u t r i e n t b i o a v a i l a b i l i t y and m e t a b o l i s m . I n t e r a c t i o n s w i t h o t h e r m i n e r a l s , or food c o n s t i t u e n t s may p l a y a r o l e i n the r e l a t i v e b i o a v a i l a b i l i t y of a n u t r i e n t such as c a l c i u m . C a l c i u m a b s o r p t i o n and m etabolism i n r e l a t i o n to i n t e r a c t i o n s w i t h z i n c and magnesium have been d i s c u s s e d and r e v iewed i n the l i t e r a t u r e (Forbes et a1., 1979; G r e g er, 1989 ; Greger et. a l _ . , 1989). An area of p a r t i c u l a r concern i n the b i o a v a i l a b i l i t y of z i n c and c a l c i u m i s the e f f e c t of p h y t a t e on the a b s o r p t i o n of these 25 m i n e r a l s . Research i n t h i s area has i n c r e a s e d a l o n g w i t h i n t e r e s t i n soy p r o d u c t s as p r o t e i n s o u r c e s . From i n v i t r o s t u d i e s , p h y t a t e complexes of c a l c i u m and z i n c t o g e t h e r are noted to be l e s s s o l u b l e than complexes of the i n d i v i d u a l metals ( O b e r l e a s , 1973; Champagne and P h i l l i p p y , 1989). Moreover, Forbes et a1 . ( 1979) r e p o r t e d t h a t a d d i t i o n of c a l c i u m as c a l c i u m c a r b o n a t e to soy p r o d u c t s r e s u l t e d i n de c r e a s e d u t i l i z a t i o n of z i n c by r a t s . These o b s e r v a t i o n s are s u p p o r t e d by the f a c t t h a t z i n c b i o a v a i l a b i l i t y i s w e l l r e c o g n i z e d as b e i n g s u s c e p t i b l e to d i e t a r y p h y t a t e l e v e l s ( G r e g e r , 1989). T h i s m u l t i p l e i n t e r a c t i o n i s n oteworthy i n l i g h t of the o b s e r v a t i o n s of Fordyce ejt a l . (1987) who r e p o r t e d a r e l a t i o n s h i p between bone z i n c l e v e l s and p h y t a t e x c a l c i u m / z i n c r a t i o s i n the d i e t . C a l c i u m and magnesium have been r e p o r t e d to have p o s s i b l e a n t a g o n i s t i c e f f e c t s on t h e i r r e s p e c t i v e b i o a v a i l a b i l i t y and meta b o l i s m (Greger e_t_ al_. , 1987; Greger, 1989). These two e s s e n t i a l m i n e r a l s can a f f e c t the r e l a t i v e amounts of each absorbed from the d i e t ( G r e g e r , 1989). T h i s r e l a t i o n s h i p i s worthy of c o n s i d e r a t i o n due t o the p r o l i f e r a t i o n of c a l c i u m supplements which can a l s o c o n t a i n s i g n i f i c a n t amounts of magnesium. Greger and coworkers (1987) s t u d i e d the b i o a v a i l -a b i l i t y of v a r i o u s c o m m e r c i a l l y a v a i l a b l e c a l c i u m supplements, some of which a l s o c o n t a i n e d s u p p l e m e n t a l magnesium, i n r a t s . T h e i r f i n d i n g s demonstrated t h a t r a t s f e d the magnesium c o n t a i n i n g p r e p a r a t i o n s were l e s s a b l e to absorb and u t i l i z e c a l c i u m f o r bone m i n e r a l i z a t i o n than c o u n t e r p a r t s f e d m i l k . 26 A l t e r n a t i v e l y , s t u d i e s i n humans have shown t h a t the i n g e s t i o n of excess c a l c i u m can i m p a i r i n t e s t i n a l a b s o r p t i o n of magnesium and o t h e r m i n e r a l s (Greger et a l . . 1981). Thus, h i g h l e v e l s of d i e t a r y c a l c i u m i n e f f e c t i n c r e a s e the magnesium requirement ( M o r r i s and O ' D e l l , 1963; Greger e_t aj_. , 1981). I n t a k e s of c a l c i u m and magnesium are of f u r t h e r concern w i t h r e g a r d 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. In c o n d i t i o n s of magnesium d e f i c i e n c y and adequate c a l c i u m and phosphorus i n t a k e , the development of r e n a l c a l c u l i i s a p o s s i b i l i t y (Greger e_t a 1 . . 1987). However, i n s t u d i e s w i t h humans, Greger and coworkers (1981) d i d not d e t e c t a s i g n i f i c a n t e f f e c t of v a r y i n g d i e t a r y c a l c i u m and phosphorus l e v e l s on r e t e n t i o n of c a l c i u m and magnesium. L a c t o s e can a f f e c t the i n t e s t i n a l a b s o r p t i o n of not o n l y c a l c i u m , but a l s o magnesium and z i n c (Greger et a1. . 1989). In 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 v a r i o u s forms of c a l c i u m ( c a l c i u m phosphate d i b a s i c , d o l o m i t e , amino a c i d c a l c i u m c h e l a t e and n o n f a t dry m i l k ) , these workers r e p o r t e d a g r e a t e r d e p o s i t i o n of magnesium and z i n c to bone from d o l o m i t e and m i l k based d i e t s . However, c a l c i u m b i o a v a i l a b i l i t y d i f f e r e d between these d i e t a r y s o u r c e s , b e i n g absorbed and u t i l i z e d l e s s from d o l o m i t e when compared to c a l c i u m phosphate d i b a s i c and m i l k , r e s p e c t i v e l y . I t i s i n s t r u c t i v e to n o t e , t h a t d o l o m i t e i s a d i e t a r y supplement r i c h i n both c a l c i u m and magnesium carbon-a t e s . Thus, the l a c t o s e enhancing e f f e c t on c a l c i u m a b s o r p t i o n may be a l t e r e d by m i n e r a l i n t e r a c t i o n s (Greger e t a1.. 1989). 27 2h. E n d o c r i n e F a c t o r s : C a l c i u m a b s o r p t i o n and metabolism are r e g u l a t e d by a number of e n d o c r i n e hormones, namely v i t a m i n D ( 1 , 2 5 - d i h y d r o x y c h o l e -c a l c i f e r o l ) , p a r a t h y r o i d hormone (PTH) and c a l c i t o n i n ( C T). These hormones f u n c t i o n to keep c i r c u l a t i n g plasma, or serum l e v e l s of c a l c i u m r e l a t i v e l y c o n s t a n t ( F i g u r e 1 ) . The 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 abolism has been e x t e n -s i v e l y r e v i e w e d ( D e l u c a , 1981; D e l v i n , 1986; E r a s e r , 1988). V i t a m i n D l a r g e l y has i t s e f f e c t s on 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 , s p e c i f i c a l l y i n the p r o x i m a l r e g i o n of the s m a l l i n t e s t i n e , the duodenum and upper jejunum. Thus, v i t a m i n D r e g u l a t e s the a c t i v e , t r a n s c e l l u l a r component of c a l c i u m a b s o r p t i o n . V i t a m i n D can be o b t a i n e d t h rough two r o u t e s , the d i e t or by exposure to sun or u l t r a v i o l e t (UV) l i g h t . D i e t a r y v i t a m i n D, termed v i t a m i n D2 ( e r g o s t e r o l ) i s o b t a i n a b l e from f o r t i f i e d m i l k , f a t t y f i s h , cheese, b u t t e r , eggs and l i v e r ( A l l e n , 1982). UV l i g h t d e r i v e d v i t a m i n D i s the p r o d u c t of the c o n v e r s i o n of a p r e c u r s o r , 7-d e h y d r o c h o l e s t e r o l , t o v i t a m i n D 3 ( c a l c i f e r o l ) i n the s k i n . V i t a m i n D2 and D 3 are c i r c u l a t e d to the l i v e r where h e p a t o c y t e microsomal v i t a m i n D 3-25-hydroxylase c o n v e r t s them to 25-h y d r o x y v i t a m i n D. T h i s m e t a b o l i t e i s then t r a n s l o c a t e d to the k i d n e y f o r c o n v e r s i o n to the f i n a l a c t i v e form of the v i t a m i n , 1 , 2 5 - d i h y d r o x y c h o l e c a l c i f e r o l ( c a l c i t r i o l ) by m i t o c h o n d r i a l 1-a-h y d r o x y l a s e . The importance of s u n l i g h t exposure i s apparent as 84% of c i r c u l a t i n g serum 2 5 - h y d r o x y - v i t a m i n D i s d e r i v e d from the c a l c i f e r o l p r e c u r s o r , not e r g o s t e r o l . 28 C a l c i t r i o l a c t s to s t i m u l a t e 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 at the l e v e l of CaBP s y n t h e s i s . C e l l s of the i n t e s t i n a l mucosa have c y t o p l a s m i c r e c e p t o r s i t e s f o r t h i s hormone to s i g n a l f o r the i n i t i a t i o n of CaBP s y n t h e s i s (Norman, 1979). T h i s r o l e of c a l c i t r i o l i s not y e t c l a r i f i e d , as a d m i n i s t r a t i o n of c a l c i t r i o l may induce enhanced a c t i v e t r a n s p o r t of c a l c i u m from the i n t e s t i n a l lumen p r i o r to i n c r e a s e d c y t o p l a s m i c l e v e l s of CaBP (Spencer et a l . . 1978). N e v e r t h e l e s s , the importance of v i t a m i n D i s c l e a r i n t h a t c i r c u l a t i n g l e v e l s of c a l c i t r i o l i n c r e a s e i n c o n d i t i o n s of c a l c i u m d e f i c i e n c y and pregnancy and l a c t a t i o n where the c a l c i u m r e q u i r e m e n t i s h i g h ( A l l e n , 1982). P a r a t h y r o i d hormone (PTH) i s a c t i v e at a number of l e v e l s of c a l c i u m m e tabolism. S t i m u l a t i o n of s e c r e t i o n of PTH o c c u r s under c o n d i t i o n s marked by h y p o c a l c e m i a ( P a t t and L u c k h a r d t , 1942). In t h i s way, PTH r e g u l a t e s the c a l c i u m - p h o s p h a t e b a l a n c e between the b l o o d and o t h e r t i s s u e s . 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 c a l c i u m b a l a n c e by PTH t a k e s p l a c e i n the k i d n e y s , bone and s e c o n d a r i l y , the i n t e s t i n e ( A urbach, 1988). Renal c a l c i u m e x c r e t i o n i s i n h i b i t e d by PTH which b i n d s to r e c e p t o r s i t e s i n the d i s t a l t u b u l e which s i g n a l s r e a b s o r p t i o n of c a l c i u m from the g l o m e r u l a r f i l t r a t e t o be i n c r e a s e d (Agus et_ a l . . 1981). C a l c i u m t r a n s -l o c a t i o n from the s k e l e t o n to i n c r e a s e e x t r a c e l l u l a r c a l c i u m l e v e l s i s another f u n c t i o n of PTH. The r a t e of bone r e s o r p t i o n i s enhanced by PTH as e v i d e n c e d by i n c r e a s e d numbers and a c t i v i t y of o s t e o c l a s t s , the bone r e s o r b i n g c e l l s (Peck and K l a h r , 1979). S i n c e bone c a l c i u m i s a s s o c i a t e d w i t h phosphate, plasma l e v e l s of 29 phosphate i n c r e a s e w i t h t h a t of plasma c a l c i u m . The excess of phosphate i s compensated f o r by PTH mediated s t i m u l a t i o n of r e n a l phosphate e x c r e t i o n (Agus et. a 1 . . 1981). The i n c r e a s e i n 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 by PTH i s secondary to i t s e f f e c t s on r e n a l h y d r o x y l a t i o n of 2 5 - h y d r o x y v i t a m i n D ( F r a s e r , 1980). Thus, PTH i n c r e a s e s the a c t i v i t y of the r e n a l 1-a-hydroxylase enzyme t o produce c a l c i t r i o l which then has i t s e f f e c t s on the c e l l s of the i n t e s t i n a l mucosa to i n c r e a s e c a l c i u m a b s o r p t i o n . These f u n c t i o n s of PTH are r e s p o n s i b l e f o r the m o b i l i z a t i o n of c a l c i u m from body p o o l s and i n t e s t i n a l c o n t e n t s to e q u i l i b r a t e the p o o l of e x t r a c e l l u l a r c a l c i u m . C a l c i t o n i n ( C T ) , a 32-amino a c i d p o l y p e p t i d e hormone, i s s e c r e t e d by the t h y r o i d g l a n d ( Aurbach, 1988). The p r i m a r y f u n c t i o n of CT i s h y p o c a l c e m i c i n n a t u r e , due to i t s i n h i b i t i o n of bone r e s o r p t i o n and t h e r e f o r e , i n h i b i t i o n of c a l c i u m r e l e a s e from the bone (Talmage et a l . , 1983). Thus, CT i n h i b i t s the e f f e c t s of PTH on the 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 p r e s e r v e s k e l e t a l i n t e g r i t y . 2 i . Impact of 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 : I n d i v i d u a l n u t r i t i o n a l s t a t u s can a l s o impact on c a l c i u m a b s o r p t i o n and m e t a b o l i s m . 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 and v i t a m i n D d e f i c i e n c i e s , r e s p e c t i v e l y , as w e l l as m a l a b s o r p t i o n syndromes. C a l c i u m d e f i c i e n c y , or n e g a t i v e c a l c i u m b a l a n c e , i s c h a r a c t e r i z e d by an i n c r e a s e i n the e f f i c i e n c y of 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 (Pansu et a l . . 1981). The a c t i v e , t r a n s -c e l l u l a r t r a n s p o r t of c a l c i u m w i l l be u p - r e g u l a t e d i f the host i s 30 v i t a m i n D r e p l e t e ( B r o n n e r , 1987). T h i s mechanism i s mediated by PTH s e c r e t i o n i n c o n d i t i o n s of h y p o c a l c e m i a to s t i m u l a t e c a l c i t r i o l s y n t h e s i s (Norman, 1979). F u r t h e r m o r e , s t u d i e s have shown t h a t the c o l o n may have an i n c r e a s e d r o l e i n c a l c i u m a b s o r p t i o n i n s t a t e s of c a l c i u m d e f i c i e n c y (Favus et a l . . 1980). The c o l o n can p l a y an i m p o r t a n t r o l e i n c a l c i u m a b s o r p t i o n as o bserved by H y l ander and coworkers (1980) i n s u b j e c t s who had undergone i n t e s t i n a l r e s e c t i o n s . A d a p t a t i o n to a c a l c i u m d e f i c i e n t d i e t i s p o s s i b l e over an extended p e r i o d of t i m e , a l b e i t not to an e x t e n t t h a t would p r e v e n t a n e g a t i v e balance (Spencer et_ a l . . , 1969). C l a s s i c a l l y , v i t a m i n D d e f i c i e n c y has been l i n k e d to i n s u f f i c i e n t s u n l i g h t exposure and the bone d i s e a s e , r i c k e t s ( A l l e n , 1982). V i t a m i n D d e f i c i e n c y has been thought to be a c a u s a l agent of bone m a l f o r m a t i o n and d e f i c i e n t m i n e r a l i z a t i o n i n c h i l d r e n and a d u l t s . In v i t a m i n D-dependent r i c k e t s , admin-i s t r a t i o n of the v i t a m i n w i l l r e s u l t i n i n c r e a s e d s y n t h e s i s of CaBP w i t h i n 6 to 8 hours (Norman, 1979). The e f f i c a c y of v i t a m i n D i n the p r e v e n t i o n of r i c k e t s l e a d to the f o r t i f i c a t i o n of f l u i d m i l k w i t h v i t a m i n D2 (400 IU per l i t r e ; A l l e n , 1982). A c a u t i o n a r y note about v i t a m i n D h y p e r a l i m e n t a t i o n i s n e c e s s a r y because of the t o x i c i t y of excess v i t a m i n D. T h i s i s e s p e c i a l l y p e r t i n e n t i n r e f e r e n c e to c a l c i u m supplements which a l s o c o n t a i n s u p p l e m e n t a l v i t a m i n D. Excess v i t a m i n D i n t a k e can l e a d to h y p e r c a l c e m i a , excess bone c a l c i f i c a t i o n and c a l c i f i c a t i o n of the s o f t t i s s u e s ( F r a s e r , 1988). Moreover, s t u d i e s have shown t h a t 31 v i t a m i n D, s u f f i c i e n t f o r s e v e r a l months i s produced i n the s k i n w i t h o n l y a few hours of summer s u n l i g h t exposure ( P o s k i t t .et_ a l . . 1979). C a l c i u m a b s o r p t i o n and metabolism may be i m p a i r e d by m a l a b s o r p t i o n syndromes secondary to f a t m a l a b s o r p t i o n , c h r o n i c a l c o h o l i s m , and s u r g i c a l p r o c e d u r e s . The e f f e c t s of f a t m a l a b s o r p t i o n on 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 have been d i s c u s s e d above. C h r o n i c a l c o h o l i s m has been l i n k e d t o s k e l e t a l d e m i n e r a l i z a t i o n and d e c r e a s e d bone mass ( S a v i l l e , 1965). C h r o n i c a l c o h o l i s m may mediate c a l c i u m m a l a b s o r p t i o n through f a t m a l a b s o r p t i o n and subsequent e f f e c t s on c a l c i u m and v i t a m i n D s t a t u s (Hepner et a_l_. , 1976). Furthermore, these workers demonstrated t h a t l i v e r c i r r h o s i s may have a n e g a t i v e impact on the a c t i v i t y of h e p a t i c v i t a m i n D 3 - 2 5 - h y d r o x y l a s e . S u r g i c a l p rocedures t h a t can r e s u l t i n m a l a b s o r p t i o n of n u t r i e n t s , and c a l c i u m i n p a r t i c u l a r , are g a s t r o i n t e s t i n a l t r a c t r e s e c t i o n s . The e f f e c t i s secondary to a r e d u c t i o n i n a b s o r p t i v e a r e a , and m a l a b s o r p t i o n of f a t and v i t a m i n D (Compston et a1. . 1980). Over t i m e , c a l c i u m m a l a b s o r p t i o n i s compensated f o r by i n c r e a s e d i l e a l and c o l o n i c c o n t r i b u t i o n s t o c a l c i u m a b s o r p t i o n ( H y l a n d e r et a l . . 1980). 2 j . P h y s i o l o g i c a l S t a t u s : C a l c i u m r e q u i r e m e n t s and a b s o r p t i v e c a p a c i t y can be seen to v a r y at s p e c i f i c s t a g e s of l i f e , namely i n f a n c y , a d o l e s c e n c e , pregnancy and l a c t a t i o n , and menopause and o l d age ( A l l e n , 1982). In i n f a n c y , both a n i m a l s and humans e x h i b i t a r e l i a n c e on the 32 d i f f u s i o n a l , p a r a c e l l u l a r component of c a l c i u m a b s o r p t i o n ( B a r l t r o p et_ al_. , 1977 ; Ghishan et_ al_. , 1980). In the r a t , the s a t u r a b l e , t r a n s c e l l u l a r t r a n s p o r t of c a l c i u m i s not o p e r a t i v e u n t i l the t h i r d week of l i f e ( Ghishan et a l • . 1980). Evidence f o r a d i f f u s i o n a l component i n c a l c i u m a b s o r p t i o n by human i n f a n t s was p r o v i d e d by s t u d i e s of B a r l t r o p et a1. ( 1 9 7 7 ) , who observed a l i n e a r dose response from m i l k - b a s e d f o r m u l a . A d o l e s c e n c e r e p r e s e n t s a p e r i o d of a c c e l e r a t e d growth and bone t i s s u e development. D u r i n g t h i s t i m e , the l o n g bones i n c r e a s e i n l e n g t h , w i d t h as w e l l as d e n s i t y (Shahani and Kaup, 1989). The r e q u i r e m e n t f o r c a l c i u m and n u t r i e n t s f o r bone development i s i n c r e a s e d at t h i s time l e a d i n g to recommendations of 1 t o 1.5g c a l c i u m per day to f a c i l i t a t e r a p i d s k e l e t a l growth i n a d o l e s c e n c e ( G r i m s t o n and Hanley, 1990). Pregnancy and l a c t a t i o n p l a c e a d d i t i o n a l demands f o r c a l c i u m on the mother, due to f e t a l and m i l k p r o d u c t i o n r e q u i r e m e n t s , r e s p e c t i v e l y . The i n c r e a s e d demand f o r c a l c i u m i s c o u p l e d w i t h changes i n the e f f i c i e n c y and a b s o r p t i v e s u r f a c e c a p a c i t y of i n t e s t i n a l c a l c i u m t r a n s p o r t . S t u d i e s by K o s t i a l et a1. (1969) u s i n g a l a c t a t i n g r a t a n i m a l model, i n d i c a t e d t h a t both the t r a n s c e l l u l a r as w e l l as p a r a c e l l u l a r components of c a l c i u m a b s o r p t i o n were i n c r e a s e d . S i m i l a r l y , u s i n g an i n v i t r o e v e r t e d i n t e s t i n a l sac methodology, Wrobel and Nagel (1980) were a b l e to demonstrate i n c r e a s e d i n t e s t i n a l c a l c i u m t r a n s p o r t by r a t s . T h i s i n c r e a s e was i n i t i a t e d d u r i n g the second week of g e s t a t i o n , peaked at p a r t u r i t i o n and d e c r e a s e d d u r i n g l a c t a t i o n . F u r t h e r , 33 changes i n the a b s o r p t i v e s u r f a c e area of the i n t e s t i n e are a l s o observed to o c c u r ; the l e n g t h of jejunum a v a i l a b l e f o r a c t i v e t r a n s p o r t of c a l c i u m was i n c r e a s e d . Taken t o g e t h e r , these s t u d i e s i n d i c a t e t h a t m a t e r n a l c a l c i u m a b s o r p t i o n mechanisms are a b l e to compensate f o r the i n c r e a s e d c a l c i u m demands of pregnancy and l a c t a t i o n p r o v i d e d t h a t c a l c i u m i n t a k e i s adequate. C a l c i u m a b s o r p t i o n appears to be i m p a i r e d i n the e l d e r l y , and more p a r t i c u l a r l y , i n the post-menopausal female. Both men and women d i s p l a y a d e c r e a s e d a b i l i t y t o absorb c a l c i u m once past the age of 60 y e a r s . F u r t h e r , those between the ages of 70 to 90 years absorb one t h i r d as much c a l c i u m as c o u n t e r p a r t s between the ages of 20 to 59 y e a r s (Shahani and Kaup, 1989). A g e - r e l a t e d c a l c i u m m a l a b s o r p t i o n appears to be the r e s u l t of a d e c r e a s e i n the t r a n s c e l l u l a r a b s o r p t i o n component ( N o r d i n e_t_ a_l_. , 1976 ; Armbrecht e_t_ al_. , 1979). In the case of the postmenopausal female, the decrease i n c i r c u l a t i n g l e v e l s of e s t r o g e n t h a t o c c u r s at the menopause, i s c o n s i d e r e d to be r e s p o n s i b l e f o r the r e d u c t i o n i n c a l c i u m a b s o r p t i o n ( N o r d i n et a1. . 1976). The r o l e of e s t r o g e n i n c a l c i u m metabolism and homeostasis has been suggested i n e i t h e r of two t h e o r i e s ( A l l e n , 1986). In the f i r s t , e s t r o g e n a c t s to reduce the r a t e of bone r e s o r p t i o n which would l e a d to a d e c l i n e i n serum c a l c i u m and t h e r e f o r e , an i n c r e a s e i n PTH. 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 would be enhanced by the e f f e c t of PTH on c a l c i t r i o l s y n t h e s i s . In the second, e s t r o g e n would have a d i r e c t s t i m u l a t o r y e f f e c t on c a l c i t r i o l s y n t h e s i s and t h u s , on 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 . Compounding the 34 e f f e c t of de c r e a s e d 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 i n the p o s t -menopausal female are the c o e x i s t i n g c o n d i t i o n s of decrea s e d r e n a l c a l c i u m r e t e n t i o n and f i n a l l y a n e g a t i v e c a l c i u m b a l a n c e (Heaney et a 1 . . 1978). The n e g a t i v e c a l c i u m b a l a n c e t h a t o c c u r s i n the post-menopausal female makes her e s p e c i a l l y prone to bone r e s o r p t i o n i n the m o b i l i z a t i o n of c a l c i u m to m a i n t a i n serum l e v e l s . Bone r e s o r p t i o n can l e a d t o a c c e l e r a t e d l o s s of bone mass and e v e n t u a l l y , t o o s t e o p o r o s i s i n the post-menopausal female ( A l o i a et a l . . 1985). 3. D a i r y Foods D a i r y p r o d u c t s r e p r e s e n t an e x c e l l e n t source of c a l c i u m w i t h c o n c e n t r a t i o n s r a n g i n g from 50 mg/100 g i n c o t t a g e cheese, to 113 mg/100 g i n f l u i d m i l k and 716 mg/100 g i n cheddar cheese (Wong and L a C r o i x , 1980). D a i r y p r o d u c t s are unique i n c o n t a i n i n g not o n l y a r e l a t i v e l y l a r g e amount of c a l c i u m , but a l s o c o n s t i t u e n t s t h a t enhance the b i o a v a i l a b i l i t y of t h a t c a l c i u m , namely the m i l k sugar l a c t o s e ( d i s c u s s e d above) and the c a s e i n p r o t e i n s . 3a. 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 : The c a l c i u m i n m i l k i s p r e s e n t as phosphate and c i t r a t e s a l t s d i s t r i b u t e d i n s o l u b l e and c o l l o i d a l phases (Pyne, 1962). McMahon and Brown (1984) reviewed the d i s t r i b u t i o n of c a l c i u m i n m i l k (32mM), t w o - t h i r d s (22mM) are i n an amorphous c o l l o i d a l form, the r e m a i n i n g t h i r d (10mM) i s s o l u b l e , a p o r t i o n of which i s f r e e i o n i z e d Ca 2* (3mM). The c o l l o i d a l c a l c i u m f r a c t i o n c o n s i s t s of complexes w i t h phosphate e s t e r s or c a r b o x y l groups of c a s e i n p o l y p e p t i d e s , or bound w i t h phosphates and c i t r a t e s i n 35 a s s o c i a t i o n w i t h the c a s e i n m i c e l l e s . The s o l u b l e c a l c i u m f r a c t i o n i s comprised of the f r e e l y h y d r a t e d c a t i o n and phosphate, c i t r a t e or serum p r o t e i n complexes. An e q u i l i b r i u m e x i s t s between the s o l u b l e and c o l l o i d a l m i c e l l a r c a l c i u m phosphate (Swaisgood, 1985). 3b. C a s e i n M i c e l l e s : A c l o s e r e l a t i o n s h i p e x i s t s between the c o l l o i d a l c a l c i u m phosphate and c a s e i n p o l y p e p t i d e s of the m i l k m i c e l l e s . The 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 d i s p e r s i o n of i n t e r a c t i n g c a s e i n p r o t e i n s s t a b i l i z e d i n t h e i r a s s o c i a t i o n by c a l c i u m phosphate b r i d g e s . The c o n s t i t u e n t 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 the aa and fl-caseins f o r m i n g a r e l a t i v e l y h y d r o p h o b i c core w i t h K~ c a s e i n c o n t r i b u t i n g t o a r e l a t i v e l y p o l a r o u t e r s u r f a c e of the m i c e l l e . The crs and p - c a s e i n s are the c a l c i u m - s e n s i t i v e c a s e i n s due to t h e i r numerous s i t e s of p o s t - t r a n s l a t i o n a l p h o s p h o r y l -a t i o n . The r o l e of the phosphate e s t e r m o i e t i e s i n the c a l c i u m s e n s i t i v i t y of the c a s e i n s has been i n v e s t i g a t e d u s i n g both n a t i v e , as w e l l as e n z y m a t i c a l l y d e p h o s p h o r y l a t e d c a s e i n s i n s o l u b i l i t y and p r e c i p i t a t i o n s t u d i e s (Yamauchi et a l . . 1967; Bingham et a l . . 1972 ; A o k i e_t. a_l_. , 1985). D e p h o s p h o r y l a t i o n of whole c a s e i n and a s - c a s e i n s d e c r e a s e d t h e i r c a l c i u m b i n d i n g c a p a c i t y to one t h i r d of t h a t of n a t i v e c a s e i n s , thus demon-s t r a t i n g t h a t the o r g a n i c phosphorus component i s i m p o r t a n t f o r c a l c i u m b i n d i n g (Yamauchi et a l . . 1967). C a l c i u m s e n s i t i v i t y of d e p h o s p h o r y l a t e d whole c a s e i n and a 8 i - c a s e i n i s d e c r e a s e d s i n c e these two c a s e i n s p e c i e s w i l l c o a g u l a t e at a lower c r i t i c a l 36 c a l c i u m c o n c e n t r a t i o n than t h e i r n a t i v e c o u n t e r p a r t s (Yamauchi et a l . . 1967 ; Bingham .et. aj_. , 1972 ; A o k i et. al_. , 1985). The m i c e l l e s formed by d e p h o s p h o r y l a t e d aal~, K - c a s e i n and C a 2 + were t h r e e times l a r g e r and fewer i n number than n a t i v e m i c e l l e s , s u g g e s t i n g t h a t t h e r e are c a l c i u m b i n d i n g s i t e s o t h e r than the o r g a n i c phosphate e s t e r m o i e t i e s i n d e p h o s p h o r y l a t e d a j i - c a s e i n m i c e l l e s (Bingham .§_t. a 1 . . 1972). F u r t h e r support f o r a l t e r n a t e c a l c i u m b i n d i n g s i t e s i s found i n the work of A o k i and coworkers (.1985), who demonstrated t h a t a S 2 - c a s e i n behaves q u i t e d i f f e r -e n t l y from a B l , when d e p h o s p h o r y l a t e d . The i n s o l u b i l i t y of d e p h o s p h o r y l a t e d a S 2 - c a s e i n at n e u t r a l pH compared t o i t s a 8 1 c o u n t e r p a r t was a t t r i b u t e d to the l o s s of n e g a t i v e l y charged phosphate groups which n e u t r a l i z e d the net charge of a S 2 ~ c a s e i n f a c i l i t a t i n g h y d r o p h o b i c i n t e r a c t i o n and p r o t e i n p r e c i p i t a t i o n . On the o t h e r hand, n a t i v e a 8 l - c a s e i n r e t a i n s a n e g a t i v e charge at n e u t r a l pH, even when phosphate groups are n e u t r a l i z e d by Ca 2* , due to i o n i z e d c a r b o x y l groups of the p r o t e i n ; t h i s n e g a t i v e charge i s m a i n t a i n e d by the d e p h o s p h o r y l a t e d p r o t e i n when the phosphate m o i e t i e s have been removed. Taken t o g e t h e r , these data suggest t h a t the o r g a n i c phosphorus component of the v a r i o u s c a s e i n p o l y p e p t i d e s p e c i e s i s e s s e n t i a l f o r t h e i r c a l c i u m - b i n d i n g p o t e n t i a l , and s t a b i l i t y i n m i c e l l e f o r m a t i o n . M i c e l l e s t a b i l i t y i s i n t e r n a l l y c o n f e r r e d by s o l v e n t and hy d r o p h o b i c i n t e r a c t i o n s , r e s p e c t i v e l y of the c a s e i n m o l e c u l e s and the c o l l o i d a l c a l c i u m phosphate. 3c. C a l c i u m E x c h a n g e a b i l i t y : 37 The r e l a t i v e l y open s t r u c t u r e of the c a s e i n m i c e l l e s c o n t r i b u t e s to the e q u i l i b r i u m and e x c h a n g e a b i l i t y of m i c e l l e c o n s t i t u e n t s between the c o l l o i d a l and s o l u b l e phases. T h i s e q u i l i b r i u m i s e a s i l y s h i f t e d by temperature and pH. Thus, a r e d u c t i o n i n temperature near 0°C causes r e v e r s i b l e d i s s o c i a t i o n of p - c a s e i n , K - c a s e i n and c o l l o i d a l c a l c i u m phosphate from the m i c e l l e . A l t e r n a t i v e l y , i n c r e a s i n g the t e m p e r a t u r e , as i n p a s t e u r i z a t i o n (71.7°C), w i l l i r r e v e r s i b l y i n c r e a s e the amount of c o l l o i d a l c a l c i u m phosphate. A l t e r i n g the pH of m i l k (pH 6.8), as i n the manufacture of low pH f e r m e n t a t i o n p r o d u c t s ( y o g u r t ) , w i l l s o l u b i l i z e the c o l l o i d a l c a l c i u m phosphate and l e a d to a r e l e a s e of i o n i z e d c a l c i u m . 3d. C a s e i n P h o s p h o p e p t i d e s : The p h y s i c o c h e m i c a l forms of c a l c i u m i n d a i r y p r o d u c t s p l a y an i m p o r t a n t r o l e i n the r e l a t i v e b i o a v a i l a b i l i t y of t h i s m i n e r a l between p r o d u c t s . The p r o t e i n component of m i l k has been r e p o r t e d to enhance the b i o a v a i l a b i l i t y of d a i r y c a l c i u m . The p r o t e i n of m i l k (3.6% w/v) has two main f r a c t i o n s , namely the c a s e i n s (80% of m i l k p r o t e i n ) , and the whey p r o t e i n s (18%) and l a s t l y , serum p r o t e i n s ( 2 % ; serum a l b u m i n , i m m u n o g l o b u l i n s , l a c t o f e r r i n and t r a n s f e r r i n ) . E a r l y s t u d i e s by M e l l a n d e r et a1. p o i n t e d to the e f f i c a c y of c a s e i n d i g e s t i o n phosphopeptides i n d e p o s i t i o n of c a l c i u m to the s k e l e t o n of r a c h i t i c c h i l d r e n (1950) and c h i c k s (1956), i n d e p e n d e n t l y from v i t a m i n D t r e a t m e n t . The a b i l i t y of c a s e i n d i g e s t i o n p h o s p h o p e p t i d e s , d e r i v e d from an i n  v i t r o p a n c r e a t i c d i g e s t i o n , t o keep c a l c i u m phosphate d i b a s i c i n 38 s o l u t i o n from pH 7 t o 10 was r e p o r t e d by Reeves and L a t o u r i n 1958. Support f o r these d a t a i s d e r i v e d from the knowledge t h a t the phosphate e s t e r s of c a s e i n p o l y p e p t i d e s e r y l r e s i d u e s are a s s o c i a t e d w i t h c a l c i u m phosphate w i t h i n the m i c e l l e . C a s e i n phosphopeptides (CPP) are r e l e a s e d upon d i g e s t i o n of c a s e i n w i t h t r y p s i n . These b i o a c t i v e p e p t i d e s are c l e a v e d from t h e i r p r o t e c t e d p o s i t i o n s w i t h i n the c a s e i n p o l y p e p t i d e c h a i n s , by d i g e s t i v e enzymes, to become a c t i v e w i t h i n the i n t e s t i n a l m i l i e u . S e v e r a l i n v e s t i g a t o r s have examined the r e l a t i v e c a l c i u m b i n d i n g p o t e n t i a l of the v a r i o u s c a s e i n p r o t e i n s , namely a a i - , a s 2 - and p - c a s e i n (Manson and Annan, 1971; Waugh et. a_l_. , 1971; Baumy et a l . . 1989). a s i - c a s e i n has e i g h t p h o s p h o s e r y l r e s i d u e s i n i t s p r i m a r y s t r u c t u r e , f o u r of which form a c l u s t e r ( r e s i d u e s 64 to 68); a s 2 - c a s e i n has e l e v e n p h o s p h o s e r y l r e s i d u e s , w i t h two c l u s t e r s at p o s i t i o n s 8 to 10 and 56 to 61; p - c a s e i n has f i v e p h o s p h o s e r y l r e s i d u e s , w i t h one c l u s t e r at p o s i t i o n s 15 to 19. Thus, the major s p e c i e s of CPP are a B i - C N ( 5 9 - 7 9 ) : 5 P , a s2-CN(35-70):4P, and p-CN(1-28):4P. S t u d i e s have demonstrated t h a t p r e f e r e n t i a l b i n d i n g of c a l c i u m o c c u r s at s i t e s of the o l i g o -p e p t i d e s o c c u p i e d by c l u s t e r s of p h o s p h o s e r y l r e s i d u e s , due to the f a v o u r a b l e pK v a l u e , and charge d i s t r i b u t i o n of these g r o u p i n g s (Baumy et a 1. . 1989). By s t u d y i n g the s a t u r a t i o n k i n e t i c s of c a l c i u m b i n d i n g to CPP, these workers were a b l e to d e termine t h a t C a 2 f f i r s t s a t u r a t e s the c l u s t e r b i n d i n g s i t e s b e f o r e b i n d i n g t o s i n g l e p h o s p h o s e r y l r e s i d u e s . Thus, the b i n d i n g a f f i n i t y of the p h o s p h o s e r y l groups f o r metals i s a l s o 39 dependent on f a v o u r a b l e charge e f f e c t s of n e i g h b o u r i n g amino a c i d r e s i d u e s . F u r t h e r c h a r a c t e r i z a t i o n of the b i n d i n g p o t e n t i a l of CPP demonstrated e f f e c t i v e i n h i b i t i o n of the p r e c i p i t a t i o n of i n s o l u b l e c a l c i u m phosphate at a c o n c e n t r a t i o n of lOOmg/L, and f u r t h e r , t h a t the e f f i c i e n c y of a c t i v i t y i s dependent on the 1:1 c a l c i u m : phosphorus s t o i c h i o m e t r y ( B e r r o c a l e_t_ a_l_. , 1989). The importance of the Ca:P r a t i o t o the b i n d i n g a c t i v i t y of these p e p t i d e s i s a r e f l e c t i o n of the c a l c i u m s e n s i t i v i t y of the o r i g i n a l i n t a c t p o l y p e p t i d e s . The p o t e n t i a l p h y s i o l o g i c a l v a l u e of these b i o a c t i v e p e p t i d e s can be seen from in. v i v o s t u d i e s which demonstrated the presence of phosphopeptides i n the i n t e s t i n a l lumen c o n t e n t s of c a s e i n f e d m i n i p i g s ( M e i s e l and F r i s t e r , 1989) and r a t s ( N a i t o et a l . . 1972; N a i t o and S u z u k i , 1974). Moreover, the i n h i b i t i o n of p r e c i p i t a t i o n of i n s o l u b l e c a l c i u m phosphate s a l t s i n the i n t e s t i n a l l u m i n a l c o n t e n t s by CPP has been w i d e l y r e p o r t e d ( M u l t i n g e r et. a l . , 1983; Sato et aX. , 1983a; 1986). Furthermore, N a i t o and coworkers (Lee et a_l_. , 1980; 1983; N a i t o et. .al_. , 1972, N a i t o and S u z u k i , 1974) have c o n s i s t e n t l y been a b l e to demon-s t r a t e a g r e a t e r p r o p o r t i o n of s o l u b l e c a l c i u m i n the d i g e s t a of c a s e i n f e d r a t s compared to c o u n t e r p a r t s f e d o t h e r d i e t a r y p r o t e i n s . U t i l i z a t i o n of the c a l c i u m from d i e t s which p o t e n t i a l l y y i e l d CPP upon d i g e s t i o n , was r e p o r t e d by Sato et a l . ( 1986) who observed enhanced d e p o s i t i o n of * 5 C a to the femora of r a t s g i v e n an i n t r a l u m i n a l dose of a crude t r y p t i c c a s e i n d i g e s t . S i m i l a r l y , i n v i t r o s t u d i e s have shown i n c r e a s e d c a l c i f i c a t i o n of 40 e x p l a n t e d embryonic r a t bone when c u l t u r e d i n a medium c o n t a i n i n g CPP (Gerber and J o s t , 1986). Thus, CPP may be e f f e c t i v e at enhancing the i n t e s t i n a l uptake of c a l c i u m i n humans consuming d a i r y p r o d u c t s , but f u r t h e r s t u d i e s s h o u l d be conducted to a s s e s s the e f f e c t of i n t e s t i n a l a l k a l i n e phosphatase a c t i v i t y on the s t a b i l i t y of i n t r a l u m i n a l Ca-CPP complexes. T h i s i s proposed on the b a s i s t h a t both r a t s and humans have s i g n i f i c a n t i n t e s t i n a l a l k a l i n e phosphatase a c t i v i t y which may be e f f e c t i v e i n the d e p h o s p h o r y l a t i o n of c a s e i n (Van der Meer, 1988). 3e. Soy P r o t e i n : The u t i l i z a t i o n of soybeans as an a l t e r n a t e p r o t e i n source i n p l a c e of d a i r y p r o t e i n s has been growing due to t h e i r lower c o s t ($4.50/kg soy i s o l a t e v e r s u s $7.00/kg c a s e i n ) and s i m i l a r i t y i n f u n c t i o n a l i t y ( B a r r a q u i o and van de V o o r t , 1988). P l a n t d e r i v e d p r o t e i n s o u r c e s have been c o n s i d e r e d to have poorer m i n e r a l b i o a v a i l a b i l i t y i n comparison to animal p r o t e i n s due to the presence of i n h i b i t o r y s u b s t a n c e s such as f i b r e and p h y t a t e s (Kahn and Weaver, 1989; Liebman and L a n d i s , 1989). For example, i n the manufacture of soy p r o t e i n i s o l a t e by a c i d p r e c i p i t a t i o n , complexes between the p r o t e i n and p h y t a t e are formed which p o t e n t i a l l y a f f e c t p r o t e i n s o l u b i l i t y and m i n e r a l a v a i l a b i l i t y of the d i e t (Erdman, 1979; Hartman, 1979). Soy p r o t e i n s are h i g h m o l e c u l a r w e i g h t , h i g h l y s t r u c t u r e d g l o b u l i n p r o t e i n s , c h a r a c t e r i s t i c s which make them r e l a t i v e l y r e s i s t a n t to enzymatic d i g e s t i o n (Raghunath and N a r a s i n g a Rao, 1984). P r o t e i n d i g e s t i b i l i t y and amino a c i d c o n t e n t can p l a y a 41 r o l e i n c a l c i u m b i o a v a i l a b i l i t y as w e l l as n u t r i t i o n a l v a l u e from a p r o t e i n s o u r c e , as demonstrated by the v a l u e of c a s e i n d e r i v e d p h o s p h o r y l a t e d p e p t i d e s , as w e l l as the a b i l i t y of v a r i o u s amino a c i d s ( L - l y s i n e and L - a r g i n i n e ) i n enhancing 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 (Wasserman ejt a l . , 1956 ; N a i t o et a 1 . , 1972) . S i n c e 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 i s dependent to some e x t e n t on the p e p t i d e or amino a c i d m i x t u r e s of p r o t e i n p o s t - d i g e s t i o n p r o d u c t s , the r e l a t i v e d i g e s t i b i l i t y of the p r o t e i n c o u l d r e p r e s e n t a l i m i t i n g f a c t o r of d i e t a r y c a l c i u m b i o a v a i l a b i l i t y . 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 and A b s o r p t i o n The c a l c i u m s t a t u s of an i n d i v i d u a l i s termed c a l c i u m b a l a n c e . C a l c i u m b a l a n c e i s a c h i e v e d when the amount of c a l c i u m absorbed from the d i e t i s the e q u i v a l e n t of t h a t l o s t i n the f e c e s , u r i n e and sweat. F e c a l l o s s e s of c a l c i u m have been a s c e r t a i n e d to be unabsorbed c a l c i u m p l u s endogenous f e c a l c a l c i u m e x c r e t i o n , which amounts to 100 to 130 mg/day. U r i n a r y c a l c i u m c l e a r a n c e and p e r s p i r a t i o n l o s s e s account f o r 150 and 15 mg/day, r e s p e c t i v e l y . T h e r e f o r e , i n o r d e r to m a i n t a i n a zero b a l a n c e , a 30% a b s o r p t i o n i s r e q u i r e d from the U.S. RDA i n t a k e recommendation of 800 mg/day to r e p l a c e l o s s e s of a p p r o x i m a t e l y 250 mg/day ( A l l e n , 1982). T h e r e f o r e , the b i o a v a i l a b i l i t y of c a l c i u m from the d i e t i s ve r y i m p o r t a n t , namely the u s e f u l n e s s of n u t r i e n t s from meals, s i n g l e food i t e m s , or supplements to the i n d i v i d u a l ( G r e g e r , 1988). There i s a c o n s i d e r a b l e b a t t e r y of assays t o determine the b i o a v a i l a b i l i t y of a n u t r i e n t such as c a l c i u m , examples a r e , s o l u b i l i t y t e s t s ; b a l a n c e s t u d i e s ; 42 m o n i t o r i n g of l e v e l s of n u t r i e n t s i n serum or plasma; and l a s t l y , f u n c t i o n a l t e s t s . 4a. In V i t r o S o l u b i l i t y : A s s a y i n g the s o l u b i l i t y of the c a l c i u m c o n t a i n e d i n supplements and food systems i s a q u i c k and i n e x p e n s i v e i n v i t r o t e s t of the a v a i l a b i l i t y t o an i n d i v i d u a l . However, s o l u b i l i t y of a c a l c i u m s a l t at a pH s i m i l a r to t h a t of the stomach (pH 2.0) does not i n s u r e s o l u b i l i t y i n the s m a l l i n t e s t i n e (pH > 6.5), the s i t e of c a l c i u m a b s o r p t i o n . These t e s t s are based on the premise t h a t s o l u b i l i t y of c a l c i u m i n the i n t e s t i n a l lumen i s a p r e -r e q u i s i t e to a b s o r p t i o n (Pak and A v i o l i , 1988). These s i m p l e l a b o r a t o r y bench-top t e s t s , however, cannot d u p l i c a t e the i n t e s t i n a l m i l i e u w i t h r e s p e c t to c o - i n g e s t e d n u t r i e n t s (such as amino a c i d s , d i g e s t i o n p e p t i d e s , s a l t s , and s u g a r s ) , which i n t u r n , i n f l u e n c e the s o l u b i l i t y , and thus the a v a i l a b i l i t y of c a l c i u m . A r e p o r t of a s u b s t a n t i a l m e a l - e f f e c t on c a l c i u m a b s o r p t i o n f u r t h e r i d e n t i f i e s the importance of c o - i n g e s t e d n u t r i e n t s i n c a l c i u m a b s o r p t i o n (Heaney et a l . . 1989). Recent f i n d i n g s of Heaney and coworkers (1990), have shown t h a t c a l c i u m s o u r c e s v a r y i n g i n magnitude of s o l u b i l i t y , do not d i f f e r s u b s t a n t i a l l y i n f r a c t i o n a l a b s o r p t i o n i n h e a l t h y a d u l t s . T h i s f i n d i n g f u r t h e r emphasizes the concept t h a t i n v i t r o s o l u b i l i t y i s not a p r e d i c t i v e measure of c a l c i u m a b s o r p t i o n i n v i v o . 4b. B a l a n c e S t u d i e s : C a l c i u m b a l a n c e s t u d i e s can be used to determine the apparent a b s o r p t i o n of a n u t r i e n t from e x p e r i m e n t a l d i e t s , and 43 under v a r y i n g p h y s i o l o g i c a l s t a t e s , r e s p e c t i v e l y . A l l e n (1982) has reviewed the v a r i o u s t e c h n i q u e s i n use f o r c o n d u c t i n g b a l a n c e s t u d i e s . Apparent a b s o r p t i o n of a n u t r i e n t i s d e f i n e d as the d i f f e r e n c e between the c a l c i u m i n t a k e and f e c a l c a l c i u m e x c r e t i o n ( A l l e n , 1982). The apparent a b s o r p t i o n of c a l c i u m commonly ranges from between 20 to 40% of the c a l c i u m i n t a k e . C a l c i u m b a l a n c e s t u d i e s are l i m i t e d i n the p r e c i s i o n of measurements by i n a c c u r a c i e s i n the c o l l e c t i o n of the e x c r e t a . I f samples are contaminated by f e e d , or are i n c o m p l e t e l y s e p a r a t e d , e r r o r s w i l l o c c u r i n e s t i m a t i n g the amount of c a l c i u m absorbed. More i m p o r t a n t , i s the f a i l u r e of b a l a n c e s t u d i e s to d i s t i n g u i s h the 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 i n t o the lumen, which can r e s u l t i n an u n d e r e s t i m a t i o n of the t r u e q u a n t i t y absorbed. T h i s problem can be p a r t i a l l y a l l e v i a t e d by p e r f o r m i n g a balance study u s i n g the " U r i n e - R a t i o Method", a procedure which i n v o l v e s the s i m u l t a n e o u s o r a l and i n t r a v e n o u s ( I V ) a d m i n i s t r a t i o n of two c a l c i u m i s o t o p e s ( A l l e n , 1982). The appearance of the o r a l i s o t o p e i n the u r i n e i s p r o p o r t i o n a l to the a b s o r p t i o n and d i s t r i b u t i o n of c a l c i u m i n the body. In a d d i t i o n , the appearance of the IV a d m i n i s t e r e d 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 of the o r a l i s o t o p e i n t o body p o o l s . Howe v e r , the U r i n e — R a t i o Method i s o n l y q u a l i t a t i v e . Thus, the b a l a n c e study i s s t i l l the method of c h o i c e , g i v e n t h a t i f the animal i s adapted to the d i e t , endogenous c a l c i u m c o n t r i b u t i o n i s r e l a t i v e l y c o n s t a n t . C o n s e q u e n t l y , the data can be used to determine i f a b s o r p t i o n i s adequate f o r c a l c i u m b a l a n c e ( A l l e n , 1982). 44 4 c. N u t r i t i o n a l S t a t u s : An i n d e x of the n u t r i t i o n a l s t a t u s of s u b j e c t s under study can be o b t a i n e d to some e x t e n t by m o n i t o r i n g serum or plasma l e v e l s of n u t r i e n t s and m e t a b o l i t e s of con c e r n . In a normal s i t u a t i o n however, the e n d o c r i n e system w i l l m a i n t a i n r e l a t i v e l y c o n s t a n t c a l c i u m l e v e l s , such t h a t an a c c u r a t e i n d i c a t i o n of c a l c i u m i n t a k e w i l l not be o b t a i n e d ( G r e g e r , 1988). S k e l e t a l c a l c i u m c o n t e n t can be m o n i t o r e d as e n d p o i n t d e t e r m i n a n t s of the lo n g t e r m e f f e c t s of d i e t a r y 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 ( G r e g e r , 1988). These d e t e r m i n a t i o n s a r e , of n e c e s s i t y , slow because adequate time must be g i v e n f o r a d a p t a t i o n of the animals to the d i e t s . 4d. F u n c t i o n a l T e s t s : F u n c t i o n a l t e s t s i n c l u d e the f o l l o w i n g d e t e r m i n a t i o n s , m o n i t o r i n g growth parameters such as body weight g a i n , f e e d e f f i c i e n c y and h e i g h t ; bone parameters such as bone b r e a k i n g or bending s t r e n g t h , and bone hist o m o r p h o m e t r y ; as w e l l as enzyme a c t i v i t i e s such as those w i t h m i n e r a l s or v i t a m i n s as c o - f a c t o r s ( G r e g e r , 1988). These t e s t s a re f u n d a m e n t a l l y p e r f e c t as they are e n d p o i n t d e t e r m i n a n t s of the u t i l i z a t i o n of d i e t a r y com-ponents f o r growth and maintenance of h e a l t h . These t e s t s can be d i f f i c u l t t o 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 , i s confounded by a s s o c i a t e d d i e t a r y or p h y s i o l o g i c a l f a c t o r s ( G r e g e r , 1988). 4e. Model Systems: E x p e r i m e n t a l t e c h n i q u e s t o determine c a l c i u m a b s o r p t i o n i n 45 a n i m a l models i n c l u d e the use of i s o l a t e d i n t e s t i n a l c e l l s and o r g a n e l l e s ; i n t e s t i n a l b r ush b o r d e r membrane v e s i c l e s ; e v e r t e d i n t e s t i n a l s a c s , and i n s i t u l o o p s ( A l l e n , 1982). The use of i s o l a t e d i n t e s t i n a l c e l l s , o r g a n e l l e s , or brush border membrane v e s i c l e s a l l e y s the s t r i c t c o n t r o l and d e f i n i t i o n of e x p e r i m e n t a l c o n d i t i o n s such as c a l c i u m c o n c e n t r a t i o n , i o n i c s t r e n g t h , t e mperature and pH. U s i n g t h e s e methods, s p e c i f i c k i n e t i c models can be d e r i v e d to s i m u l a t e c a l c i u m a b s o r p t i o n . However, these methods do not g i v e i n f o r m a t i o n about c o n f o u n d i n g e f f e c t s i n  v i v o . such as the presence of o t h e r d i e t a r y c o n s t i t u e n t s and c h e m i c a l and e l e c t r i c a l g r a d i e n t s . S i m i l a r l y , in_ v i t r o or i n  s i t u i n t e s t i n a l p r e p a r a t i o n s a l l o w the measurement of the f l u x of c a l c i u m , or the s e c r e t i o n p a t t e r n of c a l c i u m from a s p e c i f i c segment of the s m a l l i n t e s t i n e , but are l i m i t i n g i n i n f o r m a t i o n on the c a p a c i t y of the e n t i r e i n t e s t i n e f o r c a l c i u m a b s o r p t i o n . 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 The s k e l e t o n c o n t a i n s 99% of the body c a l c i u m c o n t e n t as i n o r g a n i c s a l t s d e p o s i t e d i n an o r g a n i c f i b r o u s c o l l a g e n m a t r i x (Gray, 1974). Throughout l i f e , bone undergoes c y c l e s of r e s o r p t i o n f o l l o w e d by r e p a i r (Niewoehner, 1988). O s t e o p o r o s i s has been d e f i n e d as a c o n d i t i o n i n which t h e r e i s a s i g n i f i c a n t l o s s i n bone mass w i t h an a s s o c i a t e d i n c r e a s e i n bone f r a g i l i t y such t h a t the r i s k of f r a c t u r e due to m i n i m a l trauma i s enhanced (Niewoehner, 1988). Because females g e n e r a l l y have a s m a l l e r s k e l e t a l mass than males, i n a d d i t i o n to a g r e a t e r r a t e of bone l o s s w i t h age, the r i s k s of o s t e o p o r o s i s are i n c r e a s e d i n the 46 female ( A v i o l i , 1981). The b a s i s of a g e - r e l a t e d bone l o s s appears to be an i n h e r e n t l y unbalanced r e m o d e l l i n g s c h e d u l e , w h e r e i n the amount of bone formed i s not s u f f i c i e n t t o r e p l a c e t h a t removed by r e s o r p t i o n (Marcus, 1987). Bone i s l a i d down d u r i n g growth by the o s t e o b l a s t c e l l s which d e p o s i t c a l c a r e o u s m a t e r i a l i n a c o l l a g e n m a t r i x g i v i n g r i s e to f r e s h l a y e r s of bone. Thus, bone i s formed and r e s o r b e d throughout l i f e at the o u t e r ( p e r i o s t e u m ) and i n n e r s u r f a c e s (endosteum), r e s p e c t i v e l y t o y i e l d a mature s k e l e t o n (Gray, 1974). Some degree of bone l o s s from the s p i n e b e g i n s to occur by about 20 to 30 years of age i n human f e m a l e s , and s u b s e q u e n t l y c o n t i n u e s at a r a t e of a p p r o x i m a t e l y 1% per year (Anonymous, 1988). Thus, i n the years p r i o r t o menopause, s m a l l d e c r e a s e s can occur i n bone mass c o n t r i b u t i n g to a g r a d u a l t h i n n i n g of compact c o r t i c a l bone (Niewoehner, 1988). At menopause, bone metabolism b a l a n c e s h i f t s towards an i n c r e a s e i n the r a t e of bone r e s o r p t i o n , which c o n t i n u e s f o r a p e r i o d l a s t i n g 2 to 5 y e a r s (Marcus, 1987). C l a s s i c a l l y , the decade f o l l o w i n g menopause i s c h a r a c t e r i z e d by an enhanced r a t e of bone l o s s , but t h e r e a f t e r the r a t e d e c l i n e s once more. The change i n bone metabolism i s accompanied by changes i n o v e r a l l c a l c i u m m e tabolism and homeostasis marked by a d e c l i n e i n r e n a l c a l c i u m r e t e n t i o n ; d e c r e a s e d 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 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 (Marcus, 1987). To a m e l i o r a t e the s e v e r i t y of the o s t e o p o r o t i c symptoms, i t f o l l o w s t h a t bone mass and m i n e r a l i z a t i o n s h o u l d be o p t i m i z e d i n 47 the f i r s t 3 decades of l i f e when bone d e p o s i t i o n i s o c c u r r i n g (Niewoehner, 1988; Shah and B e l o n j e , 1988). T h i s can be best a c c o m p l i s h e d by k e e p i n g i n mind t h a t bone mass i s a m u l t i f a c t -o r i a l f u n c t i o n b e i n g a f f e c t e d by c a l c i u m i n t a k e , v i t a m i n D n u t r i t i o n and l e v e l of a c t i v i t y (Shah and B e l o n j e , 1988). An adequate c a l c i u m i n t a k e i n the premenopausal y e a r s i s e s s e n t i a l f o r bone h e a l t h i n l i g h t of the f i n d i n g s from e p i d e m i o l o g i c a l s t u d i e s based on d i e t a r y r e c a l l which i n d i c a t e t h a t bone d e n s i t y i s p o s i t i v e l y c o r r e l a t e d to past c a l c i u m and m i l k consumption (Odland e_t al_. , 1972 ; S a n d l e r e_t al_. , 1985). V i t a m i n D n u t r i t i o n i s i m p o r t a n t f o r the p r e v e n t i o n of r i c k e t s , as w e l l as i t s r o l e i n i n t e s t i n a l a c t i v e c a l c i u m t r a n s p o r t . P h y s i c a l a c t i v i t y i s a v i t a l component of bone h e a l t h i n i t s promotion of bone f o r m a t i o n and i n c r e a s e d d e n s i t y of bone t i s s u e due to m e c h a n i c a l s t r e s s e s p l a c e d on the bone (Simonen, 1986). Thus, r e g u l a r , moderate, w e i g h t - b e a r i n g e x e r c i s e i s of b e n e f i t i n a c h i e v i n g a maximal bone mass p r i o r t o menopause (Simonen, 1986; J a w o r s k i , 1987). 5a . Treatment of Osteoporosis'. C u r r e n t t r e a t m e n t s of o s t e o p o r o s i s i n c l u d e sex hormone r e p l a c e m e n t , n u t r i t i o n a l changes, and e x e r c i s e to m a i n t a i n or i n c r e a s e bone mass (Niewoehner, 1988). Menopause i s c h a r a c t e r -i z e d by a r a p i d d e c l i n e i n l e v e l s of e s t r o g e n . Thus, e s t r o g e n t h e r a p y i s e f f e c t i v e at p r e v e n t i n g bone mass l o s s i f i n i t i a t e d soon a f t e r menopause (Simonen, 1986). Bone m i n e r a l i z a t i o n can be m a i n t a i n e d i n the 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 e s t r o g e n 48 r e p l a c e m e n t ; however, i f tr e a t m e n t i s d i s c o n t i n u e d , bone m i n e r a l l o s s w i l l o c cur (Niewoehner, 1988). The p r i m a r y n u t r i t i o n a l t h e r a p y of postmenopausal o s t e o -p o r o s i s to i n c r e a s e c a l c i u m i n t a k e i s i n d i c a t e d from the d i s t u r b e d c a l c i u m m e t a b o l i s m , bone l o s s and n e g a t i v e c a l c i u m b a l a n c e which have been r e p o r t e d i n the e l d e r l y and i n p a r t i c -u l a r , the postmenopausal fem a l e . C a l c i u m s u p p l e m e n t a t i o n of the d i e t l e a d s to s u p p r e s s i o n of p a r a t h y r o i d hormone s e c r e t i o n and bone r e m o d e l l i n g ; improved c a l c i u m b a l a n c e ; and p r o t e c t i o n of bone mass. Thus, c a l c i u m s u p p l e m e n t a t i o n may suppress biochem-i c a l and e n d o c r i n e i n d i c e s of bone m o d e l l i n g and improve c a l c i u m homeostasis i n the e l d e r l y (Marcus, 1987). I n c l u s i o n of c a l c i u m supplements, or c a l c i u m r i c h foods such as d a i r y p r o d u c t s i n the d i e t have been r e p o r t e d to improve c a l c i u m b a l a n c e w i t h 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 through m i n e r a l i z a t o n (Lee et. a_L. , 1981; H a s l i n g e_t_ a_l_. , 1986; H o r o w i t z et a_l_. , 1988; S h i h et a l . . 1988). A l t e r n a t i v e l y , the f i n d i n g s of Sinha and coworkers (1988) i n d i c a t e t h a t a h i g h c a l c i u m i n t a k e does not a f f e c t bone metabolism i n aged female r a t s , but i s e f f e c t i v e i n enhancing bone f o r m a t i o n i n young r a t s . F u r t h e r , the p o s s i b l e c o u p l i n g of de c r e a s e d bone f o r m a t i o n secondary to de c r e a s e d bone r e s o r p t i o n i n response to d i e t a r y c a l c i u m s u p p l e m e n t a t i o n may e x p l a i n why bone l o s s i s not c o m p l e t e l y p r e v e n t e d i n some p a t i e n t s ( H o r o w i t z et a1. . 1988). In view of the e q u i v o c a l e v i d e n c e a v a i l a b l e on the e f f i c a c y of i n c r e a s e d c a l c i u m i n t a k e i n 49 the t r e a t m e n t of o s t e o p o r o s i s , the be s t p r o p h y l a c t i c a c t i o n may be to i n s u r e maximal bone mass i n the ye a r s p r i o r t o menopause. E x e r c i s e has been r e p o r t e d to i n c r e a s e c i r c u l a t i n g l e v e l s of the hormone c a l c i t o n i n and to decrease plasma p a r a t h y r o i d hormone l e v e l s , i n a d d i t i o n to i t s e f f e c t s on bone f o r m a t i o n (Simonen, 1986). Thus, moderate e x e r c i s e s h o u l d c o n t i n u e to be a p a r t of the l i f e s t y l e d u r i n g o l d age. 5b. Anatomy of the Femur: The femur, or t h i g h bone, i s the l o n g e s t , l a r g e s t and s t r o n g e s t bone i n the mammalian 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 s h a f t w i t h two e x t r e m i t i e s (Gray, 1974). The s h a f t , or d i a -p h y s i s , i s e s s e n t i a l l y a h o l l o w c y l i n d e r w i t h w a l l s c o n s t r u c t e d of a dense, compact c o r t i c a l bone t i s s u e w i t h a h o l l o w e d out i n t e r i o r , the m e d u l l a r y c a n a l . The p r o x i m a l ( f e m o r a l head and g r e a t e r t r o c h a n t e r ) and d i s t a l e x t r e m i t i e s , by n a t u r e of t h e i r f u n c t i o n , are expanded f o r a r t i c u l a t i o n and to a l l o w a s u r f a c e f o r muscle attachment. The e x t r e m i t i e s are c h a r a c t e r i z e d by a spongy, t r a b e c u l a r bone t i s s u e w i t h an o u t e r c o v e r i n g of compact bone . Compact c o r t i c a l bone makes up a p p r o x i m a t e l y 75 to 80% of the s k e l e t o n w i t h the remainder t r a b e c u l a r bone (Niewoehner, 1988). Compact bone i s h i g h l y m i n e r a l i z e d w i t h a slow t u r n o v e r r a t e . The m i n e r a l c o n t e n t of t h i s bone type peaks i n the f o u r t h decade of l i f e . C o n v e r s e l y , t r a b e c u l a r bone i s c h a r a c t e r i z e d by a r e l a t i v e l y lower m i n e r a l c o n t e n t per u n i t volume of bone. 50 M i n e r a l i z a t i o n of t r a b e c u l a r bone peaks i n the t h i r d decade of l i f e . The i n t e r n a l a r c h i t e c t u r e of the femur i s d e s i g n e d t o w i t h s t a n d the p r e s s u r e i n the s u p p o r t of the weight of the body and to r e s i s t the t e n s i o n e x e r t e d by muscles and l i g a m e n t s . As w e l l , bones are r e q u i r e d to have impact r e s i s t a n c e to a l l o w s t i f f n e s s i n t e n s i o n as w e l l as s h e a r . S t i f f n e s s towards s h e a r i n g f o r c e s a l l o w bones to bear compressive l o a d s such as body w e i g h t , and to r e s i s t bending ( C u r r e y , 1984). Bones commonly f r a c t u r e due to sudden impact l o a d s as opposed to s t a t i c l o a d i n g over a p e r i o d of time (Gray, 1974). 5 c. Bone Bio m e c h a n i c s: In many e x p e r i m e n t a l s t u d i e s , c a l c i u m u t i l i z a t i o n i s e s t i m a t e d from an e n d p o i n t measurement of the femur, or t i b i a (Kusy ejt al_. , 1987 ; O r t o f f and Oxlund, 1988). Moreover, i t has been p o s t u l a t e d t h a t bone s t r e n g t h may be a s s o c i a t e d w i t h bone m i n e r a l c o n t e n t (Kusy et a1.. 1987). Bone s t r e n g t h has been d e f i n e d as the f o r c e a p p l i e d at the p o i n t of f a i l u r e , or r u p t u r e , of the bone, c o r r e c t e d f o r i t s i n i t i a l c r o s s - s e c t i o n a l a r e a , e x p r e s s e d i n terms of Newtons (N) per cm 2, or mm2. The energy or work ( a r e a under the f o r c e - d e f o r m a t i o n c u r v e ) r e q u i r e d to f r a c t u r e a bone can a l s o be d etermined by i n s t r u m e n t a l a n a l y s i s as a f u n c t i o n a l t e s t f o r 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 ( Segars and K a p s a l i s , 1987). Bones are however, by t h e i r v e r y n a t u r e , complex b i o l o g i c a l m a t e r i a l s w i t h few, i f any, c h a r a c t e r i s t i c s t h a t w i l l f o l l o w 51 t h e o r i e s of e l a s t i c b e h a v i o u r . Two approaches can t h e r e f o r e be t a ken i n the m a t e r i a l t e s t i n g of bone, namely an e m p i r i c a l approach, or a w e l l - d e f i n e d e n g i n e e r i n g t e s t (Segars and K a p s a l i s , 1987). The e m p i r i c a l approach i n v o l v e s the a p p l i c a t i o n of f o r c e s i n d e f o r m a t i o n t h a t may be i m i t a t i v e of a p a r t i c u l a r a c t i o n . Data g e n e r a t e d i n t h i s manner are u s e f u l i n comparing a s e r i e s of samples t e s t e d under i d e n t i c a l c o n d i t i o n s . The magnitude of the v a l u e s o b t a i n e d w i l l have l i t t l e s i g n i f i c a n c e from a p h y s i c a l p o i n t of view, b e i n g h i g h l y dependent on t e s t c o n d i t i o n s . In c o n t r a s t , the w e l l - d e f i n e d e n g i n e e r i n g t e s t i n v o l v e s the a p p l i c a t i o n of a pure model of d e f o r m a t i o n to the sample. Data can then be n o r m a l i z e d through the t h e o r e t i c a l a n a l y s i s of bone response to f o r c e s a p p l i e d . In bone, and i n p a r t i c u l a r the femur and t i b i a , the c r o s s -s e c t i o n a l a rea and moment of i n e r t i a can both be observed to vary a l o n g the l e n g t h of the s h a f t . Bone m a t e r i a l p r o p e r t i e s can be d e t e r m i n e d by assuming t h a t the c o n f i g u r a t i o n of the s h a f t i s u n i f o r m and approximates two c o n c e n t r i c e l l i p s e s . In a bending t e s t , t h r e e - or f o u r - p o i n t bending may be chosen. F o u r - p o i n t bending a p p l i e s a u n i f o r m s t r e s s f i e l d a l o n g the bone s u r f a c e , whereas t h r e e - p o i n t bending may a p p l y a s i n g l e s t r e s s p o i n t at the c e n t r e s u p p o r t . V a r i a b i l i t y i n bone c r o s s - s e c t i o n a l area and bone c u r v a t u r e as seen w i t h the femur and t i b i a , d e t r a c t from the apparent advantages of f o u r - p o i n t bending i n f a v o u r of t h r e e -p o i n t bending (Kusy et_ a l . . 1987). 52 In summary, t h e r e are many methods, some c h e m i c a l , p h y s i c a l or b i o p h y s i c a l , f o r 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 and subsequent u t i l i z a t i o n . The o b j e c t i v e s of the f o l l o w i n g s t u d i e s were to examine the e f f e c t of d i e t a r y f a c t o r s a s s o c i a t e d w i t h d a i r y f o o d s , i n p a r t i c u l a r , on c a l c i u m b i o a v a i l a b i l i t y and u t i l i z a t i o n u s i n g both i n t e s t i n a l and bone endpoint measurements. 53 E x p e r i m e n t 1 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 femur b i o m e c h a n i c s i n r a t s . I n t r o d u c t i o n I n a d d i t i o n t o d a i r y p r o d u c t s b e i n g w e l l r e c o g n i z e d as e x c e l l e n t s o u r c e s o f d i e t a r y c a l c i u m ( A l l e n , 1 9 8 2 ) , i t has a l s o b e e n d e m o n s t r a t e d t h a t v a r i o u s c o n s t i t u e n t s i n m i l k may e n h a n c e t h e b i o a v a i l a b i l i t y o f c a l c i u m and p o s s i b l y o t h e r m i n e r a l s ( L e n g e m a n n e t a l . , 1959 ; Lee et. a l . . , 1983; G r e g e r et. a_l_. , 1 9 8 7 ) . L a c t o s e , t h e p r i n c i p a l c a r b o h y d r a t e i n m i l k , has b e e n shown t o f a c i l i t a t e e n h a n c e d p a r a c e l l u l a r t r a n s p o r t o f c a l c i u m i n b o t h p r o x i m a l and d i s t a l s m a l l i n t e s t i n a l s e g m e n t s ( A r m b r e c h t and W a s s e r m a n , 1976; A r m b r e c h t , 1 9 8 7 ) . T h i s e f f e c t was i n d e p e n d e n t o f c a l c i u m i n t a k e , age and t h e v i t a m i n D e n d o c r i n e s y s t e m (Lengemann e_t al_. , 1959 ; B e h a r and K e r s t e i n , 1976; N e l l a n s and K i m b e r g , 1978; A r m b r e c h t , 1 9 8 7 ) . C o n t r o v e r s y s t i l l e x i s t s as to t h e r e l a t i v e s i g n i f i c a n c e o f d i e t a r y l a c t o s e , and t h e v a r i o u s f o r m s o f c a l c i u m ( c o l l o i d a l o r i o n i z e d ) p r e s e n t i n d a i r y p r o d u c t s , on c a l c i u m a b s o r p t i o n (Wong and L a C r o i x , 1980; B u c h o w s k i e t a l . . , 1 9 8 9 ) . The o b j e c t i v e s o f t h e p r e s e n t s t u d y were t o d e t e r m i n e t h e e f f e c t s 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 c a l c i u m b i o a v a i l a b i l i t y and s u b s e q u e n t u t i l i z a t i o n i n l a c t o s e t o l e r a n t and i n t o l e r a n t r a t s . M a t e r i a l s and M e t h o d s  A n i m a l s and D i e t s : M a l e W i s t a r r a t s ( C h a r l e s R i v e r , M o n t r e a l , PQ) w e i g h i n g 103 +_ 6g were r a n d o m l y s e g r e g a t e d i n t o f o u r g r o u p s o f 8 r a t s p e r 54 group and a s s i g n e d to d i e t a r y t r e a t m e n t s c o n s i s t i n g of a c o n t r o l skim m i l k p r o t e i n c o n c e n t r a t e (SMPC; 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); SMPC supplemented w i t h 20% l a c t o s e (ML); SMPC supplemented w i t h 50% l a c t o s e ( HL); and a yog u r t powder c o n c e n t r a t e (Y; Champlain I n d u s t r i e s , M i s s i s s a u g a , ON). The d i e t a r y p r o t e i n s o u r c e s were a n a l y z e d f o r p r o t e i n , f a t and ash con t e n t by proxi m a t e a n a l y s e s ; c a l c i u m and phosphorus c o n t e n t by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y ( P e r k i n Elmer-306 atomic a b s o r p t i o n s p e c t r o p h o t o m e t e r ; P e r k i n Elmer, Norwalk, CT); and l a c t o s e by a 8 - g a l a c t o s i d a s e enzymatic assay ( B o e h r i n g e r Mannheim; D o r v a l , PQ). The SMPC and y o g u r t powder c o n c e n t r a t e s c o n t a i n e d d i f f e r e n t amounts of c a l c i u m , t h e r e f o r e , d i e t s were supplemented w i t h c a l c i u m carbonate (BDH, T o r o n t o , ON) to g i v e a u n i f o r m c a l c i u m c o n t e n t of 0.7% w/w. The SMPC was found to c o n t a i n o n l y a t r a c e amount of l a c t o s e which was not c o r r e c t e d f o r i n the l a c t o s e supplemented d i e t s c o n t a i n i n g 20% and 50% l a c t o s e , r e s p e c t i v e l y . The y o g u r t powder d i e t c o n t a i n e d 8.2% l a c t o s e by a n a l y s i s . A l l d i e t s were made i s o n i t r o g e n o u s (20% p r o t e i n ) and i s o c a l o r i c ( T a b l e 1.1). Animals were f e d e x p e r i m e n t a l d i e t s ad 1 i b i t u m u n t i l body we i g h t s reached 150g, a f t e r which a m e a l - f e e d i n g s c h e d u l e was i n i t i a t e d . For a two-week p e r i o d , a n i m a l s were t r a i n e d to consume d i e t s w i t h i n a s i x hour p e r i o d (9am-3pm). D e i o n i z e d water was made a v a i l a b l e ad l i b i t u m . 55 TABLE 1.1 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 f e d to r a t s . D i e t a r y component 1 SMPC ML HL (g/lOOg) SMPC2 23 . 5 23 . 5 23 . 5 -Yogurt powder 3 — — — 55 . 6 D.L. me t h i o n i n e * 0.3 0 .3 0 .3 0.3 C o r n s t a r c h * 11.35 11 .35 11 .35 11 .35 Sucrose 50.0 30 .0 - 18 . 65 L a c t o s e - 20 .0 50 .0 -F i b r e * 5.0 5 .0 5 .0 5.0 Ve g e t a b l e o i l 4.8 4 .8 4 .8 4.8 Ca f r e e m i n e r a l mix* 3 . 5 3 . 5 3 . 5 3 . 5 V i t a m i n m i x t u r e * 1.0 1 .0 1 .0 1.0 C h o l i n e b i t a r t r a t e * 0.2 0 .2 0 . 2 0 . 2 C a l c i u m carbonate** 0.35 0 .35 0 .35 -F i n a l c o m p o s i t i o n by a n a l y s i s (%) P r o t e i n 20 .0 20 .0 20 .0 20 .0 L a c t o s e 0.6 20 .6 50 .6 8.2 M i l k f a t 0.2 0 .2 0 .2 0 . 2 Ca 0.72 0 .72 0 .72 0.72 1 SMPC = skim m i l k p r o t e i n c o n c e n t r a t e ; ML = = 20% l a c t o s e + SMPC; HL = 50% l a c t o s e + SMPC; Y = yog u r t powder. Skim m i l k p r o t e i n c o n c e n t r a t e - (85% 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 ) . Yogurt powder - (35.9% p r o t e i n , 52.3% 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% c a l c i u m ) . 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 C a l c i u m a b s o r p t i o n s t u d i e s were performed u s i n g the i n s i t u l i g a t e d i l e a l l o o p t e c h n i q u e of Lee et a l . (1983) w i t h minor m o d i f i c a t i o n s . Rats (15 weeks of age) were i n i t i a l l y a l l o w e d a c c e s s to d i e t s f o r a 1.5 hour p e r i o d on the morning of the expe r i m e n t . Animals were a n a e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l p e n t o b a r b i t a l i n j e c t i o n (50 mg/kg) 1.5 hours a f t e r food w i t h d r a w a l . A l o n g i t u d i n a l abdominal i n c i s i o n was made and the i l e u m i s o l a t e d and l i g a t e d at p o i n t s 8 cm and 20 cm from the i l e o c e c a l j u n c t i o n , t o form a c l o s e d l o o p . The i n t e s t i n a l c o n t e n t s of the l i g a t e d l oop were l e f t unwashed. A 0.3 mL a l i q u o t of 4 5 C a l c i u m (dose = 5.4 u C i , 4 5 C a C l 2 j S.A. = 10 mCi/ mg CaJ ICN B i o m e d i c a l , I r v i n e , CA) i n 0.15N s a l i n e was i n j e c t e d i n t o the l o o p . The i n t e s t i n e was r e p l a c e d and the abdomen s u t u r e d c l o s e d . Rats were s a c r i f i c e d one hour a f t e r i n t r a l u m i n a l i n j e c t i o n of 4 5 C a by e x s a n g u i n a t i o n , and the l i g a t e d i l e a l l o o p removed to f l u s h out the c o n t e n t s u s i n g 5 mL c o l d 0.15N s a l i n e . A n a l y s e s : I n t e s t i n a l c o n t e n t s were homogenized u s i n g a p o l y t r o n homogenizer (Brinkmann I n s t r . , R e x d a l e , ON) and an a l i q u o t of homogenate d i g e s t e d w i t h NCS t i s s u e s o l u b i l i z e r (Amersham, O a k v i l l e , ON) and mixed w i t h ACS s c i n t i l l a t i o n c o c k t a i l (Amersham, O a k v i l l e , ON). R a d i o a c t i v i t y measurements were made w i t h an LKB-1215 l i q u i d s c i n t i l l a t i o n s p e c t r o p h o t o m e t e r ( W a l l a c Oy, Turku, F i n l a n d ) w i t h a 90% c o u n t i n g e f f i c i e n c y f o r 4 5 C a . In s e p a r a t e e x p e r i m e n t s , i t was determined t h a t 92 +_ 3% of t o t a l r a d i o a c t i v i t y p r e s e n t i n the i n t e s t i n a l l o o p was r e c o v e r e d i n the 57 s a l i n e f l u s h . The measurement of * 0 C a i n the i l e a l homogenate was performed by atomic a b s o r p t i o n f o l l o w i n g wet a s h i n g w i t h HC I / H N O 3 (Mauer, 1977) and d i l u t i o n w i t h 0.5% L a C l 3 . L u m i n a l l a c t o s e Content was determined by p - g a l a c t o s i d a s e enzymatic h y d r o l y s i s ( B o e h r i n g e r Mannheim, D o r v a l , PQ). The d e t e c t i o n l i m i t f o r l a c t o s e was 5 ug/mL i n t h i s a s s a y . 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 of 4 5 C a i n the femur, an endpoint i n d e x of 4 5 C a i n t e s t i n a l a b s o r p t i o n (Mykkanen and Wasserman, 1980) was dete r m i n e d from the r i g h t femur i m m e d i a t e l y a f t e r s a c r i f i c i n g the r a t . Femora were weighed and then ashed at 550°C f o r 24 hours. The bone ash was d i s s o l v e d i n 3 mL of 4N HC1 and a l i q u o t s were taken f o r 4 5 C a r a d i o a c t i v i t y and bone m i n e r a l c o n t e n t , r e s p e c t -i v e l y . Femur c a l c i u m and magnesium c o n t e n t s were determined i n the a c i d i f i e d ashed samples d i l u t e d w i t h 0.5% L a C l 3 by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y . A f u r t h e r a l i q u o t of a c i d i f i e d bone ash was d i l u t e d w i t h d e i o n i z e d water and the phosphorus co n t e n t measured s p e c t r o p h o t o m e t r i c a l l y by the method of Chen et  a l . ( 1 9 5 6). B l o o d C h e m i s t r y : Plasma m i n e r a l s were measured from b l o o d samples taken by h e a r t p u n c t u r e . Plasma c a l c i u m , magnesium, sodium and p o t a s s i u m were de t e r m i n e d by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y i n the presence of 0.5% L a C l 3 • Plasma p r o t e i n was a n a l y z e d by the c o l o r i m e t r i c BCA p r o t e i n assay ( P i e r c e Chemical Co., R o c k f o r d , 58 I L ) . I o n i z e d c a l c i u m was c a l c u l a t e d u s i n g the e q u a t i o n of Z e i s l e r (1954): Ca* 2 = (6Ca - P r o t e i n / 3 ) / ( P r o t e i n + 6) where both Ca* 2 and Ca are mg/dL and P r o t e i n i s p r o t e i n c o n c e n t r a t i o n , g/dL. Plasma a-amino n i t r o g e n was determined u s i n g the p r o t o c o l of Goodwin (1968). Plasma t r i g l y c e r i d e and t o t a l c h o l e s t e r o l c o n c e n t r a t i o n s were a n a l y z e d by enzymatic 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 Mannheim, D o r v a l , PQ) . Bone Bi o m e c h a n i c s: An e m p i r i c a l measure of femur s t r e n g t h was performed u s i n g a s e r o h y d r a u l i c m a t e r i a l s t e s t i n g machine ( i n s t r o n Corp., Model 1122, Canton, MA) f i t t e d w i t h a s i n g l e - b l a d e shear attachment. The l e f t femur was c o n s i s t e n t l y used f o r t h i s measurement once c l e a n e d of s o f t t i s s u e s and e p i p h y s e s removed. Femora were p o s i t i o n e d unsupported on the l o a d p l a t f o r m r e s t i n g on the g r e a t e r t r o c h a n t e r . Bone d e f o r m a t i o n measurements were conducted by a p p l y i n g a shear f o r c e at a c o n s t a n t speed (1.0 mm/ min) to a c o n s i s t e n t a r e a , 4 mm from the d i s t a l end of the bone, perpen-d i c u l a r to the l o n g a x i s of the bone. T h i s r e g i o n of the femur was chosen because i t r e p r e s e n t s a t r a n s i t i o n area between compact c o r t i c a l and c a n c e l l o u s t r a b e c u l a r bone. The t i m e - d e f o r m a t i o n d a t a 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 . , L i t t l e t o n , CO) which was i n t e r f a c e d w i t h the I n s t r o n , through an IBM AT c o m p a t i b l e p e r s o n a l computer. Sample run time was 3 m i n u t e s , at 59 a s a m p l i n g r a t e of 5 s i g n a l s per second. The I n s t r o n s i g n a l was c a l i b r a t e d u s i n g 1.0 and 2.0 kg known w e i g h t s . Data were a n a l y z e d by t r a n s f o r m i n g the m i l l i v o l t s i g n a l output i n t o kg f o r c e . Two i n d i c e s of bone o s s i f i c a t i o n were d e t e r m i n e d , bone s t r e n g t h and bone hardness (Segars and K a p s a l i s , 1987). Bone s t r e n g t h i s d e s c r i b e d as the f o r c e (Newtons, N) a p p l i e d at the p o i n t of f r a c t u r e , d i v i d e d by the o r i g i n a l c r o s s - s e c t i o n a l a rea of the bone (mm 2). The bone hardness parameter i s d e f i n e d as the work or energy ( J o u l e s , J ) r e q u i r e d to f r a c t u r e the bone. S t a t i s t i c a l Analyses'. A l l the d a t a are e x p r e s s e d as mean +. SEM. One-way a n a l y s i s of v a r i a n c e was used to t e s t f o r d i f f e r e n c e s between the exper-i m e n t a l t r e a t m e n t s . Where d i f f e r e n c e s d i d e x i s t , the source of the d i f f e r e n c e s at a p<0.05 s i g n i f i c a n c e l e v e l was i d e n t i f i e d by the Student-Newman-Keuls m u l t i p l e range t e s t . 60 R e s u l t s Body weight g a i n s and food i n t a k e parameters were s i m i l a r i n SMPC, ML and Y f e d a n i m a l s , i n d i c a t i n g no adverse p a l a t a b i l i t y e f f e c t s of i n d i v i d u a l d i e t s ( T a b l e 1.2). In comparison, HL f e d ani m a l s e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) reduced food i n t a k e , body weight g a i n , and f e e d e f f i c i e n c y r a t i o . F u r t h e r , c a l c i u m i n t a k e was reduced i n HL f e d an i m a l s due to the lower feed i n t a k e . These p a r t i c u l a r a n i m a l s a l s o e x p e r i e n c e d s o f t s t o o l s which i n d i c a t e d some degree of d i a r r h e a and m a l a b s o r p t i o n , d u r i n g the e x p e r i m e n t a l p e r i o d . No i n d i c a t i o n s of d i a r r h e a were observed i n SMPC, ML or Y f e d a n i m a l s . Feeding r a t s e x p e r i m e n t a l d i e t s c o n t a i n i n g l a c t o s e or yo g u r t powder d i d not a f f e c t plasma p r o t e i n l e v e l s (range 5.64 +_ 0.14 to 6.63 +_ 0.21 mg/dL). Plasma m i n e r a l p r o f i l e s were a l s o not a f f e c t e d by d i e t a r y t r e a t m e n t s ( T a b l e 1.3). Plasma a-amino n i t r o g e n and t r i g l y c e r i d e c o n c e n t r a t i o n s were s i g n i f i c a n t l y (p<0.05) reduced i n an i m a l s f e d the HL d i e t , compared to SMPC and ML f e d c o u n t e r p a r t s . Plasma c h o l e s t e r o l l e v e l s were not a f f e c t e d by the e x p e r i m e n t a l d i e t s ( T a b l e 1.3). The 1 hour i n t e s t i n a l a b s o r p t i o n of 4 5 C a from the l i g a t e d i l e a l l o o p and femur d e p o s i t i o n of 4 5 C a are p r e s e n t e d i n Table 1.4. No s i g n i f i c a n t d i f f e r e n c e s i n the p e r c e n t of 4 5 C a absorbed from the i n t e s t i n a l l o op i n SMPC, ML and Y f e d an i m a l s were d e t e c t e d . The HL f e d group e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) enhanced a b s o r p t i o n of 4 5 C a from the l i g a t e d l o o p . There was no s i g n i f i c a n t d i f f e r e n c e between the SMPC c o n t r o l and ML d i e t a r y 61 TABLE 1.2 D i e t 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 . D i e t 2 I n i t i a l body F i n a l body Dry M a t t e r Feed E f f i c i e n c y wt. 3 (g) wt.* (g) I n t a k e (g) R a t i o SMPC 117 + 5« 323 37* 504 .0 20 .6* 0 .269 +_ 0 .012« ML 118 + 7* 301 39 a 477 .2 16 .8 a 0 .253 + 0 .014« HL 116 + l l a 246 +_ 46 b 366 .0 + 16 .0«> 0 . 162 0 .044b Y 111 + 10 a 304 •+ 66 a 452 . 5 +_ 25 . 6 a 0 .213 + 0 .027 a 1 Data are ex p r e s s e d as mean +. SEM. 2 SMPC = skim m i l k p r o t e i n c o n c e n t r a t e ; ML = 20% l a c t o s e + SMPC; HL = 50% l a c t o s e + SMPC; Y = y o g u r t powder. 3 5 weeks of age . 4 15 weeks of age. 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 d i f f e r e n t at p<0.05. 62 TABLE 1.3 Blood chemistry of r a t s fed experimental d i e t s . 2 +? T o t a 1 D i e t ' Ca Ca c a-amino-N T r i g l y c e r i d e s Cholesterol (mg/dL) SMPC 8.47 + 0.08 a ML 8.51 + 0.18 a HL 8.48 + 0.17 a Y 8.40 + 0.28 a 3.95 + 0.06 a 10.32 4.23 + 0.07 a 8.07 3.63 + 0.21 a 5.13 3.88 + 0.13 a 7.20 + 0.88 a 201.8 + + 0.50 a 184.4 + + 0.51 b 144.0 + + 0.66 a 118.3 + 0.5 a 94.88 + 0.02 a 0.3 a 106.38 + 0.01 a 0.4 b 103.50 + 0.02 a 0.2 C 106.38 + 0.04 a t, Data are expressed as mean + SEM. d SMPC = skim milk p r o t e i n concentrate; ML = 20% lactose + SMPC; HL = 50% lactose + SMPC; Y = yogurt powder. Means sharing 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 d i f f e r e n t at 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 femur d e p o s i t i o n of 4 5 c a l c i u m i n 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 . D i e t 2 Absorbed 4 5 C a I n t e s t i n a l S.A. Bone 4 5 C a Bone S.A. (% dose) (dpm/ mg 4 0 C a ) (% dose/g Ash) (dpm/mg 4 0 C a ) 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. 18 b 1 .678 0 .272«> 436 .9 50 . 9» Y 28 .7 +_ 2 .8* 9 .68 +_ 0 . 30«> 0 .793 + 0 . 105* 219 .8 + 24 .7« 1 Data are e x p r e s s e d as mean +_ SEM. 2 SMPC = skim m i l k p r o t e i n c o n c e n t r a t e ; ML = 20% l a c t o s e + SMPC; HL = 50% l a c t o s e + SMPC; Y = y o g u r t powder. 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 d i f f e r e n t at p<0.05. 64 group i n e i t h e r the a b s o r p t i o n of 4 5 Ca , or the amount of 4 0 C a p r e s e n t i n the l i g a t e d l o o p ; t h u s , the s p e c i f i c a c t i v i t i e s of the t r a c e r were not d i f f e r e n t . Animals f e d the HL and Y d i e t s , had s i g n i f i c a n t l y (p<0.05) lower 4 0 C a c o n t e n t s i n the i n t e s t i n a l l o o p , which c o n t r i b u t e d to h i g h e r i n t e s t i n a l s p e c i f i c a c t i v i t i e s . T h i s r e s u l t was p a r a l l e l e d by a h i g h e r 4 5 C a d e p o s i t i o n i n t o f e m o r a l t i s s u e i n o n l y the HL f e d a n i m a l s ( T a b l e 1.4). The apparent enhanced t r a n s l o c a t i o n of r a d i o l a b e l l e d c a l c i u m i n t o bone observed i n HL f e d ani m a l s l i k e l y r e s u l t e d from both an enhanced a b s o r p t i o n e f f i c i e n c y of 4 5 C a due to reduced c a l c i u m i n t a k e ; as w e l l as a s i g n i f i c a n t l y (p<0.05) h i g h e r l a c t o s e c o n t e n t of the l i g a t e d l oop ( F i g u r e 1.1). There was no s i g n i f i c a n t d i f f e r e n c e i n the femur d e p o s i t i o n of 4 5 C a i n Y f e d an i m a l s from SMPC and ML f e d a n i m a l s , d e s p i t e the h i g h e r i n t e s t i n a l s p e c i f i c a c t i v i t y o bserved i n these a n i m a l s . I t i s noteworthy t h a t the presence of l a c t o s e at both the 20% (ML) and 50% (HL) l e v e l s had no e f f e c t on o v e r a l l femur m i n e r a l i z a t i o n as ind e x e d by c a l c i u m (range 350.4 +. 10.2 to 383.8 +. 27.9 mg/g A s h ) , magnesium co n t e n t (range 11.53 +. 0.88 to 13.51 +_ 0.58 mg/g Ash) and Ca/P r a t i o (range 1.99 +_ 0.06 to 2.12 +_ 0.05). Femur p h y s i c a l and b i o m e c h a n i c a l s t r e n g t h parameters are p r e s e n t e d i n T a b l e 1.5. Femur bone l e n g t h d i d not d i f f e r between d i e t a r y t r e a t m e n t groups. Both femur weight and bone hardness were s i g n i f i c a n t l y (p<0.05) lower i n HL f e d a n i m a l s , compared to c o n t r o l s . No s i g n i f i c a n t d i f f e r e n c e i n femur weight or bone hardness were observed i n ML and Y f e d a n i m a l s compared to 65 c o n t r o l s . These parameters were however, s i g n i f i c a n t l y g r e a t e r (p<0.05) than i n a n i m a l s f e d the HL d i e t . 66 N.D. i SMPC Experimental Diets F i g . 1.1 Lacto s e content of i l e a l l o o p of r a t s f e d skim m i l k p r o t e i n c o n c e n t r a t e (SMPC), 20% l a c t o s e + SMPC (ML), 50% l a c t o s e + SMPC (HL), 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. = not 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 from ML t r e a t m e n t . Values are expressed as mean +. SEM. TABLE 1.5. Femur p h y s i c a l dimensions and b i o m e c h a n i c a l parameters i n 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 . D i e t 2 Femur Wt. Bone Length Bone S t r e n g t h Bone Hardness (g) (mm) (N/mm2) (x 1 0 " 3 J ) SMPC 0 . 681 + 0 .020- 31 .99 0 . 14" 1 .23 +_ 0 .03* 182 . 5 + 16 .6* ML 0 . 652 + 0 .047* 32 . 12 + 0 .44' 1 .28 +_ 0 .06* 161 . 9 +_ 14 .8* HL 0 . 562 + 0 .046* 30 . 19 + 0 . 67* 1 .53 +_ 0 .06* 121 .7 + 13 .8* Y 0 . 601 +_ 0 .043* 31 .04 + 0 .59* 1 .55 + 0 .04* 152 .3 + 23 .0* 1 Data are e x p r e s s e d as mean +_ SEM. 2 SMPC = skim m i l k p r o t e i n c o n c e n t r a t e ; ML = 20% l a c t o s e + SMPC; HL = 50% l a c t o s e + SMPC; Y = y o g u r t powder. 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 d i f f e r e n t at p<0.05. 68 D i s c u s s i o n P r e v i o u s r e p o r t s have e x p r e s s e d concern i n u s i n g the p o s t -weaning r a t as a model f o r s t u d y i n g l a c t o s e enhanced 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 , due to the apparent i n t o l e r a n c e of the r a t to l a c t o s e ( L e i c h t e r , 1973). No s i g n s of l a c t o s e i n t o l e r a n c e were observed i n t h i s s t u d y i n r a t s f e d e i t h e r the 20% (ML) l a c t o s e d i e t , or the Y d i e t . T h i s f i n d i n g agrees w i t h the r e c e n t study of Greger et_ al_. ( 1 9 8 9 ) , who a l s o r e p o r t e d no s i g n s of l a c t o s e i n t o l e r a n c e or d e c r e a s e d d i g e s t i b i l i t y of d i e t s c o n t a i n i n g 20% l a c t o s e . Moreover, r a t s f e d moderate amounts of l a c t o s e over an extended p e r i o d of t i m e , have been r e p o r t e d to e x h i b i t an i n c r e a s e d i n t e s t i n a l l a c t a s e a c t i v i t y ( B o l i n et a l . . 1969; 1971). Rats f e d the 50% l a c t o s e d i e t (HL) however, showed s i g n s of n u t r i e n t m a l a b s o r p t i o n , as i n d i c a t e d by s o f t s t o o l t e x t u r e , and de c r e a s e d food i n t a k e and body weight g a i n e d . These a n i m a l s were r e t a i n e d i n t h i s s t u d y , i n the absence of p a i r - f e d c o n t r o l s , because the l a c t o s e i n t o l e r a n t r a t has been r e p o r t e d to r e p r e s e n t a good a n i m a l model f o r s t u d y i n g c a l c i u m a b s o r p t i o n i n l a c t o s e 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 , 1975). The absence of a l a c t o s e mediated enhancement of c a l c i u m a b s o r p t i o n from the d i s t a l s m a l l i n t e s t i n e of r a t s f e d the ML d i e t , compared to SMPC and Y f e d a n i m a l s , r e s p e c t i v e l y , i s c o n t r a r y to r e p o r t s which have observed s i g n i f i c a n t enhancement of c a l c i u m b i o a v a i l a b i l i t y a t t r i b u t e d to l a c t o s e or i t s h y d r o l -y s i s p r o d u c t s (Pansu et al.. , 1979; Sato et_ al.. , 1983; M i l l e r e_t a l . . 1988; Greger e_t_ a_l_. , 1989). U s i n g the same .in. s i t u i l e a l 69 l o o p t e c h n i q u e f o r e s t i m a t i n g 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 , Sato and coworkers (1983) observed a l a c t o s e induced enhancement of c a l c i u m a b s o r p t i o n from the d i s t a l p o r t i o n of the s m a l l i n t e s t i n e . The d i s p a r i t y i n r e s u l t s may be e x p l a i n e d by the d i f f e r e n c e i n f e e d i n g p r o t o c o l s . The m e a l - f e e d i n g p r o t o c o l used i n t h i s s t u d y , e s p e c i a l l y p r i o r to the s u r g i c a l m a n i p u l a t i o n s , l i k e l y r e s u l t e d i n a reduced i n t e s t i n a l c o n t e n t of l a c t o s e and o t h e r n u t r i e n t s . C o n s e q u e n t l y , the l a c t o s e mediated e f f e c t 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 was d e c r e a s e d . The enhanced c a l c i u m a b s o r p t i o n observed i n the l a c t o s e i n t o l e r a n t HL f e d a n i m a l s , was l i k e l y the combined r e s u l t of an i n c r e a s e d a b s o r p t i o n e f f i c i e n c y due to the d e c r e a s e d c a l c i u m i n t a k e and i n t e s t i n a l l u m i n a l c a l c i u m c o n t e n t of these a n i m a l s ; as w e l l as a l a c t o s e mediated enhancement of 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 . I n t e s t i n a l c a l c i u m t r a n s p o r t i s known to be more e f f i c i e n t i n c o n d i t i o n s of c a l c i u m d e f i c i e n c y or n e g a t i v e c a l c i u m b a l a n c e (Pansu ejL ILL- > 1981). Moreover, the i l e u m has been r e p o r t e d to have an even g r e a t e r r o l e i n c a l c i u m a b s o r p t i o n i n c o n d i t i o n s of n u t r i e n t m a l a b s o r p t i o n ( H y l a n d e r et a l . . 1980). A l t e r n a t i v e l y , s i n c e l a c t o s e i n t o l e r a n c e i s known to i n c r e a s e g a s t r i c m o t i l i t y i n r a t s ( L e i c h t e r and T o l e n s k y , 1975), the h i g h l a c t o s e c o n t e n t of our HL d i e t , appeared to r e s u l t i n a g r e a t e r c o n c e n t r a t i o n of l a c t o s e i n the i l e u m d u r i n g the time p e r i o d of our e x p e r i m e n t a l p r o t o c o l . These r e s u l t s s u p p o r t the concept t h a t l a c t o s e f a c i l i t a t i o n of c a l c i u m a b s o r p t i o n from the i l e u m , i s an acute p r o c e s s and dependent on o p t i m a l c a l c i u m and l a c t o s e 70 c o n c e n t r a t i o n s i n t h i s r e g i o n of the i n t e s t i n e (Armbrecht and Wasserman, 1976). At the pH of m i l k (pH 6.8), a major p r o p o r t i o n (68%) of the c a l c i u m i s p r e s e n t i n c o l l o i d a l form, e i t h e r bound to phosphate e s t e r m o i e t i e s of c a s e i n p h o s p h o s e r y l r e s i d u e s , or c a l c i u m phosphate l i n k a g e s a c t i n g to s t a b i l i z e the c a s e i n m i c e l l e . Lowering the pH t o produce fermented d a i r y p r o d u c t s , such as y o g u r t , s o l u b i l i z e s the c o l l o i d a l c a l c i u m phosphate and t h u s , i n c r e a s e s the p r o p o r t i o n of i o n i c c a l c i u m (Swaisgood, 1985). 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 reduced c a l c i u m b i o a v a i l a b i l i t y a s s o c i a t e d w i t h fermented, low pH d a i r y p r o d u c t s , i n which the c a l c i u m i s p r e s e n t p r i m a r i l y i n an i o n i c form (Wong and L a C r o i x , 1980; K a n s a l and Chaudhary, 1982). However, the d i f f e r e n t 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 based d i e t s f e d to a n i m a l s i n t h i s s t u d y were observed to have no e f f e c t 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 . Our r e s u l t s agree w i t h those of Smith et a l . (1985) who r e p o r t e d no d i f f e r e n c e i n c a l c i u m b i o a v a i l a b i l i t y from whole m i l k and y o g u r t . S i m i l a r l y , c a l c i u m has been shown to be absorbed e q u a l l y e f f i c i e n t l y from y o g u r t and c o m m e r c i a l l y a v a i l a b l e c a l c i u m supplements, both of which p r o v i d e c a l c i u m i n a p r e d o m i n a n t l y i o n i c form ( B e h l i n g and Greger, 1988). The g r e a t e r i n t e s t i n a l c a l c i u m s p e c i f i c a c t i v i t y of the HL f e d a n i m a l s corresponded t o an i n c r e a s e d 4 5 C a femur d e p o s i t i o n . Y f e d a n i m a l s e x h i b i t e d a s i m i l a r d e p o s i t i o n of 4 5 C a to the bone as ML and SMPC f e d a n i m a l s . P r e v i o u s workers have used s h o r t term femur 4 3 C a d e p o s i t i o n d a t a as an i n d e x of c a l c i u m 71 b i o a v a i l a b i l i t y and u t i l i z a t i o n (Mykkanen and Wasserman, 1980; Sato et a1. . 1986). Our r e s u l t s i n d i c a t e t h a t 4 5 C a found i n the bone of HL f e d a n i m a l s does not equate w i t h an enhanced bone a c c r e t i o n , s i n c e n e i t h e r bone s t r e n g t h or c a l c i f i c a t i o n was enhanced i n these a n i m a l s . Moreover, a r e d u c t i o n i n bone hardness i n l a c t o s e m a l a b s o r b i n g r a t s c o u l d not be a t t r i b u t e d to 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 . Thus, c a u t i o n i s needed i n the c o r r e c t i n t e r p r e t a t i o n of bone 4 5 C a d e p o s i t i o n d a t a . The HL f e d a n i m a l s a l s o e x h i b i t e d a d ecrease i n both plasma a-amino n i t r o g e n and t r i g l y c e r i d e c o n c e n t r a t i o n s , which are an expected r e s u l t of reduced food i n t a k e and d i g e s t i o n . These f i n d i n g s s u pport p r e v i o u s b a l a n c e s t u d i e s conducted by L e i c h t e r and T o l e n s k y (1975) which showed both p r o t e i n and f a t , but not c a l c i u m , m a l a b s o r p t i o n i n l a c t o s e i n t o l e r a n t r a t s . Our r e s u l t s extend these f i n d i n g s to the s m a l l i n t e s t i n e , s p e c i f i c a l l y the i l e u m , and f u r t h e r m o r e add t h a t l a c t o s e or o t h e r n o n s p e c i f i c i n d i c a t o r s of m a l a b s o r p t i o n o c c u r r i n g i n HL a n i m a l s , d i d not a d v e r s e l y a f f e c t bone s t r e n g t h , a l b e i t femur weight and hardness were reduced. 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 w i l l l e a d to d e l e t e r i o u s e f f e c t s on s k e l e t a l c o r t i c a l bone t h i c k n e s s i n b o th young a d u l t s and c h i l d r e n (Garn et a1.. 1964; Crosby et  a l . . 1983). Thus, i t i s c o n c l u d e d t h a t the n u t r i e n t malabsorp-t i o n caused by the l a c t o s e 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 HL d i e t , c o n t r i b u t e d s i g n i f i c a n t l y to r e d u c i n g bone growth and a r c h i t e c t u r a l m a t r i x components, which i n t u r n c o n t r i b u t e to bone s i z e and t i s s u e c o n t e n t . T o g e t h e r , these components comprise the 72 b i o l o g i c a l mechanisms r e q u i r e d to respond to the s p e c i f i c b i o m e c h a n i c a l usages of bone ( F r o s t , 1988). F u r t h e r s t u d i e s are r e q u i r e d to c o n f i r m t h i s h y p o t h e s i s i n p a i r - f e d a n i m a l s , s i n c e the s i g n i f i c a n c e of these f i n d i n g s i s r e l e v a n t to the bone met a b o l i s m 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 , the presence of a moderate d i e t a r y l e v e l of l a c t o s e , t o g e t h e r w i t h s o l u b l e and i n s o l u b l e d a i r y forms of c a l c i u m , have been shown to have l i t t l e s i g n i f i c a n c e i n the s u s t a i n e d enhancement of 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 f o r bone d e p o s i t i o n . The r e d u c t i o n i n bone har d n e s s , but not bone s t r e n g t h observed i n l a c t o s e i n t o l e r a n t r a t s c o u l d not be a t t r i b u t e d to a d e c r e a s e d femur m i n e r a l c o n t e n t , a l t h o u g h an apparent enhancement of 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 was ob s e r v e d . 73 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 r a t s f e d s e l e c t e d d i e t a r y p r o t e i n s . I n t r o d u c t i o n In r e c e n t s t u d i e s , c a l c i u m supplements have been shown to i n c r e a s e bone mass i n post-menopausal p a t i e n t s ( S h i h et a1. . 1988). In a d d i t i o n t o the c a l c i u m a v a i l a b l e t o the i n d i v i d u a l through the d i e t , t h e r e has been c o n s i d e r a b l e study i n t o the r o l e of d i e t a r y c o n s t i t u e n t s , such as p r o t e i n , on 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 u r i n a r y c a l c i u m e x c r e t i o n ( L i n k s w i l e r et a 1. . 1981). Taken t o g e t h e r , these two f a c t o r s w i l l a f f e c t the c a l c i u m b a l a n c e of the i n d i v i d u a l . The o b j e c t i v e s of the p r e s e n t study were to determine the c a l c i u m a b s o r p t i o n from the s m a l l i n t e s t i n e i n r a t s f e d s e l e c t e d d i e t a r y p r o t e i n s . F u r t h e r , bone 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 parameters were examined as endpoint i n d i c e s of c a l c i u m u t i l i z a t i o n . M a t e r i a l s and Methods  Animals and D i e t s : T h i r t y - t w o 5 week o l d male W i s t a r r a t s , matched f o r age and sex were purchased from C h a r l e s R i v e r ( M o n t r e a l , PQ). Animals were i n d i v i d u a l l y housed i n s t a i n l e s s s t e e l cages w i t h c o n t r o l l e d temperature (25°C) and l i g h t i n g (14:10 d/n c y c l e ) . Animals were s e g r e g a t e d i n t o f o u r d i e t a r y groups w i t h r e s p e c t t o d i e t a r y p r o t e i n : c a s e i n , whey p r o t e i n c o n c e n t r a t e , m i l k p r o t e i n c o n c e n t r a t e and soy p r o t e i n i s o l a t e ( T a b l e 2.1). Animals were f e d d i e t s ad l i b i t u m u n t i l lOOg body weight was reached, whereupon m e a l - f e e d i n g was s t a r t e d . Animals were t r a i n e d to 74 consume t h e i r d i e t s w i t h i n a 6 hour p e r i o d d a i l y . D e i o n i z e d water was p r o v i d e d ad 1 i b i turn to the a n i m a l s . D a i l y f e e d i n t a k e s and weekly body w e i g h t s were r e c o r d e d . 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 was measured by the i n s i t u l i g a t e d i l e a l l o o p t e c h n i q u e based on the procedure of Lee ejt_ a l . (1983) as d e s c r i b e d i n experiment 1, when the a n i m a l s were 14 weeks of age. A dose (0.3 mL) of 4 5 C a ( 4 5 C a C l 2 , 18.2 mCi/mg Ca, ICN B i o m e d i c a l I n c . , I r v i n e , CA) i n 0.9% s a l i n e was i n j e c t e d i n t r a l u m i n a l l y i n t o the l o o p ( t o t a l dose was 5.4 u C i ) . A b s o r p t i o n of c a l c i u m was e s t i m a t e d from the amount of a d m i n i s t e r e d 4 5 C a r e m a i n i n g i n the l o o p a f t e r one hour. A n a l y s e s : I n t e s t i n a l lumen, and b l o o d plasma m i n e r a l s were a n a l y z e d as p r e v i o u s l y d e s c r i b e d i n experiment 1. Bone Bi o m e c h a n i c s : A f t e r e u t h a n a s i a , bone samples, femur and t i b i a , were e x c i s e d by b l u n t d i s s e c t i o n , c l e a n s e d of a d h e r i n g s o f t t i s s u e , and the e p i p h y s e s removed. Bone b i o m e c h a n i c a l parameters were a s s e s s e d 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 Machine (Model 1122, I n s t r o n Corp., Canton, MA). The femora were s u b j e c t e d to a s i n g l e - b l a d e shear t e s t w h i l e the t i b i a e underwent a 3 - p o i n t bending a n a l y s i s . In the s i n g l e - b l a d e shear t e s t , femora were a n a l y z e d as p r e v i o u s l y d e s c r i b e d i n experiment 1. 75 TABLE 2.1 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 f e d t o a n i m a l s . C a s e i n SPI MPC Whey D i e t a r y component 1 (g/lOOg) C a s e i n * 20 .0 - - -Soy p r o t e i n i s o l a t e * - 20 .0 - -MPC* * - - 23 . 5 -Whey p r o t e i n * * — — — 23 . 5 D.L. met h i o n i n e * 0 .3 0 .3 0 .3 0 .3 C o r n s t a r c h * 11 .35 11 .35 11 .35 11 . 35 Sucrose 53 . 16 53 .23 50 .0 50 .29 F i b r e * 5 .0 5 .0 5 .0 5 .0 Ve g e t a b l e o i l 5 .0 5 .0 4 .8 4 .8 Ca f r e e m i n e r a l mix* 3 .5 3 .5 3 .5 3 .5 V i t a m i n m i x t u r e * 1 .0 1 .0 1 .0 1 .0 C h o l i n e b i t a r t r a t e * 0 .2 0 .2 0 .2 0 .2 Ca l c i u m carbonate** 0 .49 0 .42 0 .35 0 .06 1 SPI = soy p r o t e i n i s o l a t e ; MPC = skim m i l k p r o t e i n c o n c e n t r a t e . *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. ** 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. 76 In 3 - p o i n t b e n d i n g , each t i b i a was bent u n t i l f a i l u r e o c c u r r e d , by l o w e r i n g a c e n t r a l l y p l a c e d p o i n t at a c o n s t a n t speed (1.0 mm/ min ) . T h i s t e s t a l l o w s two whole bone p r o p e r t i e s to be d e t e r m i n e d , bending f a i l u r e energy and maximum bending s t r e s s ( a ) . Bending f a i l u r e energy i s d e s c r i b e d as the work, or energy, ( a r e a under the f o r c e - d e f o r m a t i o n c u r v e ; Appendix F i g u r e 1) n e c e s s a r y t o a c h i e v e f a i l u r e of the bone i n ben d i n g , e x p r e s s e d as J o u l e s ( J ) . The maximum bending s t r e s s i s a c a l c u l a t e d v a l u e t h a t t a k e s i n t o c o n s i d e r a t i o n bone s i z e (N/mm2; O r t o f f and Oxlund, 1988): a - 8 x Maximum Bending Load (L - 1) D (D 4 - d 4 ) where L i s the d i s t a n c e between the s u p p o r t i n g p o i n t s (13 mm); D and d are the o u t e r and i n n e r d i a m e t e r s of the bone (mm). 77 Bone M i n e r a l Content A n a l y s e s : Bone m i n e r a l a n a l y s e s were performed on the femora and t i b i a e used i n the s h e a r i n g and 3 - p o i n t bending p r o c e d u r e s . Samples were d r i e d at 110°C f o r 3 days, then ashed at 550°C f o r 24 hou r s . The ash was s o l u b i l i z e d w i t h c o n c e n t r a t e d HC1 f o r d e t e r m i n a t i o n of * 5 C a by l i q u i d s c i n t i l l a t i o n c o u n t i n g , 4 0 C a and Mg by atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y and P as d e s c r i b e d i n experiment 1. S t a t i s t i c a l A n a l y s e s : A l l the data are e x p r e s s e d as mean +_ SEM. D i f f e r e n c e s between t r e a t m e n t s were t e s t e d f o r by one-way a n a l y s i s of v a r i a n c e . The Student-Newman-Keuls m u l t i p l e range t e s t was used to i d e n t i f y ' t h e source of the d i f f e r e n c e s at the p<0.05 l e v e l . 78 R e s u l t s A l l a n i m a l s 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 f o l l o w i n g meal-f e e d i n g t r a i n i n g . There were no s i g n i f i c a n t d i f f e r e n c e s between e x p e r i m e n t a l groups i n body weight g a i n e d or dry matter i n t a k e ( T a b l e 2.2). In a d d i t i o n , f e e d e f f i c i e n c y r a t i o s d u r i n g the course of the e x p e r i m e n t a l p e r i o d were not s i g n i f i c a n t l y (p<0.05) d i f f e r e n t between t r e a t m e n t s ( T a b l e 2.2). Plasma p r o t e i n l e v e l s were not a f f e c t e d by d i e t a r y t r e a t m e n t s (range 6.12 +_ 0.10 t o 6.74 +_0.27 mg/dL). Plasma m i n e r a l s were a l s o not d i f f e r e n t between d i e t a r y groups. N e i t h e r the t o t a l plasma c a l c i u m (range 8.47 +_ 0.08 to 8.72 +_ 0.13 mg/dL), nor the c a l c u l a t e d i o n i z e d plasma c a l c i u m (range 3.82 +_ 0.10 to 4.04 +_ 0.05 mg/dL) were found to be a f f e c t e d by d i e t a r y t r e a t m e n t s . The p e r c e n t of 4 5 C a absorbed from the l i g a t e d i l e a l l o o p i n c a s e i n , whey p r o t e i n , and MPC f e d r a t s was found to be s i g n i f -i c a n t l y (p<0.05) g r e a t e r than f o r anim a l s f e d soy p r o t e i n i s o l a t e ( T a b l e 2.3). With the e x c e p t i o n of whey p r o t e i n f e d a n i m a l s , i n t e s t i n a l s p e c i f i c a c t i v i t i e s were not s i g n i f i c a n t l y d i f f e r e n t between d i e t a r y groups. The s p e c i f i c a c t i v i t y of c a l c i u m p r e s e n t i n the i n t e s t i n a l l o o p of whey p r o t e i n f e d r a t s was s i g n i f i c a n t l y (p<0.05) h i g h e r . T h i s o b s e r v a t i o n was f u r t h e r r e f l e c t e d i n the 4 5 C a d e p o s i t i o n t o the bone ( T a b l e 2.3). The s p e c i f i c a c t i v i t y of the femora from the whey p r o t e i n f e d animals was s i g n i f i c a n t l y (p<0.05) g r e a t e r than t h a t of animals f e d c a s e i n , MPC, and soy p r o t e i n i s o l a t e . The s i g n i f i c a n t l y (p<0.05) 79 TABLE 2 . 2 D i e t 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 . D i e t 2 Body Weight Gain Dry M a t t e r I n t a k e Feed E f f i c i e n c y (g) (g) R a t i o C 143 .0 +_ 9 . 6 5 2 8 . 9 + 23 . 8 0 . 2 6 9 + 0 .009 s 1 3 2 . 8 4 . 0 4 9 7 . 0 + 12 . 7 0 . 2 6 8 + 0 . 0 0 7 M 1 3 6 . 4 + 9 . 9 504 .0 + 20 . 6 0 . 2 6 9 0 . 0 1 2 W 1 2 0 . 8 5 . 5 470 . 9 + 1 6 . 3 0 . 2 5 6 +_ 0 . 0 0 7 1 Data are e x p r e s s e d as mean +. SEM. 2 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 p r o t e i n 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 not s i g n i f i c a n t l y d i f f e r e n t at p<0.05. 80 TABLE 2 . 3 I n t e s t i n a l a b s o r p t i o n 4 5 c a l c i u m i n r a t s f e d and femur d e p o s i t i o n e x p e r i m e n t a l d i e t s 1 . of D i e t 2 Absorbed 4 5 C a I n t e s t i n a l S.A. Bone 4 5 C a Bone S.A. (% dose) (dpm/ mg 4 0 C a ) (% dose/g Ash) (dpm/mg 4 0Ca) C 36 .53 2 .82* 2 .91 + 0 .35* 0 .857 + 0 . 117* 204 .3 + 19 . 9* s 22 .69 3 . 17«> 3 .65 + 0 .48* 0 . 706 + 0 . 114* 187 .2 + 19 . 4* M 35 . 19 + 4 .72" 2 .37 + 0 .28* 0 .654 + 0 . 114* 161 . 1 +_ 26 . 1* W 31 . 70 + 1 . 04* • • «> 5 .04 +_ 0 . 52»> 1 . 505 +_ 0 . 1 8 7 » 342 .4 + 33 .8<> 1 Data are e x p r e s s e d as mean +_ SEM 2 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 p r o t e i n 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 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 d i f f e r e n t at p<0.05. 81 lower amount of 4 5 C a absorbed from the i l e a l l o o p i n soy f e d a n i m a l s however, was not r e f l e c t e d i n e i t h e r bone 4 5 C a depos-i t i o n , or bone s p e c i f i c a c t i v i t y ( T a b l e 2.3). The p h y s i c a l d i m e n s i o n s , m i n e r a l c o n t e n t s , and b i o m e c h a n i c a l parameters of the femora and t i b i a e of the a n i m a l s from the d i f f e r e n t d i e t a r y groups are summarized i n T a b l e s 2.4 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 f e d the c a s e i n and MPC d i e t s , compared to those f e d the soy p r o t e i n i s o l a t e and whey p r o t e i n d i e t s , no s i g n i f i c a n t d i f f e r e n c e s i n e i t h e r bone m i n e r a l i z a t i o n or bone b i o m e c h a n i c a l p r o p e r t i e s of these p a r t i c u l a r a n i m a l s were o b s e r v e d . S i g n i f i c a n t c o r r e l a t i o n s were found t o e x i s t between femur c a l c i u m content (mg Ca/g Ash) and bone hardness ( F ( 1, 42 ) = 9 . 38 , p<_ 0.003) and femur weight ( F ( l , 42)=10.43, p<. 0.0024). 82 TABLE 2.4 Femur m i n e r a l d i e t s 1 . c o m p o s i t i o n i n r a t s f e d e x p e r i m e n t a l D i e t 2 Ash Wt. C a l c i u m Ca/P Magnesium (g/femur) (mg/g Ash) (mg/g Ash) C 0 .270 + 0 .009 361 .8 + 5 .3 2 .01 0 .06 13 .350 0 .421 s 0 .274 + 0 .008 372 .4 +_ 9 . 1 2 . 10 + 0 .07 13 . 506 + 1 .068 M 0 .277 0 .004 361 . 1 +_ 9 . 1 2 .05 +_ 0 .06 13 .506 + 0 .580 w 0 .256 + 0 .013 369 .0 + 4 .8 2 .08 +_ 0 .05 9 . 948 + 0 .315 1 Data are e x p r e s s e d as mean +_ SEM 2 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 p r o t e i n 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 not s i g n i f i c a n t l y d i f f e r e n t at p<0.05. 83 TABLE 2.5 Femur p h y s i c a l d imensions and b i o m e c h a n i c a l parameters i n 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 . D i e t 2 Femur Wt. Bone Length Bone S t r e n g t h Bone Hardness (g) (mm) (N/mm2) (x 1Q- 2J) 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 1 .66 W 0 . 626 +_ 0 .020 32 .35 + 0 .40 1 . 289 + 0 .058 13 .55 + 0 .87 1 Data are e x p r e s s e d as mean +_ SEM 2 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 p r o t e i n 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 not s i g n i f i c a n t l y d i f f e r e n t at p<0.05). 84 TABLE 2.6 T i b i a calcium content and biomechanical parameters of three-point bending a n a l y s i s i n r a t s fed experimental d i e t s 1 . D i e t * T i b i a Wt. Ash Wt. Calcium Bending F a i l u r e Maximum Bend-(g) ( g / t i b i a ) (mg/g Ash) Energy ing Stress (x 10 - 3 J) (N/mm2) 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 are expressed as mean + SEM C = case i n ; S = soy pro t e i n i s o l a t e ; M = milk p r o t e i n concentrate; w = whey p r o t e i n i s o l a t e . D i s c u s s i o n The a b s o r p t i o n of c a l c i u m from the i l e u m , as w e l l as i t s subsequent u t i l i z a t i o n f o r bone m i n e r a l i z a t i o n and biomechanics were examined i n a n i m a l s f e d d i f f e r e n t d i e t a r y p r o t e i n s . D a i r y f o o d s , c o n s i d e r e d to be e x c e l l e n t s o u r c e s of c a l c i u m , were 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 as a p p l i e d to d i e t a r y c a l c i u m b i o a v a i l a b i l i t y . D a i r y p r o t e i n s , namely c a s e i n , whey p r o t e i n s and a c o m b i n a t i o n of b o t h i n a m i l k p r o t e i n c o n c e n t r a t e were compared to soy p r o t e i n , a p l a n t p r o t e i n s o u r c e . The presence of c a s e i n , whey p r o t e i n , and MPC i n the r e s p e c t i v e d i e t s was shown to r e s u l t i n a h i g h e r i l e a l a b s o r p t i o n of 4 5 C a compared to the soy p r o t e i n d i e t . In the case of whey p r o t e i n fe d a n i m a l s however, a lower l e v e l of 4 0 Ca i n the i n t e s t i n a l lumen r e s u l t e d i n a h i g h e r s p e c i f i c a c t i v i t y f o r c a l c i u m i n the l i g a t e d l o o p , and p o s s i b l y an o v e r e s t i m a t i o n i n the amount of 4 5 C a absorbed. T h i s r e s u l t may r e f l e c t the poor d i g e s t i b i l i t y of whey p r o t e i n s observed i n p r e v i o u s a n i m a l f e e d i n g s t u d i e s , which has been a t t r i b u t e d to one of i t s c o n s t i t u e n t p r o t e i n s , i n p a r t i c u l a r a - l a c t a l b u m i n ( K e i t h and B e l l , 1988). The low d i g e s t i b i l i t y of whey p r o t e i n s c o u l d f e a s i b l y d e c r ease the i n t e s t i n a l t r a n s i t time which would r e s u l t i n a l e s s e r amount of 4 0 C a b e i n g p r e s e n t i n the i l e a l l o o p at any g i v e n t i m e . A c c o r d i n g l y , the lower r e l a t i v e d i g e s t i b i l i t y of soy p r o t e i n s o u r c e s observed by o t h e r s (Raghunath and N a r a s i n g a Rao, 1984), s h o u l d have a l s o i n c r e a s e d the s p e c i f i c a c t i v i t y of 4 5 C a i n the i n t e s t i n a l l o o p , but t h i s was not the case. The lower a b s o r p t i o n 86 of 4 5 C a i n r a t s f e d the soy p r o t e i n d i e t was t h e r e f o r e not r e l a t e d to the d i g e s t i b i l i t y of t h i s p r o t e i n . Soy p r o t e i n s are h i g h l y s t r u c t u r e d p r o t e i n s and c o n s e q u e n t l y , are more r e s i s t a n t to enzymatic a t t a c k . The r e s u l t i s t h a t the y i e l d of p e p t i d e and amino a c i d p r o d u c t s d e r i v e d from soy p r o t e i n d i g e s t i o n s are c o n s i d e r a b l y d i f f e r e n t than those from c a s e i n (Raghunath and N a r a s i n g a Rao, 1984). In p r e v i o u s s t u d i e s , i t was c o n s i d e r e d t h a t the amino a c i d p r o f i l e of i n t e s t i n a l c o n t e n t s i s v i r t u a l l y c o n s t a n t , r e g a r d l e s s of the p r o t e i n s d i g e s t e d (Nasset and J u , 1961). However, r e c e n t i n v i t r o s t u d i e s have c l e a r l y shown a markedly h i g h e r L - a r g i n i n e and lower L - l y s i n e c o n t e n t from d i g e s t i o n p r o d u c t s d e r i v e d from soy p r o t e i n compared to c a s e i n (Jacques et a l . . 1986). Moreover, Wasserman and coworkers (1956) r e p o r t e d t h a t L - a r g i n i n e enhances i l e a l c a l c i u m uptake by m o d i f y i n g the n o n s a t u r a b l e , p a r a c e l l u l a r pathway of c a l c i u m a b s o r p t i o n . The f a c t t h a t a lower r e l a t i v e a b s o r p t i o n of 4 5 C a was observed i n the l i g a t e d i l e a l loop of a n i m a l s f e d soy, compared to both c a s e i n and the MPC f e d a n i m a l s , s t r o n g l y s u g g e s t s t h a t the d i g e s t i o n p r o d u c t s of soy p r o t e i n had l i t t l e e f f e c t i n enhancing c a l c i u m a b s o r p t i o n . T h i s o b s e r v a t i o n would a l s o negate any s i g n i f i c a n c e of the lower d i g e s t i b i l i t y of soy p r o d u c t s i n e l i c i t i n g t h i s r e s p o n s e . On the o t h e r hand, phosphopeptides d e r i v e d from c a s e i n (CPP) have been shown to i n c r e a s e the a v a i l a b i l i t y of i o n i c c a l c i u m , by m a i n t a i n i n g i t i n a s o l u b l e form i n the lower s m a l l i n t e s t i n e ( S a t o ejt a_l_. , 1983). Lee ejt a_l_. (1983) r e p o r t e d t h a t r a t s f e d a 87 c a s e i n d i e t had a s i g n i f i c a n t l y g r e a t e r amount of s o l u b l e c a l c i u m and d e c r e a s e d i n s o l u b l e c a l c i u m i n the lower 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 amino a c i d m i x t u r e s i m u l a t i n g c a s e i n or egg albumen, r e s p e c t i v e l y . T h i s o b s e r v a t i o n has a l s o r e c e n t l y been c o n f i r m e d i n our l a b o r a t o r y ( K i t t s et a1., 1989). T h e r e f o r 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 observed i n both the c a s e i n and MPC f e d a n i m a l s s u p p o r t s the h y p o t h e s i s t h a t c a s e i n phosphopeptides produced from t r y p t i c d i g e s t i o n of c a s e i n w i l l f a c i l i t a t e the a b s o r p t i o n of c a l c i u m from the d i s t a l s m a l l i n t e s t i n e . The r e l a t i v e importance of t h i s a c t i o n t o the o v e r a l l c a l c i u m b a l a n c e of the i n d i v i d u a l has not yet been a s c e r t a i n e d . In t h i s study we a l s o examined bone d e p o s i t i o n of 4 5 C a , bone m i n e r a l i z a t i o n and biomechanics as i n d i c e s of c a l c i u m u t i l i z a t i o n from r a t s f e d the d i f f e r e n t d i e t a r y p r o t e i n s o u r c e s . I t i s noteworthy t h a t the h i g h e r i n t e s t i n a l s p e c i f i c a c t i v i t y of c a l c i u m i n the whey p r o t e i n f e d a n i m a l s corresponded to a g r e a t e r 4 5 C a d e p o s i t i o n and s p e c i f i c a c t i v i t y i n the femora of these a n i m a l s . T h i s method i s s u b j e c t to o v e r e s t i m a t i o n when the i n t e s t i n a l c a l c i u m s p e c i f i c a c t i v i t y i s a r t i f i c i a l l y e l e v a t e d due to a s s o c i a t e d f a c t o r s c h a r a c t e r i s t i c of the d i e t . On the o t h e r hand, bone 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 p r o p e r t i e s are measurements of c a l c i u m u t i l i z a t i o n which are not s u s c e p t i b l e to s i m i l a r s o u r c e s of o v e r e s t i m a t i o n . In the p r e s e n t s t u d y , f o r c e s were a p p l i e d i n d e f o r m a t i o n to i m i t a t e a p a r t i c u l a r a c t i o n i n the s i n g l e b l a d e shear t e s t . Data g e n e r a t e d i n t h i s t e s t were u s e f u l 88 i n comparing a s e r i e s of samples t e s t e d under i d e n t i c a l c o n d i t i o n s . The magnitude of the v a l u e s o b t a i n e d had l i t t l e i m portance from an a b s o l u t e p o i n t of view however, s i n c e they were h i g h l y dependent on t e s t c o n d i t i o n s (Segars and K a p s a l i s , 1987). In a d d i t i o n , a w e l l d e f i n e d e n g i n e e r i n g t e s t i n v o l v i n g 3-p o i n t bending was used. The a p p l i c a t i o n of a pure model of d e f o r m a t i o n to the sample was employed and a f o r m u l a was used to c a l c u l a t e the maximum bending s t r e s s t h a t models the bending s t r e n g t h of tubes t h a t are c i r c u l a r i n c r o s s - s e c t i o n and possess u n i f o r m w a l l t h i c k n e s s ( O r t o f f and Oxlund, 1988). In the p r e s e n t s t u d y , s i g n i f i c a n t c o r r e l a t i o n s were o b t a i n e d between f e m o r a l hardness and femur weight and c a l c i u m c o n t e n t , r e s p e c t i v e l y . These r e s u l t s c o r r o b o r a t e the p o s t u l a t i o n t h a t bone s t r e n g t h may be a s s o c i a t e d w i t h bone m i n e r a l c o n t e n t (Kusy et a 1. . 1987), and may be a u s e f u l f u n c t i o n a l t e s t f o r a s s e s s i n g c a l c i u m u t i l i z a t i o n (Kusy e t a l . , 1987; O r t o f f and Oxlund, 1988). D e s p i t e the apparent i n c r e a s e d c a l c i u m b i o a v a i l a b i l i t y from c a s e i n c o n t a i n i n g d i e t s , no enhancement of e i t h e r plasma t o t a l and i o n i z e d c a l c i u m , or bone 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 p r o p e r t i e s i n the femur and t i b i a samples were ob s e r v e d . Other workers have shown a marked i n c r e a s e i n the u r i n a r y e x c r e t i o n r a t e of c a l c i u m i n r a t s f e d a h i g h p r o t e i n d i e t , a l b e i t no e f f e c t on i n t e s t i n a l a b s o r p t i o n was observed ( A l l e n ejc. a 1 . . 1979). T h i s h y p e r c a l c i u r i a has been a t t r i b u t e d to a decrease i n f r a c t i o n a l r e n a l t u b u l a r r e a b s o r p t i o n and an i n c r e a s e i n g l o m e r u l a r f i l t r a t i o n of c a l c i u m ( S c h u e t t e et a l . , 1981). The f i n d i n g s of 89 W h i t i n g and Draper (1981) t h a t showed p r o t e i n s u l f u r amino a c i d c o n t e n t to be r e l a t e d to a c a l c i u r e t i c a c t i o n , f u r t h e r demon-s t r a t e d t h a t the source of d i e t a r y p r o t e i n , i n a d d i t i o n t o the q u a n t i t y f e d , can a d v e r s e l y a f f e c t c a l c i u m b a l a n c e . In t h i s 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 c a l c i u m at a l e v e l adequate f o r c a l c i u m h o m e o s t a s i s . Thus, 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 due to the presence of CPP, or c e r t a i n amino a c i d s t h a t may enhance e i t h e r p a r a c e l l u l a r movement, or u r i n a r y e x c r e t i o n of t h i s m i n e r a l , had l i t t l e p h y s i o l o g i c a l s i g n i f i c a n c e i n r e g a r d to i t s u t i l i z a t i o n f o r bone metabolism. F u r t h e r s t u d i e s are r e q u i r e d t o determine i f t h i s i s the case i n c a l c i u m d e f i c i e n t s u b j e c t s . 90 Experiment 3 C a l c i u m b i o a v a i l a b i l i t y i n c a s e i n and soy f e d r a t s . I n t r o d u c t i o n D i e t a r y c a l c i u m i n t a k e i s c o n s i d e r e d t o be a f a c t o r i n bone m i n e r a l i z a t i o n , and i n p a r t i c u l a r , the i n c i d e n c e of o s t e o p o r o s i s . B i o a v a i l a b i l i t y of c a l c i u m from p l a n t p r o t e i n sources i s noted to be i n f e r i o r , when compared to animal d e r i v e d p r o t e i n s , such as c a s e i n , due t o the presence of p o t e n t i a l c h e l a t o r s , namely f i b r e and p h y t a t e (Liebman and L a n d i s , 1989; Khan and Weaver, 1989). In the p r e s e n t s t u d y , the o b j e c t i v e s were to determine the e f f e c t s of d i e t a r y c a l c i u m l e v e l and p r o t e i n source on i l e a l c a l c i u m a b s o r p t i o n , as w e l l as subsequent u t i l i z a t i o n i n bone d e p o s i t i o n and b i o m e c h a n i c a l s t r e n g t h . These s t u d i e s were performed u s i n g o s t e o p o r o s i s prone 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 normotensive c o n t r o l W i s t a r - K y o t o (WKY) r a t s . M a t e r i a l s and Methods  Animals and D i e t s : Four week-old male 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 normotensive W i s t a r Kyoto (WKY) r a t s ( C h a r l e s R i v e r , M o n t r e a l , PQ) were each d i v i d e d i n t o s i x e x p e r i m e n t a l groups (6 an i m a l s per g r o u p ) . D i e t a r y groups i n c l u d e d 20% c a s e i n or soy p r o t e i n i s o l a t e (ICN B i o c h e m i c a l s , 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 l e v e l of d i e t a r y c a l c i u m (2 w/w%); a medium l e v e l of c a l c i u m (0.5 w/w%); and a low l e v e l of c a l c i u m (0.05 w/w%), r e s p e c t i v e l y ( T a b l e 3.1). Animals were f e d ad 1 i b i t u r n u n t i l they reached lOOg body w e i g h t , a f t e r which m e a l - f e e d i n g was i n i t i a t e d . Over a two 91 week p e r i o d , a n i m a l s were t r a i n e d to consume the d i e t s w i t h i n a s i x hour p e r i o d d a i l y (9 a.m. to 3 p.m.). D e i o n i z e d water was made a v a i l a b l e t o a n i m a l s a_d_ 1 i b i t u m . D a i l y f e e d i n t a k e s and weekly body weight g a i n s were r e c o r d e d throughout the experiment. 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 was measured as p r e v i o u s l y d e s c r i b e d i n experiment 1 when an i m a l s were 14 weeks of age. In an attempt to s t a n d a r d i z e the i n j e c t e d dose of 4 5 C a to the i n t e s t i n a l c a l c i u m c o n t e n t s , 15 u C i t o t a l dose was chosen f o r a n i m a l s f e d the h i g h c a l c i u m d i e t s ; 5.4 u C i f o r a n i m a l s f e d the medium c a l c i u m d i e t s ; and 0.375 u C i f o r a n i m a l s f e d the low c a l c i u m d i e t s , r e s p e c t i v e l y . A n a l y s e s : A n a l y s e s of i n t e s t i n a l lumen, and b l o o d plasma m i n e r a l s were as p r e v i o u s l y d e s c r i b e d i n experiment 1. Bone B i o m e c h a n i c s : A f t e r s a c r i f i c e , femur and t i b i a bone samples were e x c i s e d , c l e a n s e d of a d h e r i n g s o f t t i s s u e , and e p i p h y s e s removed. Both femora and t i b i a e were s u b j e c t e d t o 3 _ p o i n t bending a n a l y s i s as p r e v i o u s l y d e s c r i b e d i n experiment 2. Bone M i n e r a l Content A n a l y s e s : Femora and t i b i a e used i n the 3 - p o i n t bending procedure were a n a l y z e d f o r bone m i n e r a l c o n t e n t as p r e v i o u s l y d e s c r i b e d i n experiment 2. Femora were a n a l y z e d f o r Ca and P, whereas t i b i a e were a n a l y z e d f o r Ca and Mg c o n t e n t . 92 TABLE 3.1 Composition of experimental d i e t s fed to animals. Casein d i e t s Soy d i e t s Dietary component 2%Ca 0.5%Ca 0.05%Ca 2%Ca 0.5%Ca 0.05%Ca (9/ 100g) Casein* + Soy p r o t e i n i s o l a t e D.L. methionine"*" Cornstarch Sucrose Fibr e Vegetable o i l Ca f r e e mineral^mix* Vitamin mixture Choline bi t a r t r a t e " 1 " Calcium carbonate"*"*" 20.0 20.0 20.0 0.3 0.3 0.3 15.0 15.0 15.0 45.02 48.77 49.89 5.0 5.0 5.0 5.0 5.0 5.0 3.5 3.5 3.5 1.0 1.0 1.0 0.2 0.2 0.2 4.98 1.23 0.11 20.0 20.0 20.0 0.3 0.3 0.3 15.0 15.0 15.0 45.08 48.83 49.96 5.0 5.0 5.0 5.0 5.0 5.0 3.5 3.5 3.5 1.0 1.0 1.0 0.2 0.2 0.2 4.92 1.17 0.04 ICN Biochemicals, Inc., Cleveland, OH. + U n i t e d States Biochemical Co., Cleveland, OH. "^BDH Chemicals, Toronto, ON. S t a t i s t i c a l A n a l y s e s : A l l d a t a are e x p r e s s e d as mean +_ SEM. D i f f e r e n c e s between t r e a t m e n t s were t e s t e d f o r by one-way a n a l y s i s of v a r i a n c e and p a i r e d T - t e s t . Where d i f f e r e n c e s d i d e x i s t , the Student-Newman-K e u l s m u l t i p l e range t e s t was used to i d e n t i f y the s o u r c e s of the d i f f e r e n c e s at a p<0.05 l e v e l of s i g n i f i c a n c e . 94 R e s u l t s There was no s i g n i f i c a n t d i f f e r e n c e i n f i n a l body w e i g h t , food i n t a k e , or fe e d e f f i c i e n c y r a t i o (PER) i n SHR and WKY r a t s f e d the same d i e t ( T a b l e 3.2). Both SHR and WKY r a t s f e d the 0.05% Ca d i e t s e x h i b i t e d a lower f i n a l body weight (pi.0.05), food i n t a k e (p<0.05) and PER (p<0.01) compared to ani m a l s f e d the 0.5% and 2.0% Ca d i e t s . D i e t a r y p r o t e i n was o n l y observed to a f f e c t a n i m al growth a t the 2.0% c a l c i u m d i e t l e v e l ; soy f e d a n i m a l s e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) lower food i n t a k e and FER which c o r r e s p o n d e d to a lower body weight g a i n than observed i n those f e d c a s e 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 WKY a n i m a l s . Plasma m i n e r a l s are p r e s e n t e d i n Table 3.3. For both SHR and WKY r a t s , d i e t a r y p r o t e i n source was not observed to have an e f f e c t on plasma m i n e r a l p r o f i l e s i n a n i m a l s f e d the h i g h and medium c a l c i u m l e v e l s . Plasma t o t a l Ca, and i o n i z e d Ca* 2 were s i g n i f i c a n t l y (p<0.01) decrea s e d i n a n i m a l s f e d the 0.05% Ca c a s e i n d i e t o n l y , whereas plasma P was s i g n i f i c a n t l y (p<0.05) i n c r e a s e d i n these a n i m a l s , compared to c o u n t e r p a r t s f e d the 0.05% Ca soy d i e t . T h i s t r e n d was observed i n both SHR and WKY a n i m a l s . Animal s t r a i n d i f f e r e n c e s were not observed to a f f e c t e i t h e r the i n t e s t i n a l 4 0 C a c o n t e n t or 4 5 C a a b s o r p t i o n ( F i g u r e 3.1, Table 3.4). I l e a l * 0 C a c o n t e n t was a l s o not a f f e c t e d by d i e t a r y p r o t e i n s o u r c e , but was d i r e c t l y c o r r e l a t e d w i t h d i e t a r y c a l c i u m l e v e l (r=0.845, p < 0 . 0 l ) . I n t e s t i n a l s p e c i f i c a c t i v i t i e s 95 TABLE 3.2 Body weight gain and food intake of experimental animals . Diet I n i t i a l body wt. F i n a l body wt.3 Dry Matter Intake Feed E f f i c i e n c y (g) (g) (g) Ratio SHR WKY SHR WKY SHR WKY SHR WKY 2% Ca Casein 116 + 2ax 125 + 4ax 284 + 6 a x 309 + l l a x 863 + 3 8 a x 838 + 1 0 a x 0. 132 + 0.015 a b x 0.157 + 0.019 a b x Soy 110 + 4ax 118 + 2 a x 262 + 7 a b x 257 + 8 a b x 793 + 2 5 a b x 750 + 1 4 b x 0. 146 + 0.015 a b x 0.102 + 0.013 b c y 0.5% Ca Casein 116 + 4ax 119 + 5ax 307 + 9 a x 259 + 8 a b x 982 + 1 5 a x 844 + l g a x 0. 173 + 0.015 a x 0.180 + 0.008 a x Soy 108 + 3ax 108 + 6ax 282 + 5 a x 252 +14 b x 888 + l g a x 850 + 2 7 a x 0. 177 + 0.307 a x 0.184 + 0.016 a x 0.05% Ca Casein 119 + 6ax 125 + 4ax 239 +13 b x 249 + 9 b x 730 + n b x 693 + 1 3 b x 0. 104 + 0.014 b x 0.092 + 0.016 c x Soy 118 + 6ax 122 + 5ax 260 ± 9 b x 253 + 9 b x 800 + 22bx 718 + 1 5 b x 0. 120 + 0.010 a b x 0.111 + 0.012 b c x Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto r a t s . 5 weeks of age. 14 weeks of age. = 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. TABLE 3.3 E f f e c t of d i e t a r y p r o t e i n source on plasma minerals of r a t s fed d i f f e r e n t l e v e l s of calcium . Diet Ca Ca"^ Mg Na K P SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY SHR WKY (mg/dL) 2% Ca Casein Soy 11.3+0.3 10.4+1.4 10.1+1.1 10.3+0.5 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 0.5% Ca Casein 9.7+0.4 9.4+0.7 4.3+0.1 4.1+0.2 1.9+0.1 2.0+0.2 311+10 290+ 9 18+1 19+1 9.3+0.5 9.0+0.4 Soy 10.6+0.6 9.9+0.4 4.4+0.1 4.3+0.1 2.1+0.3 2.5+0.5 313+11 329+13 23+3 23+6 8.7+0.7 8.4+0.5 0.05% Ca Casein Soy 5.9+0.7* 10.3+0.5 6.6+0.6* 9.8+0.6 2.2+0.4* 4.8+0.2 3.0+0.4* 4.5+0.4 1.8+0.2 2.4+0.2 2.4+0.2 2.4+0.1 265+22 289+ 3 288+ 9 295+ 3 19+5 18+1 16+2 17+1 11.8+0.7* 8.6+0.8 12.5+1.3* 8.6+0.9 Data are expressed as mean + SEM; SHR = spontaneously hypertensive r a t s , WKY = Wistar-Kyoto r a t s . C a + 2 = (6Ca-P/3)/(P+6); where Ca and C a + 2 are 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 at p<0.01. C-SHR S-SHR C-WKY S - W K Y „ 5 s O o •* a e E a u < - o 1 0 0 C-SHR S-SHR C - W K Y S-WKY » a o a O 10 •* •o e D < C-SHR S-SHR C - W K Y Protein and Animal Type S-WKY 2.0% Ca 0.5% Ca \ M 0.05% Ca F i g . 3.1 E f f e c t of d i e t a r y p r o t e i n source 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 from the i l e a l l o op of 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) r a t s f e d c a s e i n (C) and soy p r o t e i n i s o l a t e (S) d i e t s . *oCa c o n t e n t of i l e a l loop ( a ) ; 4 5 C a s p e c i f i c a c t i v i t y of lo o p ( b ) ; * 5 C a absorbed from l o o p ( c ) . 98 TABLE 3.4 E f f e c t of d i e t a r y p r o t e i n source on calcium absorption i n r a t s fed d i f f e r e n t l e v e l s of calcium 4 0 C a i n loop 4 5 C a S p e c i f i c a c t i v i t y 4 5 C a absorbed 2 (mg/ loop) (dpm/ mg 4 0Ca) (% dose) Diet SHR WKY SHR WKY SHR WKY 2% Ca Casein 10.3 + 1.2 a x 13.2 + 1.7 a x 2.4 + 0.2 a x 2.1 + 0.3 a x 36.6 + 4.4 a x 37.9 + 1.8 a x Soy 8.8 + 1.0 a x U.1 + 1.8 a x 3.3 + 0.3 a x 2.8 + 0.3 a x 40.0 + 2.6 a x 29.1 + 4.0 b y 0.5% Ca Casein 2.3 + 0.3 a x 2.5 + 0.6 a x 3.9 + 0.6 a x 4.7 + 0.9 a x 35.8 + 3.8 a x 31.9 + 0.9 a x Soy 2.7 + 0.4 a x 2.7 + 0.4 a x 3.5 + 0.5 a x 4.0 + 0.7 a x 20.8 + 5.3 b x 20.3 + 3.4 b x 0.05% Ca Casein 0.08 + 0.01 a x 0.07 + 0.01 a x 1.4 + 0.3 a x 1.5 + 0.3 a x 87.8 + 2.3 a x 80.G + 4.6 a x Soy 0.14 + 0.02 a x 0.11 + 0.01 a x 2.1 + 0.5 a x 1.9 + 0.2 a x 71.0 + 5.0 b x 70.4 + 1.6 b x ^•Data are expressed as mean + SEM; SHR = spontaneously hypertensive r a t s , WKY = Wistar-Kyoto r a t s . 2 4 5 C a absorption (% dose) = [ l - 4 5 C a (dpm) at 1.0 hr/ dose 4 5 C a (dpm) administered] x 100. a * b = s i g n i f i c a n t d i f f e r e n c e (p<0.05) between i n d i v i d u a l treatment means i n column. x , y = s i g n i f i c a n t d i f f e r e n c e (p<0.05) between i n d i v i d u a l treatment means i n row. were s u c c e s s f u l l y s t a n d a r d i z e d to e l i m i n a t e v a r i a t i o n s due to d i e t a r y p r o t e i n s o u r c e . There was no s i g n i f i c a n t d i f f e r e n c e i n 4 5 C a a b s o r p t i o n i n SHR and WKY r a t s f e d the same d i e t . I n t e s t i n a l 4 5 C a a b s o r p t i o n was however, s i g n i f i c a n t l y (p<0.05) lower i n soy f e d a n i m a l s than c a s e i n f e d c o u n t e r p a r t s at both the medium and low d i e t a r y c a l c i u m l e v e l s , r e s p e c t i v e l y . Animals f e d the 0.05% c a l c i u m l e v e l e x h i b i t e d a g r e a t e r a b s o r p t i o n e f f i c i e n c y of the 4 5 C a dose when compared to c o u n t e r p a r t s f e d the 0.5% and 2% c a l c i u m l e v e l s . The i n t e s t i n a l 4 5 C a a b s o r p t i o n d a t a was c o n f i r m e d by the s i g n i f i c a n t l y (p<0.05) g r e a t e r d e p o s i t i o n of 4 5 C a i n t o f e m o r a l t i s s u e i n the 0.05% Ca f e d anim a l s o n l y ( F i g u r e 3.2, Table 3.5). The s i g n i f i c a n t l y (p<0.05) lower amount of 4 5 C a d e p o s i t e d to the bone i n anim a l s f e d the 2% c a l c i u m d i e t i n d i c a t e d a lower e f f i c i e n c y i n d e p o s i t i o n of the absorbed c a l c i u m t o bone by these a n i m a l s . These o b s e r v a t i o n s are i n agreement w i t h the bone s p e c i f i c a c t i v i t i e s which were a l s o s i g n i f i c a n t l y (p<0.05) lower i n the 2% c a l c i u m f e d a n i m a l s and g r e a t e r f o r a n i m a l s f e d the 0.05% c a l c i u m d i e t . Femur m i n e r a l i z a t i o n i s summarized i n Table 3.6. Femur ash weight and c a l c i u m c o n t e n t were s i g n i f i c a n t l y (p<0.05) decrea s e d i n a n i m a l s 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% and 2% c a l c i u m l e v e l s . SHR a n i m a l s had a s i g n i f i c a n t l y (p<0.05) g r e a t e r femur ash weight and c a l c i u m c o n t e n t than WKY c o u n t e r p a r t s at both the 2% and 0.5% d i e t a r y c a l c i u m l e v e l s . Soy f e d a n i m a l s e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) lower femur c a l c i u m c o n t e n t than those f e d c a s e i n at the 2% and 0.5% c a l c i u m 100 C - S H R S - S H R C - W K Y S - W K Y 14-09 < 12-10-v» -0 CO 8-O "0 6 -# 4-<0 O 2-10 o - I 1 ^ t i i i i i i i i • I ilflil IP i l l i i i f8 C - S H R S - S H R C - W K Y Prote in a n d An ima l T y p e mm® S - W K Y 2.0% C a 0.6% C a 0.06% C a F i g . 3.2 Femur d e p o s i t i o n of 4 5 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) r a t s f e d c a s e i n (C) and soy p r o t e i n i s o l a t e (S) d i e t s . Bone 4 5 C a s p e c i f i c a c t i v i t y ( a ) ; Bone 4 5 C a co n t e n t ( b ) . 101 TABLE 3.5 Femur d e p o s i t i o n of 4 5 c a l c i u m i n 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 . D i e t Bone 4 5 C a Bone S.A. (% dose/g Ash) (% dose/g 4°Ca) SHR WKY SHR WKY 2% Ca C a s e i n 0.52 + 0.06 d* 0.18 + 0.04 d* 1.29 + 0.10 d* 0.41 + 0.04** Soy 0.50 +. 0.03 d* 0.22 +_ 0.04 dr 1.32 +. 0.08 d* 0.58 ± 0.03 d' 0.5% Ca C a s e i n 1.26 + 0.06°* 0.62 +. 0.03°* 3.54 +. 0.12 c* 1.85 ± 0.07=* Soy 0.91 +_ 0.04 c* 0.86 +_ 0.06 c* 2.66 ±_ 0.12«* 2.02 +_ 0.09°* 0.05% Ca C a s e i n 11.22 ± 0.09»* 13.25 + 0.10«* 28.30 +_ 0.34»* 26.80 ± 0.20»* Soy 8.13 i 0.08 b* 6.53 +. 0.08 b* 20.92 +. 0.27b» 15.46 +. 0.16<>y 1 Data are e x p r e s s e d as mean +_ SEM; SHR = Sp o n t a n e o u s l y hyper-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 - 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. *•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 t r e a t m e n t means i n rows . 102 TABLE 3.6 Femur mineral composition of r a t s fed experimental d i e t s . Diet Ash wt. Ca Ca/P (g/bone) (mg/bone) Ratio SHR WKY SHR WKY SHR WKY 2% Ca 0.006 a x 0.009 a y 2.98 a x 4.02 a y Casein 0.247 + 0.219 + 99.60 + 86.25 + 2.22 + 0.05 a x 2.00 + 0.20 a x Soy 0.226 + 0.008 a x 0.196 + 0.007 a y 88.10 + 4.70 a b x 71.55 + 5.01 b* 2.06 + 0.19 a x 1.91 + 0.06 a x 0.5% Ca 2.71 b* Casein 0.251 + 0.011 a x 0.184 + 0.006 a : y 93.09 + 4.39 a x 64.50 + 2.17 + 0.07 a x 2.11 + 0.03 a x Soy 0.218 + 0.008 a b x 0.173 + o.on a by 79.72 + 0.83 b x 68.62 + 2.36 b x 2.15 + 0.03 a x 2.19 + 0.04 a x 0.05% Ca 0.015 d x 0.006 d x 3.18 d x 2.31 d x 0.05 b x + 0.04 b x Casein 0.067 + 0.056 + 27.75 + 27.98 + 1.36 + i.44 Soy 0.095 + 0.009 c x 0.108 + 0.005 c x 39.00 + 3.68 c x 43.11 + 3.01 c x 1.39 + 0.04 b x 1.38 + 0.05 b x Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto 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 between treatment means i n columns. x»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. l e v e l s , r e s p e c t i v e l y . Femoral c a l c i u m c o n t e n t was c o r r e l a t e d w i t h d i e t a r y c a l c i u m c o n t e n t (r=0.765, p<0.01). Thus, s i g n i f -i c a n t i n t e r a c t i o n s were found to e x i s t between c a l c i u m i n t a k e and a n i m a l s t r a i n (F(2,61)=7 . 33, pOO.001; Appendix Table 1) and p r o t e i n source (F(2,61)=9 .43 , p<0.001; Appendix Table 2 ) , r e s p e c t i v e l y , f o r femur c a l c i f i c a t i o n . The femur Ca/P r a t i o was s i g n i f i c a n t l y (p<0.05) d e c r e a s e d i n a n i m a l s f e d the 0.05% c a l c i u m d i e t when compared to those f e d the 2% and 0.5% c a l c i u m d i e t s , r e s p e c t i v e l y . There were no a n i m a l s t r a i n or p r o t e i n source e f f e c t s on femur Ca/P r a t i o . Femur bone l e n g t h and bone d r y weight were not d i f f e r e n t i n SHR and WKY r a t s f e d the same d i e t ( T a b l e 3.7). Bone dry weight was s i g n i f i c a n t l y (p<0.05) d e c r e a s e d i n a n i m a l s f e d the 0.05% c a l c i u m d i e t compared to those f e d the 0.5% and 2% c a l c i u m d i e t s . However, bone l e n g t h was s i g n i f i c a n t l y (p<0.05) de c r e a s e d i n o n l y those a n i m a l s f e d the 0.05% c a l c i u m c a s e i n d i e t . Femur b i o m e c h a n i c a l parameters were not s i g n i f i c a n t l y a f f e c t e d by a n i m a l s t r a i n d i f f e r e n c e s or by d i e t a r y p r o t e i n source ( T a b l e 3.8). Femur bending f a i l u r e energy was d e c r e a s e d i n the 0.05% c a l c i u m f e d a n i m a l s o n l y , i n d i c a t i n g l e s s work energy t o break the bones of these a n i m a l s . Moreover, a s i g n i f i c a n t c o r r e l a t i o n was found between femur c a l c i u m content and bending f a i l u r e energy (F(1,54)=41.28, p^. 0.01). F u r t h e r , f e m o r a l maximum bending s t r e s s was s i g n i f i c a n t l y (p<0.05) d e c r e a s e d i n these a n i m a l s when compared t o 0.5% and 2% c a l c i u m f e d a n i m a l s . Thus, the maximum f o r c e r e q u i r e d to break the bones 104 TABLE 3.7 Femur physical parameters of r a t s fed experimental d i e t s . Diet Bone Length Bone Dry Wt. (mn) (g) SHR WKY SHR WKY 2% Ca Casein 30.39 + 0.44 a x 30.10 + 0.25 a x 0.400 + 0.011 a x 0.360 + 0.014 a x Soy 29.98 + 0.49 a x 29.54 + 0.54 a x 0.365 + 0.013 a x 0.331 + 0.012 a b x 0.5% Ca , Casein 32.00 + 0.58 a x 30.78 + 0.66 a x 0.383 + 0.016 a x 0.296 + 0.010 b y Soy 31.84 + 0.04 a x 29.56 + 0.59 a x 0.342 + 0.011 a x 0.270 + 0.014 b c y 0.05% Ca . . , Casein 26.96 + 1.36 b x 24.24 + 1.82 b y 0.167 + 0.026 c x 0.144 + 0.008 d x Soy 29.20 + 0.25 a x 29.61 + 0.35 a x 0.211 + 0.010 b x 0.235 + 0.007 c x ^ Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto r a t s . a,b,c,d _ s - i g n i f i c a n t (p<0.05) dif f e r e n c e between treatment means i n columns. x»y = s i g n i f i c a n t (p<0.05) di f f e r e n c e between treatment means i n rows. TABLE 3.8 Femur biomechanical parameters of r a t s fed experimental d i e t s Diet Bending F a i l u r e Energy Maximum Bending Stress (x 1 0 - 2 J) (N/mm2) SHR WKY SHR WKY 2% Ca Casein 7.55 + 0.46 a x 7.87 + 0.74 a x 74.18 + 6.21 a x 69.44 + 1.83 a x Soy 7.98 + 0.79 a x 6.96 + 0.31 a x 63.15 + 1.80 a x 70.04 + 1.63 a x 0.5% Ca Casein 8.80 + 1.01 a x 6.71 + 0.61 a x 60.26 + 2.28 a x 67.43 + 5.14 a x Soy 7.31 + 0.63 a x 6.05 + 0.61 a x 56.91 + 1.97 a x 69.74 + 6.06 a x 0.05% Ca Casein 3.31 + 0.15 b x 5.70 + 0.51 a x 12.10 + 2.17 b x 14.44 + 0.62 c x Soy 5.45 + 0 . 7 8 a b x 5.40 + 0.56 a x 24.81 + 2.32 b x 37.66 + 5.76 b* Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto r a t s . a,D,c _ S i g n i f i c a n t (p<0.05) dif f e r e n c e between treatment means i n columns. x » 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. of t h e s e a n i m a l s was d e c r e a s e d , a parameter which c o r r e l a t e d w e l l w i t h the de c r e a s e d bone m i n e r a l i z a t i o n a l s o seen i n these a n i m a l s (F(1,53)=106.81, p<0.01). An i n t e r a c t i o n between c a l c i u m i n t a k e and p r o t e i n source (F(2,49)=8.74, p<0.001), was found to e x i s t f o r f e m o r a l maximum bending s t r e s s . In c o n t r a s t , no s i g n i f i c a n t i n t e r a c t i o n s were found w i t h r e s p e c t to femur bending f a i l u r e energy. The t i b i a m i n e r a l c o m p o s i t i o n ( T a b l e 3.9), p h y s i c a l parameters and b i o m e c h a n i c a l s t r e n g t h parameters ( T a b l e 3.10) data f o l l o w the same t r e n d s as those observed f o r the femora. In a d d i t i o n , d i e t a r y p r o t e i n source d i d not a f f e c t t i b i a Mg co n t e n t ( T a b l e 3.9). An animal s t r a i n d i f f e r e n c e was observed o n l y i n the groups f e d the 2% c a l c i u m d i e t s . SHR anim a l s were found to have a s i g n i f i c a n t l y (p<0.05) de c r e a s e d t i b i a Mg co n t e n t when compared to WKY c o u n t e r p a r t s f e d the same d i e t . T i b i a Mg co n t e n t was s i g n i f i c a n t l y d e c r e a s e d i n both SHR and WKY an i m a l s f e d the 0.05% c a l c i u m d i e t s when compared to c o u n t e r p a r t s at the 0.5% d i e t a r y c a l c i u m l e v e l . Thus, t i b i a magnesium co n t e n t was found to be a f f e c t e d by a s i g n i f i c a n t i n t e r a c t i o n between c a l c i u m i n t a k e and an i m a l s t r a i n (F(2,54)=13.17, p<0.001; Appendix Table 3 ) . 107 TABLE 3.9 T i b i a mineral composition of r a t s fed experimental d i e t s . Diet Ash wt. Ca Mg (g/bone) (mg/bone) (mg/bone) SHR WKY SHR WKY SHR WKY 2% Ca Casein 0.167 + 0.003 a x 0.155 + 0.005 a x 62.40 + 1.23 a x 57.90 + 2.48 a x 1. 58 + 0.17 b x 2.33 + 0.08 a* Soy 0.166 + 0.006 a x 0.140 + 0.006 a y 61.86 + 1.93 a x 51.90 + 2.58 a x 1. 52 + 0.15 b x 2.42 + 0.14^ 0.5% Ca Casein 0.172 + 0.007 a x 0.133 + 0.004 a b y 63.64 + 1.94 a x 51.11 + 2.50 a* 2. 10 + 0.13 a x 2.16 + 0.10 a x Soy 0.152 + 0.002 a x 0.129 + 0.009 b x 57.82 + 0.86 a x 53.14 + 4.51 a x 2. 17 + 0.12 a x 2.24 + 0.20 a x 0.05% Ca Casein 0.044 + 0.008 c x 0.057 + 0.003 d x 19.38 + 3.17 c x 22.65 + 0.95 b x 1. 58 + 0.19 b x 1.62 + 0.11 b x Soy 0.068 + 0.006 b x 0.077 + 0.007 c x 27.54 + 2.38 b x 30.28 + 2.71 b x 1. 53 + 0.15 b x 1.42 + 0.07 b x Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto r a t s . 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 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. TABLE 3.10 T i b i a biomechanical parameters of rats fed experimental d i e t s 1 . D iet Dry wt. Bending F a i l u r e Energy Maximum Bending Stress (g/bone) (x 10" 2 J) (N/itim2) SHR WKY SHR WKY SHR WKY 2% Ca Casein 0.260 + 0.004 a x 0.242 + 0.008 a x 6.68 + 0.15 a x 4.49 + 0.42 a y 67.95 + 2.50 a x 75.50 + 4.18 a x Soy 0.261 + 0.008 a x 0.222 + 0.008 a b y 6.11 + 0.66 a x 3.42 + 0.50 a y 60.24 + 1.06 a x 77.38 + 5.53 a* 0.5% Ca Casein 0.261 + 0.009 a x 0.206 + 0.008 b y 6.04 + 0.60 a x 4.13 + 0.38 a x 56.19 + 1.96 a x 63.15 + 3.73 a x Soy 0.236 + 0.005 a x 0.198 + 0.014 b y 6.01 + 0.72 a x 4.42 + 0.24 a x 59.11 + 4.78 a x 79.17 + 4.40 a y 0.05% Ca Casein 0.109 + 0.014 c x 0.129 + 0.005 c x 2.89 + 0.44 b x 4.19 + 0.31 a x 10.66 + 2.32 b x 16.56 + 1.15 C X Soy 0.145 + 0.007 b x 0.148 + 0.009 c x 4.33 + 0.62 b x 4.97 + 0.27 a x 20.18 + 2.86 b x 34.38 + 5.69 b y Data are expressed as mean + SEM; SHR = Spontaneously hypertensive r a t s , WKY = Wistar Kyoto r a t s . a,b,c _ s - j 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. x , 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. D i s c u s s i o n P r e v i o u s s t u d i e s have i n d i c a t e d t h a t the SHR a n i m a l model may have a d i s t u r b e d 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 c a l c i u m m e t abolism. C o n f l i c t i n g r e p o r t s on the 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 i n the SHR have been confounded by the v a r i o u s a b s o r p t i o n t e c h n i q u e s and a n i m a l ages, r e s p e c t i v e l y used i n these s t u d i e s (Lau et a1. . 1984; G a f t e r et a l . . 1986; Toraason and W r i g h t , 1981). In the p r e s e n t s t u d y , SHR and WKY a n i m a l s were not observed to d i f f e r i n c a l c i u m a b s o r p t i o n from the l i g a t e d i l e a l l o o p , s u g g e s t i n g t h a t the d i f f e r e n c e i n c a l c i u m homeostasis between g e n e t i c a l l y h y p e r t e n s i v e r a t s and normotensive c o n t r o l s i s not a r e s u l t of d i f f e r e n c e s i n 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 . The l a c k of d i f f e r e n c e i n plasma i o n i z e d c a l c i u m between SHR and WKY a n i m a l s i n the p r e s e n t s t u d y , can be e x p l a i n e d by d i f f e r e n c e s i n f e e d i n g p r o t o c o l s used. In the p o s t - a b s o r p t i v e s t a t e , serum t o t a l c a l c i u m and i o n i z e d c a l c i u m have been r e p o r t e d to be s i g n i f i c a n t l y h i g h e r i n SHR than WKY a n i m a l s (Lau et a 1. . 1984; McCarron et a1.. 1981). In f a s t e d , or m eal-fed r a t s , such as those used h e r e i n , serum t o t a l c a l c i u m and i o n i z e d c a l c i u m were not d i f f e r e n t however between SHR and WKY a n i m a l s (Lau et a l . . 1984). The d e c r e a s e d growth parameters of a n i m a l s f e d the h i g h c a l c i u m d i e t s may be e x p l a i n e d by the i n t e r f e r e n c e of excess c a l c i u m i n the r e a b s o r p t i o n of b i l e s a l t s and hence, f a t d i g e s t i o n and a b s o r p t i o n . O r a l supplements of c a l c i u m carbonate have been r e p o r t e d t o i n c r e a s e f e c a l b i l e a c i d e x c r e t i o n by 110 i n h i b i t i n g the a b s o r p t i o n of b i l e s a l t s i n the c o l o n , due to the p r e c i p i t a t i o n of b i l e s a l t s by c a l c i u m i o n s (Saunders and S i l l e r y , 1989). Moreover, b i l e a c i d r e a b s o r p t i o n may have been f u r t h e r d i s t u r b e d i n a n i m a l s f e d soy p r o t e i n w i t h a h i g h l e v e l of c a l c i u m . The f i n d i n g s of S a u t i e r and coworkers (1979) suggested t h a t the presence of soy p r o t e i n i n i n t e s t i n a l lumen c o n t e n t s c o u l d i n t e r f e r e w i t h the r e a b s o r p t i o n of b i l e a c i d s , r e s u l t i n g i n e l e v a t e d l e v e l s of b i l e a c i d e x c r e t e d i n the f e c e s , when compared to c a s e i n f e d a n i m a l s . S u p p o r t i n g e v i d e n c e i s p r o v i d e d by 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 c o n t e n t s of r a t s f e d soy p r o t e i n d i e t s , as opposed to c a s e i n c o n t a i n i n g d i e t s (Roy and Schneeman, 1981). I n t e s t i n a l c o n t e n t s c o n t a i n e d u n d i g e s t e d p e p t i d e s a c t i v e i n b i n d i n g to b i l e a c i d s , and r a t h e r than b e i n g r e a b s o r b e d , b i l e a c i d s were e x c r e t e d i n f e c a l m a tter ( S k l a n et a1.. 1979). B i l e s a l t s are e s s e n t i a l f o r the e m u l s i f i c a t i o n of f a t i n the i n t e s t i n e to f a c i l i t a t e f a t h y d r o l y s i s and d i g e s t i o n (Guyton, 1977). T h e r e f o r e , an i n h i b i t i o n of f a t d i g e s t i o n would cause f a t m a l a b s o r p t i o n , and as a r e s u l t , a r e d u c t i o n i n f e e d e f f i c i e n c y and body weight g a i n e d would be e x p e c t e d . In t h i s s t u d y , a n i m a l s f e d the low c a l c i u m d i e t s a l s o e x h i b i t e d d e c r e a s e d f e e d e f f i c i e n c y and growth c h a r a c t e r i s t i c s which were l i k e l y caused by c a l c i u m d e f i c i e n c y , r a t h e r than n u t r i e n t m a l a b s o r p t i o n . The low c a l c i u m c o n t e n t of the i n t e s t i n a l lumen and h i g h e f f i c i e n c y of * 5 C a a b s o r p t i o n are i n d i c a t i v e of the i n c r e a s e d 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 o b s e r v e d i n c a l c i u m d e f i c i e n c y and n e g a t i v e c a l c i u m balance 111 (Pansu et a l . . 1981). In c o n t r a s t , a n i m a l s f e d the h i g h c a l c i u m d i e t s e x h i b i t e d an i n c r e a s e d i n t e s t i n a l c a l c i u m c o n t e n t , but a s i m i l a r 4 5 C a uptake as those f e d medium c a l c i u m d i e t s . A c a l c i u m r e p l e t e s t a t u s i s c h a r a c t e r i z e d by d o w n - r e g u l a t i o n of a c t i v e c a l c i u m t r a n s p o r t i n the p r o x i m a l s m a l l i n t e s t i n e , and r e l i a n c e on the c o n c e n t r a t i o n dependent p a r a c e l l u l a r movement of c a l c i u m from the d i s t a l i n t e s t i n e ( B r o n n e r , 1987). Thus, s i m i l a r amounts of r a d i o l a b e l l e d c a l c i u m would be t r a n s p o r t e d p a r a c e l l u l a r l y by a n i m a l s f e d 2.0% and 0.5% c a l c i u m d i e t s . T h i s c o n c l u s i o n i s s u p p o r t e d by the f a c t t h a t a g r e a t e r a b s o l u t e amount of c a l c i u m i s absorbed from a h i g h c a l c i u m i n t a k e , even though the r e l a t i v e p e r c e n t absorbed i s l e s s than t h a t from a d i e t adequate i n c a l c i u m ( A l l e n , 1982). R a d i o l a b e l l e d c a l c i u m uptake by the bone was i n v e r s e l y r e l a t e d to d i e t a r y c a l c i u m l e v e l . In the case of a n i m a l s f e d the 0.05% c a l c i u m d i e t s , femur s p e c i f i c a c t i v i t y was h i g h , r e f l e c t i n g the enhanced e f f i c i e n c y of 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 low femur Ca c o n t e n t i n these a n i m a l s . C o n v e r s e l y , a n i m a l s f e d the 2.0% c a l c i u m d i e t s had h i g h e r bone Ca l e v e l s and took up the l e a s t 4 5 C a i n 1.0 hour, r e s u l t i n g i n reduced bone s p e c i f i c a c t i v i t i e s . These r e s u l t s r e f l e c t a p o s i t i v e c a l c i u m b a l a n c e and adequate bone c a l c i f i c a t i o n i n a n i m a l s f e d adequate or above adequate l e v e l s of c a l c i u m . P o t e n t i a l d i f f e r e n c e s i n the h a n d l i n g , or d i s t r i b u t i o n of e x t r a c e l l u l a r c a l c i u m by SHR a n i m a l s r e p o r t e d by o t h e r s (McCarron et a i . , 1981; A o k i et a l . , 1976 ; S c h e d l et a l . , 1988), were a l s o 112 observed i n the p r e s e n t s t u d y . SHR anim a l s t r a n s l o c a t e d i n c r e a s e d amounts of absorbed 45Ca to the bone, than WKY c o u n t e r p a r t s . T h i s f i n d i n g , s u p p o r t s the o b s e r v a t i o n s of o t h e r workers who have suggested t h a t SHR an i m a l s have an a l t e r e d d i s t r i b u t i o n of c a l c i u m between e x t r a c e l l u l a r and i n t r a c e l l u l a r compartments ( A o k i e t a1. . 1976; S c h e d l e t a 1. . 1988). A l t e r n a t i v e l y , the s h o r t time f o r 4 5 C a t r a n s l o c a t i o n t o the femora of SHR was l i k e l y confounded by the f a c t t h a t exchange between 4 5 C a and 4 0 C a o c c u r r e d on the s u r f a c e of the bone, r a t h e r than d e p o s i t i o n i n t o o s t e o i d t i s s u e . P h y t a t e s have been r e p o r t e d t o de c r e a s e c a l c i u m b i o a v a i l -a b i l i t y by b i n d i n g c a l c i u m w i t h i o n i z e d phosphate m o i e t i e s , making the c a l c i u m u n a v a i l a b l e f o r a b s o r p t i o n . In the manufacture of soy p r o t e i n i s o l a t e by a c i d p r e c i p i t a t i o n , complexes between the p r o t e i n and p h y t a t e are formed which may i n f l u e n c e the a v a i l a b i l i t y of m i n e r a l s from the d i e t (Hartman, 1979; Erdman, 1979). However, the r o l e of p h y t a t e s i n r e d u c 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 i s d i m i n i s h e d by r e p o r t s t h a t p h y t a t e s can be d i g e s t e d t o some e x t e n t i n the lower i n t e s t i n e , t h e r e b y r e l e a s i n g c a l c i u m f o r a b s o r p t i o n from the c o l o n ( R e i n h o l d et a1. . 1973). D e s p i t e the c o n t r i b u t i o n of the c o l o n t o 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 , a n i m a l s f e d soy p r o t e i n i s o l a t e , w i t h 2.0% and 0.5% c a l c i u m , r e s p e c t i v e l y , e x h i b i t e d d e c r e a s e d bone c a l c i f i c a t i o n . These f i n d i n g s support the r e p o r t e d development of o s t e o m a l a c i a observed i n humans who h a b i t u a l l y consumed a d i e t of u n l e a v e n e d , wholewheat bread ( B e r l y n e et a l . . 1973). 113 C a l c i u m d e f i c i e n c y t r i g g e r s a v a r i e t y of h o m e o s t a t i c mechanisms to m a i n t a i n plasma c a l c i u m l e v e l s , namely the p a r a t h y r o i d hormone (PTH) and v i t a m i n D e n d o c r i n e systems. P a r a t h y r o i d hormone s e c r e t i o n i s s t i m u l a t e d by h y p o c a l c e m i a ( P a t t and L u c k h a r d t , 1942) to r e g u l a t e the c alcium-phosphorus b a l a n c e between the b l o o d and o t h e r t i s s u e s . T h i s r e g u l a t i o n of e x t r a -c e l l u l a r c a l c i u m b a l a n c e o c c u r s i n the k i d n e y s by i n c r e a s i n g t u b u l a r r e a b s o r p t i o n of c a l c i u m ; at the bone by i n c r e a s i n g o s t e o c l a s t i c bone r e s o r p t i o n ; and s e c o n d a r i l y at the i n t e s t i n e by i n c r e a s i n g r e n a l p r o d u c t i o n of c a l c i t r i o l , which i n t u r n i n c r e a s e s a c t i v e t r a n s p o r t of c a l c i u m from the i n t e s t i n a l lumen (Aurbach, 1988). E l e v a t e d l e v e l s of plasma phosphorus, as e x h i b i t e d by the c a s e i n f e d a n i m a l s on a low c a l c i u m d i e t , have been r e p o r t e d to be a r e s u l t of PTH a c t i v i t y , s i n c e bone c a l c i u m i s a s s o c i a t e d w i t h phosphate (Agus et a1.. 1981). The excess phosphate i s e v e n t u a l l y compensated f o r by PTH mediated s t i m u l a t i o n of r e n a l phosphate e x c r e t i o n (Agus st_ a 1 . . 1981). However, i n soy f e d a n i m a l s g i v e n the low c a l c i u m d i e t , the d i s t u r b e d plasma m i n e r a l p r o f i l e noted i n c a s e i n f e d a n i m a l s was not o b s e r v e d . S i n c e e n d o c r i n e hormonal mechanisms g e n e r a l l y m a i n t a i n r e l a t i v e l y c o n s t a n t plasma c a l c i u m l e v e l s ( G r e g e r , 1988), the c a l c e m i a d i f f e r e n c e between low c a l c i u m soy and c a s e i n f e d a n i m a l s was unexpected. F u r t h e r s t u d i e s are r e q u i r e d to c o n f i r m and e l u c i d a t e the p o s s i b l e mechanism f o r t h i s o b s e r -v a t i o n . 114 The PTH mediated bone r e s o r p t i o n o c c u r r i n g i n c a l c i u m d e f i c i e n c y w i l l r e s u l t i n d e c r e a s e d bone mass, and i n the l o n g t e r m , o s t e o p o r o s i s ( S a n d l e r et a l . . 1985). Impaired bone m i n e r a l i z a t i o n i n a n i m a l s f e d the low c a l c i u m d i e t s was due to d e f i c i e n t c a l c i f i c a t i o n , as e v i d e n c e d by decre a s e d c a l c i u m c o n t e n t and Ca/P r a t i o s , r e s p e c t i v e l y . These r e s u l t s support p r e v i o u s hypotheses t h a t a d a p t a t i o n to a low c a l c i u m d i e t can o c c u r , but not to an e x t e n t which would p r e v e n t a n e g a t i v e c a l c i u m b a l a n c e and d i s t u r b e d bone metabolism (Spencer et a1.. 1969). Moreover, the importance of an adequate c a l c i u m i n t a k e e a r l y i n l i f e , t o maximize bone d e n s i t y and m i n e r a l i z a t i o n (Shah and B e l o n j e , 1988), i s emphasized by t h i s s t u d y . The f a c t t h a t bone c a l c i f i c a t i o n was not i n c r e a s e d i n ani m a l s f e d the h i g h c a l c i u m d i e t s i s ev i d e n c e t h a t c a l c i u m h o m e o s t a t i c mechanisms e x i s t t o c o n t r o l c a l c i u m metabolism at the l e v e l s of i n t e s t i n a l a b s o r p t i o n and r e n a l e x c r e t i o n . Thus, excess absorbed c a l c i u m i s e x c r e t e d i n the u r i n e , r a t h e r than over c a l c i f y i n g bone, or even the s o f t t i s s u e s (Aurbach, 1988). M i n e r a l i n t e r a c t i o n s , or a n t a g o n i s t i c r e l a t i o n s h i p s can become apparent i f a d i e t has an excess or d e f i c i e n c y of one m i n e r a l , such as i n the h i g h or low c a l c i u m d i e t s used i n the p r e s e n t s t u d y . These d i e t s were not b a l a n c e d f o r magnesium, which has been r e p o r t e d t o have a n t a g o n i s t i c i n t e r a c t i o n s w i t h c a l c i u m i n r e l a t i o n t o t h e i r r e s p e c t i v e b i o a v a i l a b i l i t i e s and metabolism (Greger e_t a_l.. , 1987 ; Greger, 1989). The decreased bone magnesium c o n t e n t i n a n i m a l s f e d h i g h c a l c i u m d i e t s may be 115 e x p l a i n e d by an i n c r e a s e i n magnesium requ i r e m e n t when f e d h i g h l e v e l s of c a l c i u m ( M o r r i s and O ' D e l l , 1963; Greger et_ al_. , 1981). Moreover, s t u d i e s have r e p o r t e d t h a t excess d i e t a r y c a l c i u m can i n h i b i t the i n t e s t i n a l a b s o r p t i o n of magnesium and o t h e r m i n e r a l s (Greger et_ <al_. , 1981). On the o t h e r hand, the d e c r e a s e d magnesium c o n t e n t of bone i n a n i m a l s f e d d i e t s d e f i c i e n t i n c a l c i u m suggests an o v e r a l l d ecrease i n m i n e r a l i z a t i o n secondary to c a l c i u m d e f i c i e n c y . The enhanced femur ash weight and c a l c i u m c o n t e n t of SHR a n i m a l s f e d the 2.0% and 0.5% c a l c i u m d i e t s , observed h e r e i n , are c o n t r a r y to the s u g g e s t i o n s of Izawa and coworkers (1985) from h i s t o m o r p h o m e t r i c d a t a , t h a t the SHR i s prone to the development of o s t e o p o r o t i c bone f r a g i l i t y . The d i s c r e p a n c y i n t h e s e o b s e r v a t i o n s may be due to the much g r e a t e r age of the a n i m a l s i n the former s t u d y , 26 weeks, as compared to 14 weeks, i n t h i s s t u d y . Thus, s i g n s of a g e - r e l a t e d o s t e o p o r o s i s may not have been m a n i f e s t e d i n the time p e r i o d of the p r e s e n t s t u d y . F i n a l l y , a r e d u c t i o n i n bone p h y s i c a l parameters and b i o m e c h a n i c a l s t r e n g t h i n a n i m a l s f e d the low c a l c i u m d i e t s i s a r e f l e c t i o n of d e c r e a s e d bone mass and bone c a l c i f i c a t i o n i n c a l c i u m d e f i c i e n c y (Niewoehner, 1988). S i n c e p h y s i c a l a c t i v i t y and weight b e a r i n g s t r e s s are a l s o components of bone f o r m a t i o n w i t h r e s p e c t to bone d e n s i t y and r e m o d e l l i n g (Simonen, 1986), the d e c r e a s e d body weight of these a n i m a l s l i k e l y c o n t r i b u t e d to the s m a l l e r bone s i z e of these a n i m a l s . 116 Experiment 4 E f f e c t of c a s e i n phosphopeptide 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 biomechanics i n c a s e i n and soy f e d r a t s . I n t r o d u c t i o n The p r i n c i p a l m i l k p r o t e i n f r a c t i o n , the c a s e i n s , y i e l d s p hosphopeptides upon d i g e s t i o n of c a s e i n w i t h t r y p s i n . These c a s e i n phosphopeptides (CPP) have been r e p o r t e d to p r e v e n t the p r e c i p i t a t i o n of i n s o l u b l e c a l c i u m phosphate s a l t s by f o r m i n g s o l u b l e complexes w i t h i o n i z e d c a l c i u m i n v i t r o (Reeves and L a t o u r , 1958 ; B e r r o c a l et a l . . 1989) as w e l l as jjn_ v i v o i n i n t e s t i n a l l u m i n a l c o n t e n t s ( N a i t o et. a_l_. , 1972 ; M u l t i n g e r et_ a l . . 1983; Sato e_t al_. , 1983; 1986). The o b j e c t i v e s of the p r e s e n t s t u d y were to f i r s t d etermine the b i n d i n g c h a r a c t e r i s t i c s 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 i n v i t r o . S e c o n d l y , the e f f e c t of c a s e i n and CPP s u p p l e m e n t a t i o n on c a l c i u m a b s o r p t i o n from the d i s t a l s m a l l i n t e s t i n e , and subsequent c a l c i u m u t i l i z a t i o n i n m i n e r a l b a l a n c e , and bone m i n e r a l i z a t i o n and b i o mechanics were determined i n o s t e o p o r o s i s prone 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) r a t s . M a t e r i a l s and Methods In V i t r o P u r i f i c a t i o n of C a s e i n Phosphopeptides (CPP): To l a b e l and exchange m i c e l l a r 4 0 C a w i t h 4 5 C a , a 3.6% m i l k p r o t e i n c o n c e n t r a t e (MPC; 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 . , D o r v a l , PQ) was e x t r i n s i c a l l y l a b e l l e d w i t h 0.545 u C i 4 5 C a / 1.1 ug 4 0 C a ( 4»CaCl2; S.A. 18.2 mCi/ mg 4 0 C a , ICN B i o m e d i c a l , I r v i n e , CA) and i n c u b a t e d f o r 30 min i n a 37°C s h a k i n g water b a t h . U l t r a c e n t r i -117 f u g a t i o n at 30,000 rpm f o r 90 min s e p a r a t e d c a s e i n from whey. C a s e i n p e l l e t s were homogenized w i t h d e i o n i z e d water to a f i n a l c o n c e n t r a t i o n of 2%. F o l l o w i n g pH adjustment to pH 8.0 w i t h IN NaOH, the c a s e i n p r e p a r a t i o n was r e a c t e d w i t h t r y p s i n (Sigma, Type X I I I ; S t . L o u i s , M0) at an enzyme t o p r o t e i n r a t i o of 1:200 f o r 23 h r , i n a 37°C s h a k i n g water b a t h . D i g e s t s were p r e c i p -i t a t e d w i t h 8% TCA o v e r n i g h t at 4°C and then c e n t r i f u g e d (9,500 rpm) f o r 20 min. The TCA s o l u b l e f r a c t i o n was e x t r a c t e d w i t h an equal volume of e t h y l e t h e r ( 4 X ) . An a l i q u o t (0.5 mL) was a p p l i e d to a Sephadex G-25 (Sigma, S t . L o u i s , MO) column (1.5 x 22 cm) and e l u t e d w i t h a 50 mM T r i s - H C l b u f f e r c o n t a i n i n g 5 mM EDTA and 0.02% Na A z i d e (pH 8.0) at a f l o w r a t e of 0.3 mL/ min. F r a c t i o n s (3.6 mL) were c o l l e c t e d and m o n i t o r e d f o r p e p t i d e absorbance (280 nm and 215 nm), o r g a n i c phosphorus, and r a d i o -a c t i v i t y . R a d i o a c t i v i t y was measured by m i x i n g a 300 uL a l i q u o t w i t h 10 mL ACS s c i n t i l l a t i o n c o c k t a i l (Amersham, O a k v i l l e , ON) and counted on a LKB-1215 L i q u i d S c i n t i l l a t i o n C ounter. F u r t h e r p u r i f i c a t i o n of CPP was performed by ion-exchange chromatography u s i n g a DEAE C e l l u l o s e (Sigma, S t . L o u i s , M0) column (2.5 x 6.3 cm). The s t a r t i n g b u f f e r was 50 mM T r i s - H C l c o n t a i n i n g 5 mM EDTA, 0.02% Na A z i d e (pH 8.0) w i t h a l i n e a r s a l t g r a d i e n t ( 0 -0.5 M NaCl) a t a f l o w r a t e of 0.17 mL/ min. F r a c t i o n s (5.0 mL) were c o l l e c t e d and a n a l y z e d as above. A s i n g l e peak c o n t a i n i n g r a d i o l a b e l 4 5 C a and phosphate was r e c o v e r e d at 0.25 M N a C l . 118 Animals and D i e t s : Four-week o l d male 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) r a t s , matched f o r age were purchased from C h a r l e s R i v e r ( M o n t r e a l , PQ). Animals were i n d i v i d u a l l y m a i n t a i n e d i n s t a i n l e s s s t e e l cages under c o n t r o l l e d temperature and l i g h t i n g c o n d i t i o n s (12:12 c y c l e ) . Animals were meal-fed 20% c a s e i n ( C ) , soy (S) (ICN B i o c h e m i c a l s , C l e v e l a n d , OH), C + 3%CPP (C-CPP) ( 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 ) , S + 3%CPP (S-CPP), r e s p e c t i v e l y ( T a b l e 4.1) f o r 10 weeks. A l l d i e t s c o n t a i n e d an adequate c a l c i u m l e v e l ( 0 . 5 % ) . D e i o n i z e d water was made a v a i l a b l e t o an i m a l s ad 1 i b i t u r n . D a i l y f e e d i n t a k e s and weekly body w e i g h t s were r o u t i n e l y r e c o r d e d . A 24 hour b a l a n c e study was performed when an i m a l s were 10 weeks of age. Randomly s e l e c t e d a n i m a l s from each d i e t a r y group were i n d i v i d u a l l y p l a c e d i n m e t a b o l i c cages 24 hr p r i o r to c o l l e c t i o n f o r a c c l i m a t i z a t i o n . M e t a b o l i c cages (Nalgene, R o c h e s t e r , NY) f o r r a t s , 150g to 300g, were equipped w i t h c o l l e c t i o n f u n n e l s and s e p a r a t i o n cones to s e p a r a t e f e c e s and u r i n e , to e l i m i n a t e u r i n e washover and c o n t a m i n a t i o n of f e c e s . S e p a r a t i o n of e x c r e t a was immediate and complete u s i n g t h i s a p p a r a t u s . U r i n e and f e c a l samples were c o l l e c t e d , weighed and f r o z e n u n t i l a n a l y s i s . Feed i n t a k e and water consumption were a l s o measured a f t e r 24 hou r s . 119 TABLE 4.1 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 f e d to a n i m a l s . D i e t s 1 D i e t a r y Component ( g / 100g) C C-CPP S S-CPP Ca s e i n * 20 .0 20 .0 Soy p r o t e i n i s o l a t e * - - 20.0 20 .0 C P P - I I I * * — 3.0 - 3.0 D.L. me t h i o n i n e * 0.3 0.3 0 . 3 0 . 3 C o r n s t a r c h * 15.0 15.0 15.0 15.0 Sucrose 46.82 44 .12 46 .82 44 . 12 F i b r e * 7.0 7.0 7.0 7.0 Ve g e t a b l e o i l 5.0 5.0 5.0 5.0 Ca f r e e m i n e r a l m i x t u r e * 3 . 5 3 . 5 3 . 5 3 . 5 V i t a m i n m i x t u r e * 1.0 1.0 1.0 1.0 C h o l i n e b i t a r t r a t e * 0.2 0.2 0.2 0.2 C a l c i u m carbonate** 1.224 0.916 1. 176 0.881 1 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. * 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 , Japan. * 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. 120 I l e a l 4 5 C a a b s o r p t i o n , femur m i n e r a l i z a t i o n and biomech-a n i c a l parameters were determined as p r e v i o u s l y d e s c r i b e d when ani m a l s were 14-weeks o l d . Animals were a n a e s t h e t i z e d u s i n g a vapour m i x t u r e of h a l o t h a n e and O 2 ' , 4% h a l o t h a n e a t a f l o w r a t e of 4 L/min f o r i n d u c t i o n , and 2.5% at a f l o w r a t e of 2 L/min to 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 p r o c e d u r e . A n a l y s e s : I n t e s t i n a l lumen, and b l o o d plasma m i n e r a l s were a n a l y z e d as p r e v i o u s l y d e s c r i b e d i n experiment 1. In the b a l a n c e s t u d y , m i n e r a l a n a l y s e s ( c a l c i u m , magnesium and phosphorus) were performed on d i e t , u r i n e and f e c a l samples (Chen et a l . . 1956; I t a y a and U i , 1966; Jones et aj^. , 1988). Bone Bio m e c h a n i c s : Femur b i o m e c h a n i c a l parameters were a s s e s s e d w i t h an I n s t r o n U n i v e r s a l T e s t i n g Machine i n 3 - p o i n t bending as p r e v i o u s l y d e s c r i b e d i n experiment 2. S t a t i s t i c a l A n a l y s e s : A l l d a ta are e x p r e s s e d as mean +_ SEM. Treatment d i f f e r e n c e s were t e s t e d f o r by one-way ANOVA. The source of a d i f f e r e n c e between means was i d e n t i f i e d by a m u l t i p l e range t e s t at the p<0.05 s i g n i f i c a n c e l e v e l . 121 R e s u l t s In V i t r o CPP P u r i f i c a t i o n : A f t e r i n c u b a t i o n of the MPC p r e p a r a t i o n w i t h the 4 5 C a and u l t r a c e n t r i f u g a t i o n , 21.52% of r a d i o a c t i v i t y was p r e s e n t i n the whey f r a c t i o n w i t h the r e m a i n i n g 78.48% b e i n g p r e s e n t i n the c a s e i n p e l l e t . F o l l o w i n g t r y p t i c d i g e s t i o n , 67.88% of c a s e i n -a s s o c i a t e d r a d i o a c t i v i t y remained i n the d i g e s t . Upon f u r t h e r p u r i f i c a t i o n s t e p s , by g e 1 - f i 1 t r a t i o n ( F i g u r e 4.1) and i o n -exchange chromatography ( F i g u r e 4.2), a s i n g l e peak c o n t a i n i n g phosphate and r a d i o a c t i v i t y was o b t a i n e d . The r e c o v e r y of r a d i o l a b e l a f t e r p u r i f i c a t i o n was 100% of t h a t p r e s e n t i n the o r i g i n a l d i g e s t i o n m i x t u r e . From m o n i t o r i n g the column f r a c t i o n s c o l l e c t e d f o r p e p t i d e absorbance and phosphorus c o n t e n t , the r e s u l t s i n d i c a t e t h a t a crude c a s e i n phosphopeptide (CPP) f r a c t i o n was o b t a i n e d . In V i v o Study: D i e t a r y p r o t e i n source had a s i g n i f i c a n t e f f e c t on both f i n a l body weight and feed e f f i c i e n c y r a t i o (FER; Table 4.2). A n i mals f e d the S d i e t e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) lower f i n a l body weight and FER than C f e d a n i m a l s . Moreover, d i e t a r y f o r t i f i c a t i o n w i t h CPP had a p o s i t i v e i n f l u e n c e on these parameters when added to the soy p r o t e i n d i e t . Feed i n t a k e was not s i g n i f i c a n t l y d i f f e r e n t between d i e t a r y groups, and t h e r e f o r e d i d not i n f l u e n c e growth parameters. Taken t o g e t h e r , these data i n d i c a t e t h a t d i e t a r y p r o t e i n source and CPP f o r t i f i c a t i o n can i n f l u e n c e a n imal growth c h a r a c t e r i s t i c s . 122 CO c © Q "5 o +* a O 6 7 8 9 10 11 12 13 14 15 16 Fraction Number 2 8 0 n m - B - 2 1 5 n m F i g . 4.1 Gel f i l t r a t i o n p u r i f i c a t i o n of c a s e i n phosphopeptide (CPP). Phosphorus and * 5 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 absorbance of f r a c t i o n s ( b ) . 123 1 I 1 I I I I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 5 10 15 20 25 Fraction Number 280 nm ~ s ~ 215 nm F i g . 4.2 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 4 5 C a r a d i o a c t i v i t y c o n tent of f r a c t i o n s ( a ) , and p e p t i d e absorbance of f r a c t i o n s ( b ) . 124 TABLE 4.2 D i e t 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 D i e t 2 I n i t i a l body F i n a l body Dry M a t t e r Feed E f f i c i e n c y wt . 3 (8) wt .* (8) I n t a k e (8) R a t i o 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 Data are e x p r e s s e d as mean +. SEM. 2 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. 3 5 weeks of age. 4 14 weeks of age. Means s h a r i n g the same l e t t e r w i t h i n the same 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 at p<0.05). 125 Plasma m i n e r a l p r o f i l e s of e x p e r i m e n t a l a n i m a l s are p r e s e n t e d i n Table 4.3. Plasma p r o t e i n was not s i g n i f i c a n t l y a f f e c t e d by d i e t a r y p r o t e i n source or CPP f o r t i f i c a t i o n (range 4.78 +_ 0.32 to 5.06 +_ 0.22 g/dL). Moreover, plasma m i n e r a l s were not s i g n i f i c a n t l y (p<0.05) a f f e c t e d by d i e t a r y t r e a t m e n t . I n t e s t i n a l 4 0 C a c o n t e n t (range 2.04 +_ 0.13 to 2.48 +.0.22 mg/ l o o p ) and 4 5 Ca s p e c i f i c a c t i v i t y (range 3.08 +_ 0.35 t o 4.64 +_ 0.32 dpm/ mg 4 0 C a ) were not s i g n i f i c a n t l y d i f f e r e n t between d i e t a r y t r e a t m e n t s . The presence of added d i e t a r y CPP i n c r e a s e d 4 5 C a a b s o r p t i o n from both the c a s e i n and soy p r o t e i n d i e t s , a l b e i t the e f f e c t was s i g n i f i c a n t (p<0.05) i n o n l y the S-CPP f e d an i m a l s ( F i g u r e 4.3). Femoral d e p o s i t i o n of 4 5 C a i s p r e s e n t e d i n Table 4.4. The acute d e p o s i t i o n of 4 5 C a to femora, as w e l l as the s p e c i f i c a c t i v i t i e s of the femora were not s i g n i f i c a n t l y a f f e c t e d by d i e t a r y p r o t e i n source or CPP f o r t i f i c a t i o n . The soy p r o t e i n f e d an i m a l s d i d however e x h i b i t both a g r e a t e r 4 5 C a d e p o s i t i o n and s p e c i f i c a c t i v i t y of the femora when compared to c a s e i n f e d a n i m a l s , i n d i c a t i n g a more e f f i c i e n t t r a n s l o c a t i o n of absorbed c a l c i u m by these a n i m a l s . M i n e r a l Balance Study: The 24 hour m i n e r a l b a l a n c e s t u d y d a t a are p r e s e n t e d i n T a b l e s 4.5 to 4.7. C a l c i u m i n t a k e and f e c a l e x c r e t i o n were not s i g n i f i c a n t l y (p<0.05) d i f f e r e n t between d i e t a r y groups (Table 4.5). However, f o r t i f i c a t i o n of the c a s e i n d i e t w i t h CPP r e s u l t e d i n a s i g n i f i c a n t l y (p<0.05) i n c r e a s e d u r i n a r y c a l c i u m 126 TABLE 4.3 Effect of diet on plasma minerals of animals fed experimental diets1. Diet* Ca Ca+* Mg Na K P (mg/dL) 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 1 Data are expressed as mean + SEM. d 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 + 2 , Ca = mg/dL; P = protein g/dL. 821 45Calcium absorbed (% dose) T l TO CO fl "B It A T) O. O w O 0> ca w 6) ro 3 H -a Cu S O cn H > C O ro i-» fl> + 3 O ro co CO I O o cr w CO ' O 1 CO H3 O rt H -O 3 O i-h 0) . to O rt g to * H -H-» fD W Cl) 3 w H» O -to co CO (->• TO 3 H -i-n CO O h3 O to .—. 3 O O O O + 3 TABLE 4.4 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 4 5 c a l c i u m d e p o s i t i o n to the f e m u r 1 . D i e t 2 4 5 C a D e p o s i t e d (% dose/ bone) Bone 4 5 C a (% dose/g Ash) 4 5 Ca S.A. dose/g 4 0 C a ) C S C-CPP S-CPP 0.148 i 0.046 0.274 +_ 0.080 0.157 + 0.041 0.264 + 0.068 0.63 +. 0.20 1.36 +_ 0.41 0.66 ± 0.17 1 .22 + 0.32 1.70 + 0.60 3.40 +_ 1.00 1.60 + 0.40 3.40 + 0.80 1 Data are e x p r e s s e d as mean +. SEM. 2 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. 129 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 hr. calcium balance 1. ? . Ca Apparent Diet Ca Intake Urine Ca Fecal Ca Ca Balance Absorption (mg/day) (mg/day) (mg/day) (mg) (%) C 68.75 + 1.25 d S 66.25 + 5.54 a C-CPP 73.12 + 5.24 a S-CPP 72.50 + 2.70 a 1.114 + 0.138 b 36.03 0.939 + 0.042 b 39.88 2.016 + 0.231 a 41.51 0.969 + 0.200 b 49.83 + 3.54 a 33.46 + + 1.46 a 28.60 + + 3.83 a 38.39 + + 4.10 a 29.26 + 2.51 a 41.11 + 1.47 3.11 a 40.00 + 2.12 2.91 a 43.15+ 0.94 a 3.08 a 32.40 + 5.791 1 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. i Ca Balance (mg) = Ca Intake (mg/day) - Urinary Ca (mg/day) - Fecal Ca (mg/day). Ca Apparent Absorption (%) = {[Ca Intake (mg/day) - Fecal Ca (mg /day)] /Ca Intake (mg/day)) x 100. Means sharing 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 d i f f e r e n t at p<0.05. e l i m i n a t i o n by t h i s group of anim a l s when compared to C, S and S-CPP f e d a n i m a l s . Apparent a b s o r p t i o n of c a l c i u m was i n c r e a s e d i n the C-CPP f e d an i m a l s o n l y , but not s i g n i f i c a n t l y so. Ca b a l a n c e was not d i f f e r e n t between d i e t a r y t r e a t m e n t groups, i n d i c a t i n g t h a t the i n c r e a s e i n c a l c i u m absorbed by the C-CPP f e d anim a l s was not u t i l i z e d but r a t h e r , was e x c r e t e d . Magnesium i n t a k e was not s i g n i f i c a n t l y (p<0.05) d i f f e r e n t between d i e t a r y groups ( T a b l e 4.6). F e c a l e x c r e t i o n of Mg was s i g n i f i c a n t l y (p<0.05) g r e a t e r i n a n i m a l s f e d the CPP f o r t i f i e d d i e t s when compared to c o u n t e r p a r t s f e d the C and S d i e t s . 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 (p<0.05) g r e a t e r i n c a s e i n f e d a n i m a l s compared to those f e d the soy p r o t e i n d i e t s . However, CPP f o r t i f i c a t i o n of the c a s e i n d i e t a c t u a l l y r e s u l t e d i n a s i g n i f i c a n t l y (p<0.05) lower u r i n a r y e x c r e t i o n of Mg than w i t h c a s e i n a l o n e . D e s p i t e these d i f f e r e n c e s i n Mg e x c r e t i o n , Mg ba l a n c e and apparent a b s o r p t i o n were not d i f f e r e n t between d i e t a r y t r e a t m e n t groups ( T a b l e 4.6). Apparent a b s o r p t i o n of Mg appeared to be d e c r e a s e d i n those a n i m a l s f e d the CPP f o r t i f i e d d i e t s . Taken t o g e t h e r , these d a t a suggest t h a t the enhanced b i o a v a i l a b i l i t y of c a l c i u m seen w i t h CPP f o r t i f i c a t i o n may have a d v e r s e l y a f f e c t e d i n t e s t i n a l magnesium a b s o r p t i o n i n these a n i m a l s . F o r t i f i c a t i o n of d i e t s w i t h CPP r e s u l t e d i n a s i g n i f i c a n t l y (p<0.05) h i g h e r i n t a k e of phosphorus by these a n i m a l s s i n c e the d i e t s were not b a l a n c e d f o r phosphorus ( T a b l e 4.7). T h i s i s r e f l e c t e d i n the f e c a l and u r i n a r y phosphorus e x c r e t i o n data 131 TABLE 4.6 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 hr. magnesium balance 1. o Mg Apparent Diet Mg Intake Urine Mg Fecal Mg Mg Balance Absorption 4 (mg/day) (mg/day) (mg/day) (mg) (%) c 6.88 + 0.12 a 1.91 + 0.03 a 1.49 + 0.10 c 3.18 + 0.31 a 71.16 + 7.08 a s 6.62 + 0.55 a 0.58 + 0.06 c 2.29 + 0.03 b 3.81 + 0.27 a 67.76 + 2.52 a c--CPP 7.31 + 0.52 a 1.45 + 0.08 b 2.92 + 0.18 a 3.65 + 0.17 a 62.53 + 3.21 a s--CPP 7.25 + 0.27 a 0.63 + 0.03 c 3.12 + 0.23 a 3.06 + 0.24 a 56.69 + 3.93 a 1 Data are expressed as mean + SEM. ^ C = casein; 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) - Urinary Mg (mg/day) - Fecal Mg (mg/day). Mg Apparent Absorption (%) = {[Mg Intake (mg/day) - Fecal Mg (mg /day)] /Mg Intake (mg/day)} x 100. Means sharing 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 d i f f e r e n t at p<0.05. TABLE 4.7 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 hr. phosphorus balance 1. - P Apparent D i e t ^ P Intake Urine P Fecal P P Balance 1* Absorption (mg/day) (mg/day) (mg/day) (mg) (%) C 79.48 + 1.44b 21.74 + 1.05c 23.61 + 2.18b 34.12 + 3.54a 68.40 + 3.00a s 84.38 + 5.48a b 17.82 + 1.36c 22.18 + 1.31b 44.38 + 4.39a 72.52 + 2.24a C-CPP 99.89 + 7.16a 32.58 + 2.13a 37.80 + 3.78a 29.51 + 3.79a 61.67 + 4.47a S-CPP 100.63 + 3.75a 27.60 + 0.92b 39.71 + 2.69a 34.82 + 3.01a 62.62 + 4.83a , Data are expressed as mean + SEM. i C = cas 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) - Urinary P (mg/day) - Fecal P (mg/day). q P Apparent Absorption (%) = {[P Intake (mg/day) - Fecal P (mg /day)] /P Intake (mg/day)} x 100. Means sharing 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 d i f f e r e n t at p<0.05. which are both s i g n i f i c a n t l y (p<0.05) g r e a t e r i n C-CPP and S-CPP fe d a n i m a l s when compared to C and S f e d c o u n t e r p a r t s . These d a t a complement those of phosphorus b a l a n c e and apparent a b s o r p t i o n which i n d i c a t e no s i g n i f i c a n t (p<0.05) d i f f e r e n c e s between d i e t a r y t r e a t m e n t s ( T a b l e 4.7). Taken t o g e t h e r , these d a t a i n d i c a t e t h a t the i n c r e a s e i n d i e t a r y phosphorus of the CPP f o r t i f i e d d i e t s d i d not r e s u l t i n a g r e a t e r a b s o r p t i o n or u t i l i z a t i o n of phosphorus by a n i m a l s f e d these d i e t s . Bone M i n e r a l i z a t i o n : Femur m i n e r a l i z a t i o n d a t a are summarized i n Table 4.8. Femur ash weight and c a l c i f i c a t i o n were s i g n i f i c a n t l y (p<0.05) a f f e c t e d by d i e t a r y p r o t e i n . s o u r c e . D i e t a r y f o r t i f i c a t i o n w i t h CPP however, d i d not s i g n i f i c a n t l y a f f e c t femur m i n e r a l i z a t i o n p a r a m e t e r s . Soy p r o t e i n f e d an i m a l s had a s i g n i f i c a n t l y (p<0.05) lower femur ash weight and bone c a l c i u m c o n t e n t than those f e d the c a s e i n d i e t s . The f e m o r a l Ca/P r a t i o was not s i g n i f i c a n t l y a f f e c t e d by d i e t a r y CPP f o r t i f i c a t i o n . Femur Mg c o n t e n t was a l s o not a f f e c t e d by d i e t a r y t r e a t m e n t , a l t h o u g h a n i m a l s f e d the c a s e i n d i e t s had a h i g h e r femur Mg c o n t e n t than soy p r o t e i n f e d a n i m a l s ; the d i f f e r e n c e however, was a g a i n , not s i g n i f i c a n t . Bone P h y s i c a l and B i o m e c h a n i c a l Parameters: N e i t h e r d i e t a r y p r o t e i n source nor CPP f o r t i f i c a t i o n had a s i g n i f i c a n t (p<0.05) e f f e c t on femur p h y s i c a l parameters (Table 4.9). Bone d r y w e i g h t s and l e n g t h s were s i m i l a r between a l l f o u r d i e t a r y groups. The b i o m e c h a n i c a l parameter, femur bending f a i l u r e energy was s i g n i f i c a n t l y (p<0.05) lower i n soy p r o t e i n 134 TABLE 4.8 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 femur m i n e r a l i z a t i o n 1 . D i e t 2 Ash wt. Ca Ca/P Mg (g) (mg/bone) r a t i o (mg/bone) c 0 .237 + 0 .006* 94 .60 + 3 .85" 2 .00 + 0 .01 a 1 . 84 +_ 0 .06 a s 0 .206 + 0 .006b 78 .75 + 3 .21<> 1 . 97 + 0 .05 a 1 . 62 +_ 0 .07 a C-CPP 0 .238 +_ 0 .002* 98 .75 + 1 .88* 2 .02 +_ 0 .02" 1 .88 + 0 .04 a S-CPP 0 .218 +_ 0 .004b 80 .35 +_ 6 .83b 1 .99 +_ 0 • 05a 1 .59 + 0 . 10a 1 Data are e x p r e s s e d as mean +_ SEM. 2 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. S u p e r s c r i p t s w i t h d i f f e r e n t a l p h a b e t s are s i g n i f i c a n t l y (p<0.05) d i f f e r e n t . 135 TABLE 4.9 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 femur phy s i c a l and biomechanical parameters 1. _ Bending F a i l u r e Maximum Diet Dry wt. Length Energy Bending Stress (g) (mm) (x 1 0 - 2 J) (N/mm2) c 0.387 + 0.010 a 30.61 + 0.47 a 9.24 + 0.93 a 68.94 + 3.06 a b s 0.360 + 0.009 a 30.49 + 0.21 a 6.12 + 0.60 b 65.82 + 1.45 a b c--CPP 0.393 + 0.0U a 30.95 + 0.50 a 8.37 + 0.52 a b 74.61 + 4.80 a s--CPP 0.360 + 0.012 3 30.02 + 0.65 a 6.78 + 0.80 a b 62.23 + 2.08 b 0 Data are expressed as mean + SEM. 1 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 sharing 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 d i f f e r e n t at p<0.05. f e d a n i m a l s compared to those f e d c a s e i n . D i e t a r y CPP f o r t i f i c a t i o n had l i t t l e e f f e c t on femur biomechanics i n soy and c a s e i n f e d a n i m a l s . 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 femur maximum bending s t r e s s . A s i m i l a r r e s u l t was observed i n CPP f o r t i f i e d d i e t s , compared to n o n - f o r t i f i e d d i e t s . 137 D i s c u s s i o n The importance of the o r g a n i c phosphate m o i e t i e s t o the c a l c i u m b i n d i n g a c t i v i t y of CPP was e s t a b l i s h e d i n s t u d i e s w i t h d e p h o s p h o r y l a t e d c a s e i n ( S a t o et. a j . . , 1983). In the f i r s t s e t of experiments conducted i n the p r e s e n t s t u d y , a crude CPP prep-a r a t i o n was o b t a i n e d , which p o s s e s s e d c a l c i u m b i n d i n g a c t i v i t y a s s o c i a t e d w i t h o r g a n i c phosphate. The r e c o v e r y of 4 5 C a a f t e r p u r i f i c a t i o n , was eq u a l t o t h a t i n the enzymatic d i g e s t , which suggested t h a t c a l c i u m i s s t r o n g l y bound to c a s e i n d i g e s t i o n p e p t i d e s . Baumy and coworkers (1989) r e c e n t l y r e p o r t e d s a t u r a t i o n k i n e t i c s d a t a which demonstrated t h a t p r e f e r e n t i a l b i n d i n g of c a l c i u m o c c u r s at s i t e s on CPP o c c u p i e d by c l u s t e r s of p h o s p h o s e r y l r e s i d u e s , due to the f a v o u r a b l e pK v a l u e , and charge d i s t r i b u t i o n of these g r o u p i n g s . These workers were a b l e t o e s t a b l i s h t h a t Ca 2* s a t u r a t i o n of p h o s p h o s e r y l c l u s t e r s o c c u r s p r i o r t o b i n d i n g t o s i n g l e p h o s p h o s e r y l r e s i d u e s . P r e v i o u s s t u d i e s have i n d i c a t e d the p o t e n t i a l p h y s i o l o g i c a l v a l u e of the b i o a c t i v e CPP i n d e m o n s t r a t i n g , not o n l y t h e i r presence i n the i n t e s t i n a l c o n t e n t s of c a s e i n f e d ani m a l s ( N a i t o ejt aj,. , 1972; N a i t o and S u z u k i , 1974; M e i s e l and F r i s t e r , 1989), but a l s o the a f f i n i t y of CPP f o r c a l c i u m i o n s , t o i n c r e a s e the p r o p o r t i o n of s o l u b l e c a l c i u m i n the d i g e s t a of c a s e i n f e d a n i m a l s when compared to c o u n t e r p a r t s f e d o t h e r d i e t a r y p r o t e i n s ( N a i t o et. a i - , 1972; N a i t o and S u z u k i , 1974; Lee et a_l. , 1980; 1983). P r o t e i n c o m p o s i t i o n and d i g e s t i b i l i t y can i n f l u e n c e the p r o f i l e of p e p t i d e s and amino a c i d s r e l e a s e d d u r i n g the d i g e s t i v e 138 p r o c e s s . A reduced p r o t e i n q u a l i t y , and thus n u t r i t i o n a l v a l u e , can be a t t r i b u t e d to d e c r e a s e d d i g e s t i b i l i t y and the r a t i o of r e l e a s e d e s s e n t i a l amino a c i d s to n o n e s s e n t i a l amino a c i d s (Mauron, 1973; Raghunath and N a r a s i n g a Rao, 1984). The decreased body weight g a i n and FER e x h i b i t e d h e r e i n by soy p r o t e i n f e d a n i m a l s , may be e x p l a i n e d by the r e l a t i v e l y lower d i g e s t i b i l i t y of the h i g h m o l e c u l a r w e i g h t , h i g h l y s t r u c t u r e d , g l o b u l i n soy p r o t e i n s (Raghunath and N a r a s i n g a Rao, 1984). A d d i t i o n of CPP to soy p r o t e i n d i e t s r e s u l t e d i n i n c r e a s e d f i n a l body weight and FER i n these a n i m a l s . T h i s response may be a t t r i b u t e d to an improved q u a l i t y of the p l a n t p r o t e i n d i e t when supplemented w i t h animal p r o t e i n d e r i v e d p e p t i d e s . The enhanced i l e a l a b s o r p t i o n of * 5 C a i n a n i m a l s f e d soy p r o t e i n d i e t s supplemented w i t h CPP extends the f i n d i n g s of Sato et a l . (1986) t h a t CPP can a c t t o enhance c a l c i u m b i o a v a i l a b i l i t y from the i n t e s t i n a l lumen. The e f f e c t i v e n e s s of s u p p l e m e n t a l d i e t a r y CPP would be determined by i t s r e s i s t a n c e to p r o t e o l y t i c enzymes i n the d i g e s t i v e chyme. T h i s r e s u l t , o b t a i n e d 1.0 hr a f t e r i n t r a l u m i n a l 4 5 C a a d m i n i s t r a t i o n , i s s u p p o r t e d by p r e v i o u s s t u d i e s by Lee and coworkers ( 1 9 8 0 ) , who were a b l e to d e t e c t the presence of macrophosphopeptides i n the d i g e s t a of c a s e i n f e d r a t s e a r l y a f t e r m e a l - f e e d i n g , but not at 13.5 or 25.5 h r . F u r t h e r m o r e , CPP have e x h i b i t e d s u s c e p t i b i l i t y t o i n t e s t i n a l a l k a l i n e phosphatase a c t i v i t y (Reeves and L a t o u r , 1958; M e l l a n d e r , 1963) which may be of s i g n i f i c a n c e i n both r a t animal model and human s t u d i e s (Van der Meer, 1988). P r e v i o u s work of 139 M e l l a n d e r (1947), demonstrated the r e s i s t a n c e of a h i g h m o l e c u l a r weight p e p t i d e r e s i d u e from an i n v i t r o c a s e i n d i g e s t i o n , to f u r t h e r a t t a c k from i n t e s t i n a l p r o t e o l y t i c enzymes. The t r a n s l o c a t i o n of 4 5 C a r a d i o l a b e l to the femora was i n v e r s e l y r e l a t e d t o the i n t e s t i n a l a b s o r p t i o n d a t a . The decr e a s e d uptake of 4 5 C a by the femora of C and C-CPP f e d a n i m a l s , r e s p e c t i v e l y was l i k e l y due to c a l c i u m h o m e o s t a t i c mechanisms c o n t r o l l i n g the e v e n t u a l u t i l i z a t i o n of the g r e a t e r amount of c a l c i u m absorbed by these a n i m a l s . A l t e r n a t i v e l y , a 1 hr p e r i o d was too s h o r t f o r 4 5 C a bone d e p o s i t i o n to o c c u r , but r a t h e r , r e f l e c t s o n l y the exchange of 4 5 C a w i t h 4 0 C a on the s u r f a c e of the bone m a t r i x . C a l c i u m r e p l e t i o n i s a s s o c i a t e d w i t h c a l c i u m b a l a n c e and adequate bone c a l c i f i c a t i o n , t h u s , homeo-s t a t i c mechanisms w i l l a l s o d i r e c t excess c a l c i u m towards e x c r e t i o n , r a t h e r than u t i l i z a t i o n ( A l l e n , 1982). I t i s noteworthy t h e r e f o r e , t h a t i n the p r e s e n t s t u d y , a n i m a l s f e d the C-CPP d i e t e x c r e t e d e l e v a t e d l e v e l s of c a l c i u m i n the u r i n e ; f e c a l c a l c i u m e x c r e t i o n , however, was not d i f f e r e n t . T h i s r e s u l t e d i n an enhanced apparent a b s o r p t i o n of c a l c i u m from t h i s d i e t , but an unchanged c a l c i u m b a l a n c e . C a l c i u m and magnesium are o f t e n c o n s i d e r e d t o g e t h e r i n s t u d i e s of m i n e r a l b i o a v a i l a b i l i t y , s i n c e one i s o f t e n a f f e c t e d by the o t h e r . Greger and coworkers have r e p o r t e d t h a t excess c a l c i u m can i m p a i r a b s o r p t i o n of magnesium or o t h e r m i n e r a l s (Greger et a l . . 1981); and c o n v e r s e l y , t h a t magnesium can i m p a i r the a b s o r p t i o n of c a l c i u m from supplements (Greger et a l . . 1987). 140 In the p r e s e n t s t u d y , a n i m a l s f e d the CPP supplemented d i e t s e x h i b i t e d i n c r e a s e d f e c a l Mg e x c r e t i o n , r e s u l t i n g i n a lower apparent a b s o r p t i o n of Mg, s u g g e s t i n g t h a t the i n c r e a s e i n c a l c i u m b i o a v a i l a b i l i t y i n these a n i m a l s had an i n h i b i t o r y e f f e c t on the i n t e s t i n a l a b s o r p t i o n of Mg. A c c o r d i n g l y , the decrea s e d u r i n a r y Mg e l i m i n a t i o n by C-CPP anim a l s r e f l e c t s c o n s e r v a t i o n of Mg, due to an i n c r e a s e d Mg requ i r e m e n t which i n t u r n accompanies i n c r e a s e d c a l c i u m a b s o r p t i o n ( M o r r i s and O ' D e l l , 1963; Greger et  a l . . 1981). The de c r e a s e d u r i n a r y e x c r e t i o n of Mg i n soy f e d a n i m a l s , compared to c a s e i n f e d c o u n t e r p a r t s was l i k e l y due to the r e l a t i v e l y g r e a t e r f e c a l e x c r e t i o n of Mg by these a n i m a l s s i n c e o v e r a l l Mg b a l a n c e was not d i f f e r e n t . S i m i l a r l y , phos-phorus b a l a n c e and apparent a b s o r p t i o n were not a f f e c t e d by d i e t a r y CPP s u p p l e m e n t a t i o n . The i n c r e a s e d phosphorus i n t a k e s of C-CPP and S-CPP f e d an i m a l s were p a r a l l e l e d by both i n c r e a s e d u r i n a r y P and f e c a l P e x c r e t i o n . Bone m i n e r a l i z a t i o n and p h y s i c a l parameters were not a f f e c t e d by the enhanced c a l c i u m b i o a v a i l a b i l i t y of the CPP supplemented d i e t s . These r e s u l t s extend those of o t h e r s who have r e p o r t e d t h a t a d i e t w i t h h i g h c a l c i u m b i o a v a i l a b i l i t y may improve c a l c i u m b a l a n c e and i n h i b i t bone r e m o d e l l i n g i n e l d e r l y s u b j e c t s , but does not a f f e c t bone metabolism ( H o r o w i t z et a 1. . 1988; S i n h a et a l . . 1988). In the p r e s e n t e x p e r i m e n t , the s i g n i f i c a n t l y lower bending f a i l u r e energy of femora from soy fed an i m a l s c o n f i r m s the t r e n d observed i n the soy f e d group i n experiment 3. Bone b i o m e c h a n i c a l parameters were s i m i l a r l y 141 u n a f f e c t e d by d i e t a r y CPP s u p p l e m e n t a t i o n . Taken t o g e t h e r , these d a t a i n d i c a t e t h a t h o m e o s t a t i c mechanisms c o n t r o l the metabolism of i n c r e a s e d absorbed c a l c i u m i n e x c r e t i o n , r a t h e r than i n c r e a s e d bone d e p o s i t i o n i n c a l c i u m r e p l e t e a n i m a l s . F u r t h e r s t u d i e s are needed to determine i f t h i s i s the case i n o s t e o p o r o t i c s u b j e c t s , and c a l c i u m d e f i c i e n t i n d i v i d u a l s . 142 Experiment 5 E f f e c t of p r o t e i n heat 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 biomechanics i n r a t s f e d c a s e i n and soy p r o t e i n s . I n t r o d u c t i o n P r o t e i n q u a l i t y may be a d e t e r m i n a n t of c a l c i u m b i o a v a i l -a b i l i t y from the d i e t . The c o m p o s i t i o n and d i g e s t i b i l i t y of p r o t e i n s w i l l i n f l u e n c e the r e l e a s e of p o t e n t i a l l y b i o a c t i v e p e p t i d e s and amino a c i d s w i t h i n the i n t e s t i n a l lumen, which i n t u r n , may a f f e c t 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 . A r e d u c t i o n i n the n u t r i t i o n a l v a l u e of heat t r e a t e d p r o t e i n s can be e x p l a i n e d by a d e c r e a s e d d i g e s t i b i l i t y and a v a i l a b i l i t y of amino a c i d s (Mauron, 1972). Moreover, p r o t e i n s , and i n p a r t i c u l a r d a i r y p r o t e i n s , are u s u a l l y heat t r e a t e d b e f o r e consumption or d u r i n g p a s t e u r i z a t i o n . In the p r e s e n t s t u d y , a severe heat t r e a t m e n t to both c a s e i n and soy p r o t e i n s o u r c e s performed a c c o r d i n g to the procedure of P e r c i v a l and Schneeman (1979) r e s u l t e d i n extreme heat d e n a t u r a t i o n . T h i s was performed t o e l i m i n a t e the t r y p t i c d i g e s t i o n of c a s e i n and t h u s , p r o d u c t i o n of c a s e i n phospho-p e p t i d e s . The o b j e c t i v e s of t h i s e xperiment were to c o n t r o l f o r p o s s i b l e CPP p r o d u c t i o n by p a n c r e a t i c enzymes and e l i m i n a t e the e s t e r i f i e d c a l c i u m phosphate a s s o c i a t e d w i t h the m i c e l l e and to s u b s e q u e n t l y determine these e f f e c t s on c a l c i u m a b s o r p t i o n and bone 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 s t r e n g t h . The spon-t a n e o u s l y h y p e r t e n s i v e (SHR) and n ormotensive c o n t r o l W i s t a r -Kyoto (WKY) r a t s were a g a i n used. 143 M a t e r i a l s and Methods In V i t r o D i g e s t i b i l i t y Study: An i n v i t r o two-step p r o t e o l y s i s method u s i n g p e p s i n , f o l l o w e d by p a n c r e a t i n enzymatic h y d r o l y s i s was used i n a m o d i f i c a t i o n of the method of Jacques et a l . ( 1986). P r o t e i n s , namely c a s e i n , soy p r o t e i n i s o l a t e , and heat denatured c a s e i n and soy p r o t e i n were suspended i n d e i o n i z e d w a t e r , r e s p e c t i v e l y . S o l u t i o n s were a d j u s t e d to pH 1.9 w i t h d i l u t e HC1 and i n c u b a t e d w i t h p e p s i n ( p o r c i n e stomach mucosa 1:10,000) at 37°C i n a s h a k i n g water b a t h . F o l l o w i n g a 30 minute p e p s i n d i g e s t i o n , samples were i n c u b a t e d once more w i t h p a n c r e a t i n ( p o r c i n e p a n c r e a s ; Grade I I , Sigma). A l i q u o t s were removed at f r e q u e n t i n t e r v a l s , d e p r o t e i n i z e d w i t h 20% TCA and r e a c t e d w i t h TNBS (Kwan et a l . . 1983). The i n i t i a l s l o p e of p r o t e i n d i g e s t i o n was o b t a i n e d by l i n e a r r e g r e s s i o n a n a l y s i s (Maga et a1.. 1973) and used t o compare the r e l a t i v e i n i t i a l p r o t e o l y s i s r a t e s between n a t i v e c a s e i n and heat dena t u r e d p r o t e i n s . Animals and D i e t s : Four-week o l d male 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 normotensive W i s t a r Kyoto (WKY) r a t s ( C h a r l e s R i v e r , M o n t r e a l , PQ) were each d i v i d e d i n t o f o u r e x p e r i m e n t a l d i e t a r y groups (6 a n i m a l s per g r o u p ) . D i e t a r y t r e a t m e n t s i n c l u d e d 20% c a s e i n ( C ) , soy p r o t e i n i s o l a t e (S) (ICN B i o c h e m i c a l s , C l e v e l a n d , OH) and heat dena t u r e d C (DC) and S (DS) d i e t s , r e s p e c t i v e l y ( T a b l e 5.1). 144 TABLE 5.1 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 f e d to a n i m a l s . C a s e i n D i e t s Soy D i e t s D i e t a r y Component 20% H.D. 20% H .D. (g/100 g) Ca s e i n * 20 .0 20.0 Soy p r o t e i n i s o l a t e * — — 20 .0 20 .0 D.L. me t h i o n i n e * 0.3 0.3 0.3 0 .3 C o r n s t a r c h * 15.0 15.0 15.0 15 .0 Sucrose 48 .77 48 .77 48.77 48 .77 F i b r e * 7.0 7.0 7.0 7 .0 V e g e t a b l e o i l 5.0 5.0 5.0 5 .0 Ca f r e e m i n e r a l m i x t u r e * 3 . 5 3.5 3 . 5 3 . 5 V i t a m i n m i x t u r e * 1.0 1.0 1.0 1 .0 C h o l i n e b i t a r t r a t e * 0.2 0.2 0.2 0 .2 C a l c i u m c a r b o n a t e * * 1 .17 1. 17 1 . 17 1 .17 H.D. = heat denatu r e d d i e t . *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. 145 Thermal d e n a t u r a t i o n of d i e t a r y p r o t e i n s was performed a c c o r d i n g to the method of P e r c i v a l and Schneeman (1979), by a u t o c l a v i n g p r o t e i n s at 121°C, 2 atm f o r 24 h r , f o l l o w e d by c o o l i n g f o r i n c o r p o r a t i o n i n t o d i e t s . A l l d i e t s c o n t a i n e d an adequate c a l c i u m l e v e l ( 0 . 5 % ) . The SHR a n i m a l s f e d the C and S p r o t e i n d i e t s were the same as i n experiment 4. Feeding 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 3. A 24 hour b a l a n c e s t u d y was performed, as p r e v i o u s l y d e s c r i b e d i n experiment 4, when a n i m a l s were 10 weeks of age. 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 was measured as p r e v i o u s l y d e s c r i b e d i n experiment 1 when a n i m a l s were 14 weeks of age. A n a l y s e s : A n a l y s e s of i n t e s t i n a l lumen, b l o o d plasma, f e e d , u r i n e , f e c e s and femur m i n e r a l i z a t i o n and biomechanics were c a r r i e d out as p r e v i o u s l y d e s c r i b e d i n experiment 4. S t a t i s t i c a l A n a l y s e s ! A l l d a ta are e x p r e s s e d as mean +_ SEM. Treatment d i f f e r e n c e s were t e s t e d f o r by one-way ANOVA. Where d i f f e r e n c e s d i d o c c u r , the source of the d i f f e r e n c e was i d e n t i f i e d u s i n g a m u l t i p l e range t e s t at a p<0.05 l e v e l of s i g n i f i c a n c e . 146 R e s u l t s In V i t r o D i g e s t i b i l i t y Study: The i n i t i a l time course 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 of c a s e i n , soy p r o t e i n i s o l a t e , and heat d e n a t u r e d c a s e i n and soy p r o t e i n i s shown i n F i g u r e 5.1. No attempt was made to remove the p r o d u c t s of d i g e s t i o n , which are known to accumulate and u l t i m a t e l y i n h i b i t the p r o c e s s of enzymatic d i g e s t i o n ( R o b b i n s , 1978). However, the use of the i n i t i a l s l o p e method overcomes t h i s d i s a d v a n t a g e . A s l o w e r r a t e of p r o t e o l y s i s was observed f o r soy p r o t e i n compared to c a s e i n . Heat d e n a t u r a t i o n of both p r o t e i n s o u r c e s r e s u l t e d i n a marked decrease i n t h e i r r e s p e c t i v e d i g e s t i b i l i t i e s . In V i v o Study: At the i n i t i a t i o n of the e x p e r i m e n t , WKY a n i m a l s had a s i g n i f i c a n t l y (p<0.05) g r e a t e r body weight than SHR c o u n t e r p a r t s fe d the same d i e t ( T a b l e 5.2). T h i s d i f f e r e n c e between animal s t r a i n s remained s i g n i f i c a n t at the c o n c l u s i o n of the experiment. D i e t a r y p r o t e i n source and heat d e n a t u r a t i o n of d i e t a r y p r o t e i n s had s i g n i f i c a n t (p<0.05) e f f e c t s on a n i m a l growth c h a r a c t e r -i s t i c s . A n i mals f e d the S d i e t had both a s i g n i f i c a n t l y (p<0.05) lower f i n a l body weight and fe e d e f f i c i e n c y r a t i o (FER), r e s p e c t i v e l y , when compared to C f e d c o u n t e r p a r t s . Moreover, heat d e n a t u r a t i o n of d i e t a r y p r o t e i n s had an adverse e f f e c t on both body weight g a i n and FER as i n f l u e n c e d by the reduced feed i n t a k e of these a n i m a l s . The DC and DS f e d ani m a l s a l s o e x p e r i e n c e d s i g n s of d i a r r h e a , i n d i c a t i n g n u t r i e n t m a l a b s o r p t i o n 147 c S D C Dietary Proteins D S F i g . 5.1 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 p r o t e i n i s o l a t e ( S ) , heat d e n a t u r e d C (DC), and heat denatured S (DS) c a l c u l a t e d from th£ i n i t i a l r e a c t i o n r a t e s (0 - 10 m i n u t e s ) . R e s p e c t i v e s l o p e s were: C = 35.5 x 10-3; S = u,5 x 10-3; D c = 5.5 x 10"3; DS = 7.5 x 1 0 " 3 ; r e g r e s s i o n range = 0.996>_ rjC0.944. TABLE 5.2 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 . D i e t 2 I n i t i a l body wt. 3 F i n a l body wt. 4 Dry M a t t e r Intake Feed E f f i c i e n c y (g) (g) (g) Ra t i o SHR WKY SHR WKY SHR WKY SHR WKY c 77 + 2ax 97 + 5 a y 259 + 8 a x 298 + gay 867 + 23 a x 865 + 26 a x 0.211 + 0.004ax 0.234 + s 79 + 2&x. 97 + 2 a y 225 + 5 b x 267 + 2*>y 861 + 1 4 a x 821 + 13 a b x 0.170 + 0.004bx 0.208 + DC 77 + 3 a x 98 + 4 a y 179 + 3C X 213 + 7 c y 733 + 1 7 b x 787 + 20 b c x 0.140 + 0.004cx 0.146 + DS 78 + 3 a x 96 + 3 a y 120 + 4 d x 155 + 3 d y 652 + 36 c x 720 + 21 c y 0.065 + 0.005dx 0.082 + 1 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 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 denatured S. 5 weeks of age. 14 weeks o f age. 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. x»' 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 tr e a t m e n t means i n rows. d u r i n g the e x p e r i m e n t a l p e r i o d . Animals f e d the DC and DS d i e t s e x h i b i t e d s i g n i f i c a n t l y (p<0.05) d e c r e a s e d f e e d i n t a k e s , f i n a l body w e i g h t s and FER, r e s p e c t i v e l y than c o u n t e r p a r t s f e d C and S d i e t s . These r e s u l t s were s i m i l a r f o r both SHR and WKY a n i m a l s . Plasma p r o t e i n l e v e l s were not a f f e c t e d by d i e t a r y p r o t e i n s ource or heat d e n a t u r a t i o n (range 4.70 +_ 0.40 to 6.03 +_ 0.30 g/dL). Plasma m i n e r a l s were not d i f f e r e n t between d i e t a r y groups. F u r t h e r m o r e , n e i t h e r the t o t a l plasma c a l c i u m (range 9.0 +_ 0.3 to 9.9 +_ 0.3 mg/dL), nor the c a l c u l a t e d i o n i z e d plasma c a l c i u m (range 4.6 +. 0.1 to 5.2 +_ 0.4 mg/dL) were found to be a f f e c t e d by d i e t a r y t r e a t m e n t . D i f f e r e n c e s i n an i m a l s t r a i n and d i e t a r y t r e a t m e n t s d i d not i n f l u e n c e the 4 0 C a c o n t e n t of the i l e a l l o op ( T a b l e 5.3). I n t e s t i n a l 4 5 C a s p e c i f i c a c t i v i t i e s were a l s o not a f f e c t e d by animal s t r a i n d i f f e r e n c e s or d i e t a r y p r o t e i n s o u r c e . However, an i m a l s f e d the denatur e d p r o t e i n d i e t s e x h i b i t e d h i g h e r i n t e s t i n a l l oop 4 5 C a s p e c i f i c a c t i v i t i e s when compared to those fe d n a t i v e c a s e i n and soy d i e t s ( T a b l e 5.3). A b s o r p t i o n of 4 5 C a from the l i g a t e d i l e a l l o o p was a f f e c t e d by d i e t a r y p r o t e i n s ource as w e l l as heat d e n a t u r a t i o n of the p r o t e i n s i n SHR and WKY a n i m a l s f e d the same d i e t ( T a b l e 5.3; F i g u r e 5.2). Soy p r o t e i n f e d anim a l s e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) lower i l e a l 4 5 C a a b s o r p t i o n than those f e d c a s e i n ; t h i s o b s e r v a t i o n was common to both SHR and WKY a n i m a l s . Both the heat denatured c a s e i n and soy p r o t e i n d i e t s r e s u l t e d i n de c r e a s e d a b s o r p t i o n of 150 TABLE 5.3 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 . D i e t 2 40Ca i n loop 4 5 C a S p e c i f i c n A c t i v i t y 4 5 C a A b s o r b e d 3 (mg/loop) (dpm/mg ^ uCa) (% dose) SHR WKY SHR WKY SHR WKY C 2.25 + 0 . 2 2 a x 2.36 + 0 . 2 0 a x 4.30 + 0 . 7 0 a x 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 a x 4.60 + 0 . 3 0 a x 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 a x 5.08 + 0 . 6 5 a x 5.03 + 0 . 6 5 a x 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 a x 5.39 + 0 . 4 9 a x 5.56 + 0 . 4 4 a x 22.6 + 4 . 8 b x 18.4 + 3 . 4 b x 1 Data are ex 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 . 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 denatured C; DS = heat denatured S. 6 ^ s C a a b s o r p t i o n (% dose) = [1- Ca(dpm) a t 1 hr/dose q t 5Ca(dpm) a d m i n i s t e r e d ] x 100. a' b = 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. x' 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. F i g . 5.2 I n t e s t i n a l a b s o r p t i o n o f * 5 C a f r o m i l e a l l o o p o f 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) 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 (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 . * 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 f r o m c a s e i n t r e a t m e n t s . 4 5 C a i n a n i m a l s f e d these d i e t s compared to n a t i v e c a s e i n and soy p r o t e i n f e d a n i m a l s . D e p o s i t i o n of 4 5 C a to the femur i s summarized i n Table 5.4. There were no s i g n i f i c a n t 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 the femur due to d i e t a r y t r e a t m e n t i n SHR and WKY ani m a l s f e d the same d i e t . Animal s t r a i n d i f f e r e n c e s were shown to have an e f f e c t on 4 5 C a d e p o s i t i o n to the bone i n o n l y n a t i v e soy p r o t e i n f e d a n i m a l s . Soy f e d WKY ani m a l s d e p o s i t e d l e s s (p<0.05) 4 5 C a to the femora, r e s u l t i n g i n lower (p<0.05) femur s p e c i f i c a c t i v i t i e s than SHR c o u n t e r p a r t s . Heat d e n a t u r a t i o n of d i e t a r y p r o t e i n s appeared to a l s o i n f l u e n c e the acute d e p o s i t i o n of 4 5 C a t o the bone, but these r e s u l t s were not s i g n i f i c a n t . The amount of r a d i o a c t i v i t y and s p e c i f i c a c t i v i t y of the femora of a n i m a l s f e d the d e n a t u r e d c a s e i n and soy p r o t e i n d i e t s appeared to be h i g h e r than t h a t of c o u n t e r p a r t s f e d n a t i v e p r o t e i n s o u r c e s , r e s p e c t -i v e l y . M i n e r a l Balance Study: The 24 hour m i n e r a l b a l a n c e study d a t a are p r e s e n t e d i n T a b l e s 5.5 to 5.10. The reduced fe e d i n t a k e of a n i m a l s f e d the de n a t u r e d p r o t e i n d i e t s r e s u l t e d i n a s i g n i f i c a n t (p<0.05) decrease i n c a l c i u m ( T a b l e 5.5) i n t a k e , of SHR and WKY ani m a l s f e d the same d i e t . F e c a l Ca e x c r e t i o n complements the i n t a k e d a t a , i n asmuch as a n i m a l s f e d the denatu r e d p r o t e i n d i e t s e x c r e t e d s i g n i f i c a n t l y (p<0.05) l e s s c a l c i u m i n the f e c e s than c o u n t e r p a r t s f e d n a t i v e p r o t e i n d i e t s . U r i n a r y c a l c i u m e l i m i n a t i o n was i n f l u e n c e d by 153 TABLE 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 on 4 5 c a l c i u m d e p o s i t i o n to the f e m u r 1 . D i e t 2 4 3 C a D e p o s i t e d (%dose/bone) 4 5 C a S p e c i f i c A c t i v i t y (% dose/g 4 0 C a ) SHR WKY SHR WKY C 0 . 148 +_ 0 .046* X 0 . 101 0 .039" 1 . 7 + 0 . 6* X 1 . 1 +_ 0 .4* X s 0. 274 +_ 0 . 0 8 0 ° X 0 .098 + 0 . 019* y 3 .4 1 .0* X 1 .2 +_ 0 .2* y DC 0 . 250 +_ 0 .090* X 0 . 174 +_ 0 .053** 4 .0 + 1 .6* X 2 . 8 +_ 0 . 9* X DS 0 . 180 + 0 .069* X 0 .111 + 0 .018** 3 .2 + 0 .8* X 2 . 1 + 0 .4* X 1 Data are e x p r e s s e d as mean +_ SEM; SHR = Spon t a n e o u s l y hyper-t e n s i v e r a t s , WKY = W i s t a r Kyoto r a t s . 2 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 den a t u r e d C; DS = heat den a t u r e d S. *> b = 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. * • 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 t r e a t m e n t means i n rows . 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 . D i e t ^ Ca Intake Urine Ca F e c a l Ca (mg/ day) SHR WKY SHR WKY SHR WKY c 68.75 + 1.25ax 71.25 + 4.62a x 1.11 + 0.14bx 2.03 + 0.50 a y 38.95 + 3.84 a x 49.09 + 5.97 a x s 66.25 + 5.54ax 68.12 + 3.12a x 0.94 + 0.04 b c x 1.78 + 0.35 a y 39.88 + 1.46ax 41.47 + 6.49 a b x DC 46.88 + 4.13b x 50.00 + 2.04b x 1.93 + 0.11 a x 2.13 + 0.21 a x 19.83 + 2.07b x 22.62 + 2.61 c x DS 45.00 + 2.28bx 41.88 + 2.13b x 0.67 + 0.08 c x 1.63 + 0.38 a y 24.34 + 3.08b x 27.74 + 3.59 b c x 1 Data a re 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 Kyoto r a t s . d 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 denatured S. a,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 columns. X,'V = 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. both animal s t r a i n d i f f e r e n c e s and d i e t a r y p r o t e i n s o u r c e . SHR an i m a l s e x c r e t e d s i g n i f i c a n t l y (p<0.05) lower l e v e l s of c a l c i u m i n the u r i n e than WKY c o u n t e r p a r t s f e d the same d i e t . Soy p r o t e i n f e d a n i m a l s tended to e x c r e t e l e s s Ca i n the u r i n e than c a s e i n f e d c o u n t e r p a r t s . T h i s o b s e r v a t i o n was s i m i l a r f o r anima l s f e d both the n a t i v e and denatur e d p r o t e i n d i e t s , r e s p e c t i v e l y . Animals f e d the n a t i v e soy p r o t e i n d i e t e x h i b i t e d a lower Ca b a l a n c e than c o u n t e r p a r t s f e d c a s e i n ( T a b l e 5.6). Moreover, Ca b a l a n c e was s i g n i f i c a n t l y (p<0.05) reduced i n both SHR and WKY a n i m a l s f e d the de n a t u r e d p r o t e i n d i e t s . C o n v e r s e l y , c a l c i u m apparent a b s o r p t i o n was g r e a t e r (p<0.05) i n the s e same an i m a l s . Magnesium i n t a k e was a l s o s i g n i f i c a n t l y (p<0.05) reduced i n SHR and WKY a n i m a l s f e d the denatur e d p r o t e i n d i e t s compared to c o u n t e r p a r t s f e d n a t i v e p r o t e i n d i e t s ( T a b l e 5.7). The i n t a k e d a t a taken t o g e t h e r w i t h the s i g n i f i c a n t l y (p<0.05) reduced f e c a l Mg e x c r e t i o n observed i n these a n i m a l s , i n d i c a t e d a s i g n i f i c a n t l y (p<0.05) enhanced apparent a b s o r p t i o n of Mg by denatured p r o t e i n f e d a n i m a l s ( T a b l e 5.8). U r i n a r y Mg e l i m i n a t i o n was i n f l u e n c e d by both d i e t a r y p r o t e i n source and p r o t e i n heat d e n a t u r a t i o n , r e s p e c t i v e l y i n SHR and WKY anim a l s f e d the same d i e t ( T a b l e 5.7). Soy p r o t e i n f e d ani m a l s e x h i b i t e d a s i g n i f i c a n t l y (p<0.05) lower u r i n a r y Mg e x c r e t i o n than c o u n t e r p a r t s f e d c a s e i n . Moreover, a n i m a l s f e d the heat denatured p r o t e i n s e x p e r i e n c e d a s i g n i f i c a n t l y (p<0.05) g r e a t e r u r i n a r y e l i m i n a t i o n of t h i s m i n e r a l 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 . Thus, Mg 156 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 . D i e t ' SHR Ca Intake (mg/ day) WKY Ca Balance" 3 (mg) SHR WKY Ca Apparent A b s o r p t i o n SHR WKY C 68.75 + 1.25 a x 71.25 + 4.62 a x 33.46 + 2 . 5 1 a x 33.42 + 2 . 4 7 a x 41.11 + 1 . 4 7 a x 38.84 + 5 . 5 0 b x S 66.25 + 5 . 5 4 a x 68.12 + 3.12 a x 28.60 + 3 . 1 1 a b x 27.15 + 3 . 0 7 a b x 40.00 + 2 . 1 2 a x 40.75 + 1.79 b x DC 46.88 + 4 . 1 3 b x 50.00 + 2 . 0 4 b x 23.11 + 3 . 2 2 b x 24.92 + 1 . 9 3 b c x 54.14 + 5 . 3 3 a x 57.60 + 3 . 5 6 a x DS 45.00 + 2 . 2 8 b x 41.88 + 2 . 1 3 b x 19.92 + 1.55 b x 18.54 + 1.02 c x 53.96 + 4 . 5 8 a x 54.86 + 3 . 9 5 a x * 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 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 denatured C; DS = heat denatured S. Ca Balance (mg) = Ca Intake (mg/day) - U r i n a r y Ca (mg/day) - F e c a l Ca (mg/day). 4 Ca Apparent A b s o r p t i o n (%) = {[Ca Intake (mg/day) - Fe c a l Ca (mg / d a y ) ] / C a Intake (mg/day)}x 100. a,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 tr e a t m e n t means i n columns. x » 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. 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 . D i e t ^ Mg Intake U r i n e Mg F e c a l Mg (mg/ day) SHR WKY SHR WKY SHR WKY c 6.88 + 0 . 1 2 a x 7.12 + 0 . 4 6 a x 1.91 + 0 . 0 3 b x 1.18 + 0 . 0 8 b y 1.49 + 0 . 1 0 b x 2.07 + 0 . 0 8 a y s 6.62 + 0 . 5 5 a x 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 a x 2.87 + 0 . 2 5 a x DC 4.69 + 0 . 4 1 b x 5.00 + 0 . 2 0 b x 2.67 + 0 . 1 8 a x 2.94 + 0 . 1 9 a x 0.48 + 0 . 0 5 c x 0.64 + 0 . 0 5 b x DS 4.50 + 0 . 2 3 b x 4.19 + 0 . 3 0 b x 1.35 + 0 . 1 2 c x 2.76 + 0 . 1 8 a y 0.66 + 0 . 0 5 c x 0.89 + 0 . 0 6 b x 1 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 = W i s t a r Kyoto r a t s . d 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 denatured S. a » 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 columns. x » v = 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. TABLE 5.8 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 balance . D i e t * Mg Intake Mg Balance" 3 Mg Apparent A b s o r p t i o n (mg/ day) (mg) (%) SHR WKY SHR WKY SHR WKY c 6.88 + 0 . 1 2 a x 7.12 + 0 . 4 6 a x 3.18 + 0 . 3 1 a b x 4.24 + 0 . 1 9 a x 71.16 + 7 . 0 8 b x 66.41 + 4 . 4 4 b x S 6.62 + 0 . 5 5 a x 6.81 + 0 . 3 1 a x 3.81 + 0 . 2 7 a x 3.60 + 0 . 1 3 b x 67.76 + 2 . 5 2 b x 72.99 + 5 . 5 0 a b x DC 4.69 + 0 . 4 1 b x 5.00 + 0 . 2 0 b x 1.51 + 0 . 2 4 c x 1.37 + 0 . 1 1 d x 89.38 + 0 . 8 4 a x 86.07 + 1.43 a x DS 4.50 + 0 . 2 3 b x 4.19 + 0 . 3 0 b x 2.45 + 0 . 1 9 b x 1.92 + 0 . 0 5 c x 87.43 + 1 . 6 7 a x 80.78 + 3 . 6 9 a b x Data a re 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 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 denatured S. Mg Bala 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 Apparent A b s o r p t i o n (%) = {[Mg Intake (mg/day) - Fe c a l Mg (mg /day)]/Mg Intake (mg/day)}x 100. 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 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. b a l a n c e was s i g n i f i c a n t l y (p<0.05) reduced i n SHR and WKY a n i m a l s f e d the denatur e d c a s e i n and soy d i e t s , r e s p e c t i v e l y i n comparison to n a t i v e c a s e i n and soy f e d a n i m a l s ( T a b l e 5.8). As a r e s u l t , a s i g n i f i c a n t i n t e r a c t i o n between an i m a l s t r a i n and p r o t e i n heat t r e a t m e n t (F(3,24)=5.85, p<0.004; Appendix Table 4) was found to e x i s t f o r Mg b a l a n c e . Phosphorus i n t a k e s p a r a l l e l e d the f e e d i n t a k e d a t a , and were s i g n i f i c a n t l y (p<0.05) reduced i n a n i m a l s f e d the denatured p r o t e i n d i e t s compared to c o u n t e r p a r t s f e d n a t i v e c a s e i n and soy d i e t s ( T a b l e 5.9). F e c a l e x c r e t i o n of P was s i g n i f i c a n t l y (p<0.05) reduced o n l y i n WKY a n i m a l s f e d denatur e d c a s e i n and soy p r o t e i n d i e t s , compared to 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 o u r c e s . Apparent a b s o r p t i o n of P however, was not s i g n i f i c a n t l y a f f e c t e d by a n i m a l s t r a i n d i f f e r e n c e s , or d i e t a r y treatment ( T a b l e 5.10). U r i n a r y e l i m i n a t i o n of P was i n f l u e n c e d by source of d i e t a r y p r o t e i n as w e l l as by p r o t e i n heat d e n a t u r a t i o n . Soy p r o t e i n f e d a n i m a l s e x c r e t e d s i g n i f i c a n t l y (p<0.05) l e s s P i n u r i n e than c o u n t e r p a r t s f e d c a s e i n . T h i s o b s e r v a t i o n was s i m i l a r i n SHR and WKY a n i m a l s . Heat d e n a t u r a t i o n of d i e t a r y p r o t e i n s r e s u l t e d i n a s i g n i f i c a n t l y (p<0.05) reduced u r i n a r y e x c r e t i o n of P i n SHR and WKY a n i m a l s f e d the same d i e t . For a l l d i e t a r y t r e a t m e n t s , WKY ani m a l s e x c r e t e d s i g n i f i c a n t l y (p<0.05) more P i n the u r i n e than SHR c o u n t e r p a r t s . P b a l a n c e i n soy f e d anim a l s was h i g h e r than i n c o u n t e r p a r t s f e d c a s e i n , a l t h o u g h the d i f f e r e n c e was not s i g n i f i c a n t ( T a b l e 5.10). Furthermore, P b a l a n c e was s i g n i f i c a n t l y (p<0.05) reduced i n SHR and WKY animals 160 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 e x c r e t i o n 1 . D i e t ^ P Intake Urine P F e c a l P (mg/ day) SHR WKY SHR WKY SHR WKY c 79.5 + 1.4ax 82.4 + 5.3 a x 21.74 + 1.05 a x 31.45 + 3.17 a y 23.61 + 2.18 a x 30.02 + 2.41 a y s 84.4 + 5.5 a x 84.2 + 3.9 a x 17.82 + 1.36 a b x 22.14 + 0.81 b x 22.18 + 1.31 a x 27.54 + 2.07 a x DC 55.6 + 3.8 b x 57.8 + 2.4 b x 15.21 + 2.07 b x 29.65 + 1.75 a y 23.31 + 2.19 a x 13.24 + 1.61 b y DS 56.4 + 2.3 b x 51.8 + 2.6 b x 16.21 + 0.95 b x 22.52 + 1.09 b y 17.09 + 1.22 a x 15.28 + 2.01 b x 1 Data a re 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 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 denatured S. a,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 columns. . x » 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. TABLE 5.10 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 . D i e t ' P Intake P B a l a n c e 0 P Apparent A b s o r p t i o n (mg/ day) (mg) (%) SHR WKY SHR WKY SHR WKY c 79.5 + 1.4 a x 82.4 + 5 . 3 a x 34.12 + 3 . 5 4 a b x 29.57 + 3 . 3 5 a x 68.40 + 3 . 0 0 a x 68.29 + 3 . 4 0 a x s 84.4 + 5 . 5 a x 84.2 + 3 . 9 a x 44.38 + 4 . 3 9 a x 37.79 + 3 . 4 4 a x 72.52 + 2 . 2 4 a x 70.68 + 4 . 7 6 a x DC 55.6 + 3 . 8 b x 57.8 + 2 . 4 b x 26.75 + 3 . 2 5 b x 16.70 + 2 . 2 2 b x 72.49 + 3 . 4 8 a x 77.18 + 2 . 4 6 a x DS 56.4 + 2 . 3 b x 51.8 + 2 . 6 b x 22.97 + 1 . 8 7 b x 18.95 + 1.86 b x 69.75 + 1.44 a x 69.78 + 5 . 1 6 a x 1 Data a re 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 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 denatured S. P B a l a n c e (mg) = P Intake (mg/day) - U r i n a r y P (mg/day) - Fe c a l P (mg/day). q P Apparent A b s o r p t i o n (%) = {[P Intake (mg/day) - Fe c a l P (mg / d a y ) ] / P Intake (mg/day)}x 100. a » 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 columns. x' 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. f e d the denatu r e d p r o t e i n d i e t s compared to those f e d n a t i v e p r o t e i n s . Taken t o g e t h e r , the fee d i n t a k e and m i n e r a l b a l a n c e d a t a i n d i c a t e t h a t the m i n e r a l a b s o r p t i o n of ani m a l s 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 , a l i k e l y r e s u l t of the n u t r i e n t m a l a b s o r p t i o n a s s o c i a t e d w i t h the e x t e n s i v e heat p r o c e s s i n g of d i e t a r y p r o t e i n s . The l o n g t e r m d e f i c i e n t m i n e r a l i n t a k e and 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 i n reduced m i n e r a l b a l a n c e s when compared t o c o u n t e r p a r t s f e d n a t i v e p r o t e i n s . The reduced m i n e r a l b a l a n c e s of these a n i m a l s r e s u l t e d i n enhanced apparent a b s o r p t i o n of m i n e r a l s f o r c a l c i u m and magnesium, but not phosphorus. While source of d i e t a r y p r o t e i n c o u l d be observed to i n f l u e n c e u r i n a r y m i n e r a l e l i m i n a t i o n , these e f f e c t s d i d not d i s t u r b m i n e r a l b a l a n c e . Bone P h y s i c a l P arameters! Femur ash weight and dry weight were reduced i n ani m a l s f e d soy p r o t e i n compared to c o u n t e r p a r t s f e d c a s e i n , f o r both SHR and WKY a n i m a l s ( T a b l e 5.11). Animals f e d the heat denatured p r o t e i n d i e t s e x h i b i t e d s i g n i f i c a n t l y (p<0.05) d e c r e a s e d femur ash we i g h t s and d r y w e i g h t s i n comparison to those f e d n a t i v e p r o t e i n . Femur bone l e n g t h was not s i g n i f i c a n t l y a f f e c t e d by a n i m a l s t r a i n d i f f e r e n c e s or source of d i e t a r y p r o t e i n . However, a n i m a l s f e d the heat de n a t u r e d d i e t a r y p r o t e i n s had s i g n i f i c a n t l y (p<0.05) d e c r e a s e d femur l e n g t h s compared to c o u n t e r p a r t s f e d n a t i v e p r o t e i n s . The r e d u c t i o n i n o v e r a l l bone s i z e i n SHR and WKY a n i m a l s f e d the d e n a t u r e d 163 TABLE 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 on femur p h y s i c a l p a r a m e t e r s 1 . D i e t * Ash Wt. Dry Wt. Length (g) (g) (mm) SHR WKY SHR WKY SHR WKY c 0.237 + 0.006 a x 0.228 + 0 . 0 0 6 a x 0.387 + 0.010 a x 0.358 + 0 . 0 1 2 a x 30.6 + 0 . 5 a x 31.2 + 0 . 6 a x s 0.206 + 0.006 b x 0.215 + 0 . 0 0 5 a x 0.360 + 0.009 b x 0.347 + 0 . 0 1 6 a x 30.5 + 0 . 2 a x 31.2 + 0 . 4 a x DC 0.176 + 0.004 c x 0.167 + 0 . 0 0 7 b x 0.323 + 0.006 c x 0.292 + 0 . 0 1 3 b x 29.2 + 0 . 2 b x 29.0 + 0 . 5 b x DS 0.121 + 0.003 d x 0.145 + 0 . 0 1 2 b x 0.235 + 0.007 d x 0.263 + 0 . 0 2 2 b x 25.6 + 0 . 4 C X 27.9 + 1 . 0 b v 1 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 Kyoto r a t s . i 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 denatured 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 treatment means i n columns. x » v = 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. p r o t e i n d i e t s was a r e s u l t of r e t a r d e d animal growth c h a r a c t e r -i s t i c s as i n f l u e n c e d by the reduced f e e d i n t a k e and FER of these a n i m a l s . T h i s i s observed when femur ash weight i s e x p r e s s e d as a per cent of f i n a l body weight (range 0.076 +. 0.001 to 0.093 +_ 0.006 % F i n a l body weight f o r WKY; and 0.091 + 0.001 to 0.101 + 0.001 % F i n a l body weight f o r SHR). Bone M i n e r a l i z a t i o n : Bone m i n e r a l i z a t i o n d a t a are summarized i n T a b l e 5.12. Bone c a l c i f i c a t i o n was lower i n soy p r o t e i n f e d ani m a l s than c a s e i n f e d c o u n t e r p a r t s i n SHR and WKY a n i m a l s f e d the same d i e t . Moreover, a n i m a l s f e d the denatu r e d p r o t e i n d i e t s had s i g n i f -i c a n t l y (p<0.05) reduced bone c a l c i u m c o n t e n t s when compared to those f e d n a t i v e p r o t e i n s o u r c e s . S i m i l a r l y , femur magnesium c o n t e n t and Ca/P r a t i o were both s i g n i f i c a n t l y (p<0.05) reduced i n a n i m a l s f e d the d e n a t u r e d p r o t e i n s as compared to those f e d n a t i v e p r o t e i n s , r e s p e c t i v e l y . T h i s o b s e r v a t i o n was common to both SHR and WKY a n i m a l s . Bone B i o m e c h a n i c a l Parameters: Femur b i o m e c h a n i c a l parameters from the 3 - p o i n t bending procedure are p r e s e n t e d i n Table 5.13. Femur bending f a i l u r e energy was reduced i n SHR and WKY a n i m a l s f e d the denatu r e d p r o t e i n d i e t s when compared t o those f e d the n a t i v e p r o t e i n s . The d i f f e r e n c e however, was o n l y s i g n i f i c a n t i n SHR a n i m a l s f e d the den a t u r e d c a s e i n and soy d i e t s . D e s p i t e these d i f f e r e n c e s i n the work energy r e q u i r e d to break the femora of these a n i m a l s , the femur maximum bending s t r e s s was not s i g n i f i c a n t l y a f f e c t e d 165 TABLE 5.12 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 . D i e t 2 Ca Mg Ca/P (mg/bone) (mg/bone) R a t i o SHR WKY SHR WKY SHR WKY C 94.60 + 3 . 8 5 a x 92.20 + 2 . 9 1 a x 1.84 + 0 . 0 6 a x 1.92 + 0 . 0 6 a x 2.00 + 0 . 0 1 a x 2.04 + 0 . 0 2 a x S 78.75 + 3 . 2 1 b x 82.35 + 4 . 9 3 a x 1.62 + 0 . 0 7 a x 1.78 + 0 . 0 7 a x 1.97 + 0 . 0 5 a x 1.88 + 0 . 0 7 a b x DC 64.60 + 4 . 0 3 c x 63.45 + 2 . 6 3 b x 1.32 + 0 . 0 6 b x 1.42 + 0 . 0 5 b x 1.77 + 0 . 1 4 a x 1.83 + 0 . 0 2 b x DS 51.20 + 4 . 2 6 d x 53.55 + 4 . 7 1 b x 1.14 + 0 . 1 3 b x 1.16 + 0 . 1 0 c x 1.82 + 0 . 0 4 a x 1.91 + 0 . 0 4 a b x 1 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 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 denatured 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 treatment means i n columns. x » 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 tr e a t m e n t means i n rows. TABLE 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 b i o m e c h a n i c a l p a r a m e t e r s 1 . on femur D i e t 2 Bending F a i l u r e Energy (x 10-2 J ) SHR WKY Maximum Bending S t r e s s (N/mm2) SHR WKY c 9 .24 + 0 . 92a * 6 .65 0 .85" X 68 . 94 + 3 .06 a X 75 .31 + 4 . 95a ' s 6 . 12 + 0 .60»>* 6 .51 + 0 .41° X 65 .82 + 1 • 45a X 78 .77 + 1 . 89a y DC 5 . 72 +_ 1 .21«>* 5 .89 + 0 . 55a X 73 .22 + 2 .30 a X 75 . 15 + 3 . 45a * DS 3 .09 + 0 . 3 3 ° * 4 .24 + 0 . 57a X 66 .46 + 2 . l l a X 71 .09 + 1 . 71«y 1 Data are e x p r e s s e d as mean +_ SEM. 2 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 de 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 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. 167 by d i e t a r y t r e a t m e n t . The femur maximum bending s t r e s s was s i g n i f i c a n t l y (p<0.05) g r e a t e r i n WKY anim a l s when compared to •SHR c o u n t e r p a r t s f e d the same d i e t i n d i c a t i n g a g r e a t e r f o r c e needed to break the femora of these a n i m a l s . T h i s o b s e r v a t i o n may be due to the g r e a t e r f i n a l body weight of these a n i m a l s i n comparison to SHR c o u n t e r p a r t s . 168 D i s c u s s i o n P e r c i v a l and Schneeman (1979) r e p o r t e d t h a t a n i m a l s f e d a heat damaged c a s e i n d i e t e x p e r i e n c e d weight l o s s , reduced fe e d i n t a k e and d i a r r h e a , s i m i l a r t o the r e s u l t s r e p o r t e d h e r e i n . The reduced body weight g a i n and PER of ani m a l s f e d the DC and DS d i e t s may be e x p l a i n e d by the reduced n u t r i t i v e v a l u e of the p r o t e i n s o u r c e s . T h i s r e s u l t i s s u p p o r t e d by the work of o t h e r s (Ikegami ejt. a_l_. , 1975 ; P e r c i v a l and Schneeman, 1979) who have r e p o r t e d i n c r e a s e d i n t e s t i n a l c o n t e n t s , and reduced p a n c r e a t i c e x o c r i n e s e c r e t i o n i n r a t s f e d p o o r l y d i g e s t i b l e p r o t e i n s o u r c e s . S t u d i e s have shown t h a t p a n c r e a t i c enzyme c o m p o s i t i o n and t u r n o v e r of p a n c r e a t i c p r o t e i n can be i n f l u e n c e d by d i e t a r y p r o t e i n q u a l i t y (Rebound et a l . . 1966; Ikegami et a1. . 1975; P e r c i v a l and Schneeman, 1979). Thus, reduced p r o t e i n d i g e s t -i b i l i t y , as demonstrated by the i n v i t r o s t u d y h e r e i n , may have r e s u l t e d i n a g r e a t e r i n t e s t i n a l r e s i d u e , and t h e r e f o r e , a p r o t e c t i v e e f f e c t on i n t e s t i n a l enzymes from a u t o d i g e s t i o n . T h i s would r e s u l t i n a s u p p r e s s i o n of f u r t h e r p a n c r e a t i c output ( i k e g a m i e_t_ a_l_. , 1975). A p r o d u c t of p a n c r e a t i c enzymatic p r o t e o l y s i s , are the c a s e i n phosphopeptides (CPP), r e l e a s e d by t r y p t i c d i g e s t i o n of c a s e i n . These p e p t i d e s have been e x t e n s i v e l y r e p o r t e d to s e q u e s t e r c a l c i u m i o n s w i t h i n the i n t e s t i n a l m i l i e u , to i n c r e a s e the i n t r a l u m i n a l s o l u b l e c a l c i u m c o n c e n t r a t i o n ( N a i t o et a l . . 1972; N a i t o and S u z u k i , 1974; Lee e_t al.. , 1980; 1983). These o b s e r v a t i o n s were extended by the f i n d i n g s of experiments 3 and 169 4, which showed enhanced 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 i n r a t s f e d c a s e i n and CPP c o n t a i n i n g d i e t s , r e s p e c t i v e l y . Other workers have a l s o shown t h a t amino a c i d s , such as L - a r g i n i n e and L-l y s i n e , enhance c a l c i u m a b s o r p t i o n from the s m a l l i n t e s t i n e (Wasserman ejb. a_l.. , 1956). Thus, s i n c e 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 can be a f f e c t e d by the p e p t i d e or amino a c i d p r o f i l e s of p r o t e i n d i g e s t i o n ; f a c t o r s a f f e c t i n g the d i g e s t i b i l i t y of d a i r y p r o t e i n s , c o u l d i n f l u e n c e d i e t a r y c a l c i u m b i o a v a i l a b i l i t y . The absence of an observed enhancing e f f e c t of p o s t - d i g e s t i o n CPP on 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 i n d e n a t u r e d c a s e i n f e d a n i m a l s i s f u r t h e r e v i d e n c e of the poor d i g e s t i b i l i t y of t h i s d i e t . P e r c i v a l and Schneeman (1979) have suggested t h a t the d i g e s t i o n of heat damaged p r o t e i n s i s i m p a i r e d ; t h u s , the r e l e a s e of CPP from d e n a t u r e d c a s e i n d i e t s would be s i m i l a r l y d e c r e a s e d . F u r t h e r , heat t r e a t m e n t of m i l k has been shown to r e s u l t i n s u b s t a n t i a l d e p h o s p h o r y l a t i o n of the c a s e i n p r o t e i n s at the p h o s p h o s e r y l r e s i d u e s (Howat and W r i g h t , 1934), which n o r m a l l y b i n d m i c e l l a r c o l l o i d a l c a l c i u m phosphate. D e p h o s p h o r y l a t i o n of c a s e i n s r e s u l t e d i n a reduced b i n d i n g c a p a c i t y f o r c a l c i u m to c a s e i n phosphopeptides (S a t o et a1.. 1983), which i n t u r n would decrease the c a l c i u m s o l u b i l i z i n g p o t e n t i a l of the p e p t i d e s i n the i n t e s t i n a l lumen. The subsequent e f f e c t t h a t t h i s would have on c a l c i u m b i o a v a i l a b i l i t y i s u n c e r t a i n , g i v e n the f i n d i n g s of Weeks and K i n g (1985) which showed no adverse e f f e c t s of t h e r m a l p r o c e s s i n g of m i l k on c a l c i u m a b s o r p t i o n . A l t e r n a t i v e l y , the reduced i l e a l a b s o r p t i o n of 4 5 C a i n r a t s f e d the heat denatured 170 p r o t e i n d i e t s may be e x p l a i n e d by a de c r e a s e d i n t e s t i n a l t r a n s i t t i m e . An i n c r e a s e i n the t r a n s i t r a t e of l u m i n a l c o n t e n t s would a l s o r e s u l t i n the observed s i g n s of n u t r i e n t m a l a b s o r p t i o n , as seen e a r l i e r i n experiment 1. D e s p i t e the observed n u t r i e n t m a l a b s o r p t i o n of ani m a l s f e d the heat d e n a t u r e d p r o t e i n s h e r e i n , plasma p r o t e i n and m i n e r a l l e v e l s were not a f f e c t e d . T h i s r e s u l t may be a t t r i b u t e d t o a d a p t a t i o n by a n i m a l s to the r e s p e c t i v e d i e t s over the 10 week e x p e r i m e n t a l p e r i o d . For example, plasma c a l c i u m l e v e l s are m a i n t a i n e d r e l a t i v e l y c o n s t a n t by the p a r a t h y r o i d e n d o c r i n e system, which r e g u l a t e s e x t r a c e l l u l a r c a l c i u m by m o b i l i z i n g bone c a l c i u m r e s e r v e s and r e d u c i n g u r i n a r y c a l c i u m l o s s ( G r e g e r , 1988). A p o s s i b l e i n d i c a t i o n of t h i s h o m e o s t a t i c mechanism may be seen w i t h the t r a n s l o c a t i o n of 4 5 C a to the femora i n animals f e d the denatur e d p r o t e i n d i e t s , which was i n v e r s e l y r e l a t e d to the 4 5 C a a b s o r p t i o n from the i l e a l l o o p . C o n s i d e r i n g the r e l a t i v e l y s h o r t time p r o t o c o l used i n t h i s s tudy to measure 4 5 C a f e m o r a l d e p o s i t i o n , i t i s expected t h a t 4 5 C a would be d e p o s i t e d o n l y on the s u r f a c e of bone. T h e r e f o r e , the s i m i l a r plasma c a l c i u m l e v e l s observed i n a n i m a l s f e d denatured and n a t i v e p r o t e i n s are l i k e l y a t t r i b u t a b l e t o a decrease i n bone 4 0 C a c o n t e n t , r e s u l t i n g i n the g r e a t e r f e m o r a l 4 5 C a a c t i v i t i e s of the former group of a n i m a l s . In an acute study such as t h i s , i t i s i m p o r t a n t to emphasize t h a t f e m o r a l 4 5 C a a c t i v i t y i s not n e c e s s a r i l y a p r e d i c t i v e i n d e x of c a l c i u m u t i l i z a t i o n . 171 A l t e r n a t i v e l y , under c o n d i t i o n s of c a l c i u m d e f i c i e n c y , e x t r a c e l l u l a r c a l c i u m homeostasis i s r e g u l a t e d by reduced u r i n a r y c a l c i u m e x c r e t i o n (Agus et a l . . 1981). However, t h i s e f f e c t was not observed i n the p r e s e n t s t u d y , as u r i n a r y , but not f e c a l c a l c i u m l o s s e s , were a c t u a l l y i n c r e a s e d i n a n i m a l s f e d d e n a t u r e d p r o t e i n d i e t s . I t i s known t h a t a phosphorus d e f i c i e n c y can r e s u l t i n i n c r e a s e d u r i n a r y c a l c i u m e x c r e t i o n and n e g a t i v e c a l c i u m b a l a n c e (Rader et_ a l _ . , 1979). Thus, i t i s noteworthy t h a t a n i m a l s f e d the heat d e n a t u r e d p r o t e i n d i e t s e x h i b i t e d reduced c a l c i u m , as w e l l as phosphorus b a l a n c e s , r e s p e c t i v e l y . S t u d i e s have shown t h a t phosphorus has a d i r e c t p o s i t i v e e f f e c t on r e n a l t u b u l a r c a l c i u m r e a b s o r p t i o n (Zemel, 1985); t h e r e f o r e , phosphorus d e f i c i e n c y would r e s u l t i n d e c r e a s e d t u b u l a r r e a b s o r p t i o n of c a l c i u m and c o n s e q u e n t l y , i n c r e a s e d u r i n a r y c a l c i u m e x c r e t i o n . The s i m i l a r i t y between the magnesium b a l a n c e d a t a and c a l c i u m d a t a , i n d i c a t e s t h a t a d e c r e a s e d i n t e s t i n a l a b s o r p t i o n and i n c r e a s e d u r i n a r y e x c r e t i o n a l s o o c c u r r e d f o r magnesium i n a n i m a l s f e d the d e n a t u r e d c a s e i n and soy d i e t s . As a r e s u l t , magnesium b a l a n c e was a l s o i n f l u e n c e d by the i n t e r -a c t i o n between a n i m a l s t r a i n and p r o t e i n heat t r e a t m e n t . P r e v i o u s s t u d i e s have i n d i c a t e d t h a t the SHR a n imal model e x h i b i t s both an a l t e r e d c a l c i u m m e t a b o l i s m , and d i s t r i b u t i o n of e x t r a c e l l u l a r c a l c i u m ( A o k i ejt JLL- , 1976 ; McCarron .et. a_L. , 1981; S c h e d l .et. aj_. , 1988). In the p r e s e n t s t u d y , i l e a l c a l c i u m a b s o r p t i o n was not d i f f e r e n t between SHR and WKY c o u n t e r p a r t s , c o n f i r m i n g p r e v i o u s r e s u l t s from our l a b o r a t o r y ( K i t t s e_t_ al_. , 172 1989). The r e l a t i v e amount of 4 5 C a t r a n s l o c a t e d to the femora of SHR a n i m a l s was g r e a t e r than i n WKY c o u n t e r p a r t s , s i m i l a r to the r e s u l t s of experiment 3. F u r t h e r e v i d e n c e f o r an a l t e r e d m e tabolism of e x t r a c e l l u l a r c a l c i u m i n SHR ani m a l s i s the dec r e a s e d u r i n a r y c a l c i u m e x c r e t i o n at 10 weeks of age, compared to WKY c o u n t e r p a r t s ; however, o v e r a l l c a l c i u m b a l a n c e was not a f f e c t e d . S i m i l a r l y , o t h e r workers employing l o n g e r b a l a n c e s t u d i e s , have r e p o r t e d a d e c r e a s e d u r i n a r y e x c r e t i o n of c a l c i u m w i t h an o v e r a l l more p o s i t i v e c a l c i u m b a l a n c e i n young SHR an i m a l s (Lau et a 1. . 1986 ; Jones et a1. . 1988). The g r e a t e r p o s i t i v e c a l c i u m b a l a n c e i n the young SHR was i n d i c a t i v e of an abnormal r e n a l c a l c i u m r e t e n t i o n (Lau et a l . . 1986). A l t e r -n a t i v e l y , u r i n a r y 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 i n o l d e r SHR have been r e p o r t e d to be both i n c r e a s e d and unchanged, r e s p e c t i v e l y compared to age-matched WKY c o n t r o l s (McCarron et a l . . 1981; Jones ejt. al . . , 1988). Taken t o g e t h e r , these data suggest t h a t a b n o r m a l i t i e s i n c a l c i u m m e t a b o l i s m , and p a r t i c -u l a r l y e x t r a c e l l u l a r c a l c i u m h a n d l i n g , occur e a r l y , b e f o r e the development of h y p e r t e n s i o n i n the SHR. The r e s u l t s of the p r e s e n t s t u d y i n d i c a t e t h a t d i f f e r e n c e s i n 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 , a s s o c i a t e d w i t h d i e t a r y p r o t e i n source measured by the i n s i t u l i g a t e d i l e a l l o op t e c h n i q u e , are not n e c e s s a r i l y d e t e c t e d u s i n g the 24 hr b a l a n c e method i n both the SHR and WKY s t r a i n s . T h e r e f o r e , 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 does not appear to e i t h e r p l a y a s i g n i f i c a n t , or s p e c i f i c r o l e i n the a l t e r e d SHR c a l c i u m m e tabolism. 173 In the p r e s e n t s t u d y , a n i m a l s f e d the denature d c a s e i n and soy d i e t s e x h i b i t e d d e c r e a s e d bone m i n e r a l i z a t i o n and p h y s i c a l p a r a m e t e r s . Decreased bone l e n g t h and c o r t i c a l t h i c k n e s s are c o n s i d e r e d to be symptomatic of p r o t e i n m a l n u t r i t i o n (Garn et a l . . 1964; Adams and B e r r i d g e , 1969; Crosby e_t al.. , 1983; P a r f i t t , 1983). P r o t e i n m a l a b s o r p t i o n has been a t t r i b u t e d to the presence of browning r e a c t i o n p r o d u c t s and t h e i r e f f e c t on p r o t e i n d i g e s t i o n (Oste and S j o d i n , 1984). Thus, the m a l a b s o r p t i o n observed h e r e i n w i t h the denature d c a s e i n and soy or presence of M a i l l a r d R e a c t i o n p r o d u c t s , l i k e l y c o n t r i b u t e d t o the d e c r e a s e d bone growth and s i z e . A l s o , bone m i n e r a l i z a t i o n has 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 i n phosphorus d e f i c i e n t a n i m a l s ( B r u i n et a l . . 1975), which s u p p o r t s the o b s e r v a t i o n s made i n t h i s s t u d y . Decreased d e p o s i t i o n of c a l c i u m to the s k e l e t o n has been shown to c o n t r i b u t e to the acute development of h y p e r c a l c e m i a i n P d e f i c i e n c y (Rader e t a1. . 1979). T h i s h y p e r c a l c e m i c response 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 c a l c i u m l e v e l s i n the denatured p r o t e i n f e d a n i m a l s , which o t h e r w i s e would have been h y p o c a l c e m i c . A l t e r n a t i v e l y , plasma c a l c i u m l e v e l s have been r e p o r t e d to be i n c r e a s e d due to enhanced bone m i n e r a l m o b i l i z a t i o n i n P d e f i c i e n c y ( C a s t i l l o ejt. a l . . 1975). In the p r e s e n t s t u d y , d e f i c i e n c i e s i n Ca, Mg, and P caused by m a l a b s o r p t i o n i n anim a l s f e d the denatured c a s e i n and soy d i e t s , r e s p e c t i v e l y , r e s u l t e d i n decrea s e d bone c a l c i u m and magnesium c o n t e n t s and low Ca/P r a t i o s . T h e r e f o r e , a decrease i n b i o m e c h a n i c a l femur bending f a i l u r e energy, but not maximum 174 bending s t r e s s , was caused by reduced bone growth and bone m a t r i x components, secondary to n u t r i e n t m a l a b s o r p t i o n . Thus, a n i m a l s f e d the heat d e n a t u r e d p r o t e i n s e x h i b i t e d d e c r e a s e d femur b i o m e c h a n i c a l parameters as i n f l u e n c e d by reduced body growth and m i n e r a l d e f i c i e n c i e s . 175 Experiment 6 C a l c i u m b a l a n c e and femur biomechanics 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 . I n t r o d u c t i o n In a d d i t i o n to the importance of source and q u a l i t y of d i e t a r y p r o t e i n to c a l c i u m b i o a v a i l a b i l i t y , the l e v e l of p r o t e i n i n t a k e must a l s o be c o n s i d e r e d . I n c r e a s i n g p r o t e i n i n t a k e , w h i l e l e a v i n g l e v e l s of c a l c i u m and phosphorus unchanged, has been shown t o r e s u l t i n i n c r e a s e d u r i n a r y c a l c i u m e x c r e t i o n and e v e n t u a l l y , n e g a t i v e c a l c i u m b a l a n c e ( A l l e n e t a l . . 1979 ; L i n k s w i l e r et aj_. , 1981; S c h u e t t e et a l _ . , 1981). Thus, i n d i v i d u a l s concerned w i t h c a l c i u m homeostasis may l i m i t t h e i r p r o t e i n i n t a k e . However, p r o t e i n d e f i c i e n c y has been r e p o r t e d to a d v e r s e l y a f f e c t bone s t r u c t u r e and s i z e (Garn et a1.. 1964; Adams and B e r r i d g e , 1969; Crosby et. a_l. , 1983; P a r f i t t , 1983), which may be cause f o r concern to i n d i v i d u a l s prone to o s t e o -p o r o s i s . In the p r e s e n t s t u d y , the o b j e c t i v e s were to determine the e f f e c t of d i e t a r y p r o t e i n l e v e l , which may i n f l u e n c e the p r o d u c t i o n of b i o a c t i v e p e p t i d e s on i l e a l c a l c i u m a b s o r p t i o n as w e l l as subsequent u t i l i z a t i o n f o r m i n e r a l b a l a n c e and bone 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 s t r e n g t h . These s t u d i e s were performed u s i n g o s t e o p o r o s i s prone 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) r a t s . M a t e r i a l s and Methods  An i m a l s and D i e t s : Four-week o l d male 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) r a t s ( C h a r l e s R i v e r , M o n t r e a l , PQ) were d i v i d e d i n t o f o u r e x p e r i m e n t a l 176 d i e t a r y groups (6 a n i m a l s per g r o u p ) . D i e t a r y t r e a t m e n t s i n c l u d e d 20% c a s e i n , soy p r o t e i n i s o l a t e (ICN B i o c h e m i c a l s , C l e v e l a n d , OH) and 6% c a s e i n and soy 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 ( T a b l e 6.1). A l l d i e t s c o n t a i n e d an adequate c a l c i u m l e v e l ( 0 . 5 % ) . The anim a l s f e d the 20% c a s e i n and 20% soy p r o t e i n i s o l a t e d i e t s were the same as i n experiments 4 and 5. Feeding 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 3. A 24 hour b a l a n c e study was performed, as p r e v i o u s l y d e s c r i b e d i n experiment 4, when an i m a l s were 10 weeks of age. 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 was measured as p r e v i o u s l y d e s c r i b e d i n experiment 1 when an i m a l s were 14 weeks of age. A n a l y s e s : A n a l y s e s of i n t e s t i n a l lumen, b l o o d plasma, f e e d , u r i n e , f e c e s and femur m i n e r a l i z a t i o n and biomechanics were performed as p r e v i o u s l y d e s c r i b e d i n experiment 4. S t a t i s t i c a l A n a l y s e s : A l l d a t a are e x p r e s s e d as mean +_ SEM. Treatment d i f f e r e n c e s were t e s t e d f o r by one-way ANOVA. Where d i f f e r e n c e s d i d o c c u r , the source of the d i f f e r e n c e was i d e n t i f i e d u s i n g a m u l t i p l e range t e s t at a p<0.05 l e v e l of s i g n i f i c a n c e . 177 TABLE 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 f e d to a n i m a l s . D i e t a r y Component (g/100 g) C a s e i n D i e t s Soy D i e t s 20% 6% 20% 6% Ca s e i n * 20.0 6.0 Soy p r o t e i n i s o l a t e * — — 20.0 6.0 D.L. me t h i o n i n e * 0.3 0.3 0.3 0.3 C o r n s t a r c h * 15.0 15.0 15.0 15.0 Sucrose 48 .77 60 .0 48 .77 60 .0 F i b r e * 7.0 7.0 7.0 7.0 V e g e t a b l e o i l 5.0 5.0 5.0 5.0 Ca f r e e m i n e r a l m i x t u r e * 3 . 5 3 . 5 3 . 5 3 . 5 V i t a m i n m i x t u r e * 1.0 1.0 1.0 1.0 C h o l i n e b i t a r t r a t e * 0.2 0.2 0.2 0.2 C a l c i u m c a r b o n a t e * * 1 . 17 1 . 17 1 . 17 1 . 17 *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. 178 R e s u l t s Both the source and l e v e l of p r o t e i n i n the d i e t , were found to have an e f f e c t on f i n a l body weig h t s and fee d e f f i c i e n c y r a t i o s (FER; Table 6.2). Animals f e d 20% soy p r o t e i n e x h i b i t e d a lower (p<0.05) f i n a l body weight and FER than 20% c a s e i n f e d a n i m a l s . A s i m i l a r t r e n d was a l s o observed i n the 6% soy v e r s u s c a s e i n f e d a n i m a l s . These r e s u l t s were not however, due to a reduced i n t a k e of the soy d i e t s compared to c a s e i n . Animals f e d the 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 (p<0.05) d e c r e a s e d f i n a l body w e i g h t , d r y m a t t e r i n t a k e and FER when compared to c o u n t e r -p a r t s f e d 20% p r o t e i n d i e t s . Plasma p r o t e i n l e v e l s were not a f f e c t e d by d i e t a r y t r e a t -ments (range 4.70 +. 0.32 to 4.91 +_ 0.16 g/dL). Plasma t o t a l c a l c i u m was d e c r e a s e d (p<0.05) i n a n i m a l s f e d the 6% p r o t e i n d i e t s compared to c o u n t e r p a r t s f e d 20% p r o t e i n ( T a b l e 6.3). C o n v e r s e l y , plasma phosphorus l e v e l s were s i g n i f i c a n t l y (p<0.05) i n c r e a s e d i n these a n i m a l s . There was no e f f e c t of d i e t a r y p r o t e i n l e v e l or s o u r c e , on plasma Mg, Na or K ( T a b l e 6.3). I n t e s t i n a l 4 0 Ca c o n t e n t (range 2.16 +_ 0.20 to 2.46 +_ 0.31 mg/loop) and 4 5 Ca s p e c i f i c a c t i v i t y (range 3.39 +_ 0.44 to 4.64 +. 0.32 dpm/ mg 4 0 C a ) were both not a f f e c t e d by d i e t a r y p r o t e i n source or l e v e l i n the d i e t . A b s o r p t i o n of 4 5 C a from the i l e a l l o o p was i n f l u e n c e d by d i e t a r y p r o t e i n source moreso, than p r o t e i n l e v e l as 4 5 C a a b s o r p t i o n from the 20% p r o t e i n d i e t s was s i m i l a r to t h a t from the 6% p r o t e i n d i e t s ( F i g u r e 6.1). Soy p r o t e i n f e d a n i m a l s absorbed s i g n i f i c a n t l y (p<0.05) l e s s 4 5 C a 179 TABLE 6.2 E f f e c t of p r o t e i n l e v e l on d i e t e f f i c i e n c y 1 D i e t I n i t i a l body F i n a l body Dry M a t t e r Feed E f f i c i e n c y wt. 2 (g) wt. 3 (g) I n t a k e (g) R a t i o 20% p r o t e i n C a s e i n 76.7 +_ 2 . 0 a Soy 78.7 +_ 2.2* 259.3 + 8.4* 225 .2 + 5 .2«> 867 + 23* 861 + 14* 0.211 ± 0.004* 0.170 + 0.004>> 6% p r o t e i n C a s e i n 81.6 +_ 1.7* Soy 88.6 +_ 2.9* 187.0 + 8.2« 159.0 + 6.3° 659 + 14»> 733 + 31b 0.160 + 0.004b 0.097 + 0.004° 1 Data are e x p r e s s e d as mean +_ SEM. 2 5 weeks of age. 3 14 weeks of age. 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 d i f f e r e n t p<0.05. 180 TABLE 6.3 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 . D i e t Ca C a ^ Mg Na K P (mg/dL) 20% p r o t e i n C a s e i n Soy 9.4 + 0.2 a 5.1 + 0.1 a 1.9 + 0.1 a 389 + 1 6 a 15.5 + 1.9 a 7.1 + 0.8 b 9.2 + 0.4 a 4.9 + 0.1 a 1.9 + 0.1 a 424 + 1 2 a 19.2 + 4.6 a 7.1 + 0.6 b 7.2 + 0.8 b 4.2 + 0.3 a 2.4 + 0.1 a 352 + 6 a 32.2 + 2.8 a 12.8 + 1.3 a 8.5 + 0 . 4 a D 4.8 + 0.2 a 2.7 + 0.2 a 346 + 1 0 a 30.8 + 2.6 a 12.5 + 1.4 a 1 Data are exp 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. Means s h a r i n g t he 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 at p<0.05. 5 0 2 0 % C 2 0 % S 6% C 6% S Experimental Diets F i g - 6.1 I n t e s t i n a l a b s o r p t i o n of * 5 C a from i l e a l f e d 20% and 6% c a s e i n and soy p r o t e i n r e s p e c t i v e l y . * denotes s i g n i f i c a n t (p<0 l o o p of r a t s i s o l a t e d i e t s , 05) d i f f e r e n c e . than c a s e i n f e d c o u n t e r p a r t s at both the 20% and 6% p r o t e i n l e v e l s . The i n t e s t i n a l 4 5 C a a b s o r p t i o n d a t a d i d not c o r r e s p o n d to the subsequent d e p o s i t i o n of r a d i o l a b e l t o the femur ( F i g u r e 6.2). A l t h o u g h the amount of 4 5 C a a c t i v i t y measured i n the femora was not d i f f e r e n t between the 20% and 6% d i e t a r y p r o t e i n groups, a n i m a l s f e d soy p r o t e i n d i e t s had a g r e a t e r femur 4 5 C a a c t i v i t y than 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 per cent dose of 45Ca i n the femur. M i n e r a l Balance Study: The 24 hour m i n e r a l b a l a n c e study d a t a are p r e s e n t e d i n T a b l e s 6.4 to 6.6. C a l c i u m i n t a k e and f e c a l e x c r e t i o n were not s i g n i f i c a n t l y d i f f e r e n t between 6% and 20% d i e t a r y p r o t e i n groups ( T a b l e 6.4). U r i n a r y c a l c i u m e x c r e t i o n was s i g n i f i c a n t l y (p<0.05) d e c r e a s e d i n anim a l s f e d the 6% p r o t e i n d i e t s i n comparison to c o u n t e r p a r t s f e d 20% p r o t e i n d i e t s . The apparent a b s o r p t i o n of Ca from the 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 the 20% p r o t e i n d i e t s . D e s p i t e these d i f f e r e n c e s , c a l c i u m b a l a n c e was s i m i l a r among the d i e t a r y t r e a t m e n t groups i n d i c a t i n g t h a t a g r e a t e r p r o p o r t i o n of absorbed c a l c i u m was 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 d i e t s . Magnesium i n t a k e was not a f f e c t e d by d i e t a r y t r e atment ( T a b l e 6.5). However, f e c a l Mg e x c r e t i o n was i n f l u e n c e d by both d i e t a r y p r o t e i n source and the l e v e l of p r o t e i n i n the d i e t . Soy p r o t e i n f e d a n i m a l s e x c r e t e d s i g n i f i c a n t l y (p<0.05) g r e a t e r amounts of Mg i n 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 at both the 20% and 6% p r o t e i n l e v e l s , r e s p e c t i v e l y . T h i s i n d i c a t e d a 183 2 0 % C 2 0 % S 6% C Experimental Diets absorbed * 5 C a to the femora of r a t c a s e i n and soy p r o t e i n i s o l a t e F i g . 6.2 D e p o s i t i o n of 20% and 6% r e s p e c t i v e l y . oo TABLE 6.4 E f f e c t of p r o t e i n l e v e l on 24 hr. c a l c i u m b a l a n c e 1 . . Ca Apparent D i e t Ca Intake U r i n e Ca Fecal Ca Ca B a l a n c e ^ Absorption" 3 (mg/day) (mg/day) (mg/day) (mg) (%) 20% p r o t e i n C a s e i n 68.75 + 1.25 a 1.11 + 0.14 a 38.95 + 3.84 a 33.46 + 2.51 a 41.11 + 1.47° Soy 66.25 + 5.54 a 0.94 + 0.04 a 39.88 + 1.46 a 28.60 + 3.11 a 40.00 + 2.12 b 6% p r o t e i n C a s e i n 61.25 + 4.27 a 0.45 + 0.04? 31.69 + 0.71 a 33.35 + 3.52 a 52.62 + 4.26 a. Soy 76.88 + 2.77 a 0.54 + 0.06 b 38.30 + 2.28 a 36.74 + 2.03 a 49.23 + 1 . 1 7 a b 1 Data are expressed as mean + SEM. ^ Ca Balance (mg) = Ca Intake (mg/day) - U r i n a r y Ca (mg/day) - F e c a l Ca (mg/day). 6 Ca Apparent A b s o r p t i o n (%) = {[Ca Intake (mg/day) - F e c a l Ca(mg /da y ) ] / C a Intake (mg/day)} x 100. 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 d i f f e r e n t at p<0.05. TABLE 6.5 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 b a l a n c e 1 . co c* Mg Apparent D i e t Mg Intake U r i n e Mg Fe c a l Mg Mg B a l a n c e ^ A b s o r p t i o n 1 3 (mg/day) (mg/day) (mg/day) (mg) (%) 20% p r o t e i n C a s e i n 6.88 + 0.12 a 1.91 + 0.03 a 1.49 + 0.10 b 3.18 + 0.31 b 71.16 + 7.08 a Soy 6.62 + 0.55 a 0.58 + 0.06 b 2.29 + 0.03 a 3.81 + 0.27 b 67.76 + 2.52 a 6% p r o t e i n C a s e i n 6.12 + 0.43 a 2.08 + 0.18 a 0.97 + 0.06?; 3.82 + 0.28 b 83.69 + 2.20 a Soy 7.69 + 0.28 a 0.75 + 0.07 b 1.76 + 0.16 b 5.38 + 0.38 a 77.53 + 2.85 a 1 Data are expressed as mean + SEM. ^ Mg Balance (mg) = Mg Intake (mg/day) - U r i n a r y Mg (mg/day) - F e c a l Mg (mg/day). d 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. 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 d i f f e r e n t a t p<0.05. r e d u c e d , but not s i g n i f i c a n t , apparent a b s o r p t i o n of Mg from soy p r o t e i n d i e t s . F u r t h e r , a n i m a l s f e d the 6% p r o t e i n d i e t s e x c r e t e d l e s s Mg i n f e c e s than c o u n t e r p a r t s f e d the 20% p r o t e i n d i e t s . Only a n i m a l s f e d the 6% soy p r o t e i n d i e t e x h i b i t e d a h i g h e r (p<0.05) Mg b a l a n c e . The r e d u c t i o n i n d i e t a r y p r o t e i n c o n t e n t w i t h the 6% p r o t e i n d i e t s r e s u l t e d i n a reduced phosphorus i n t a k e , s i n c e d i e t s were not b a l a n c e d f o r t h i s m i n e r a l ( T a b l e 6.6). T h i s r e s u l t was apparent moreso i n c a s e i n f e d a n i m a l s , than those f e d soy p r o t e i n . As a r e s u l t , a n i m a l s f e d the 6% c a s e i n d i e t e x c r e t e d s i g n i f i c a n t l y (p<0.05) l e s s phosphorus i n f e c e s than c o u n t e r p a r t s f e d 20% c a s e i n ; a s i m i l a r r e s u l t was not observed w i t h soy p r o t e i n f e d a n i m a l s however. D e s p i t e t h i s d i f f e r e n c e , apparent a b s o r p t i o n of phosphorus was not d i f f e r e n t between d i e t a r y groups. U r i n a r y phosphorus e l i m i n a t i o n however, was s i g n i f -i c a n t l y (p<0.05) reduced i n a n i m a l s f e d the 6% p r o t e i n d i e t s compared to those f e d 20% p r o t e i n . F u rthermore, a n i m a l s f e d the soy p r o t e i n d i e t s e x c r e t e d l e s s u r i n a r y phosphorus than c a s e i n f e d c o u n t e r p a r t s . The phosphorus b a l a n c e was t h e r e f o r e i n f l u -enced by both the p r o t e i n source and r e s p e c t i v e l e v e l i n the d i e t . For example, a n i m a l s f e d soy p r o t e i n d i e t s r e t a i n e d more phosphorus than c a s e i n f e d c o u n t e r p a r t s . A l s o , phosphorus b a l a n c e was d e c r e a s e d i n the 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 comparison to those f e d 20% p r o t e i n . 187 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 D i e t P Intake (mg/day) Ur i n e P (mg/day) Fecal P (mg/day) P B a l a n c e ' (mg) P Apparent A b s o r p t i o n 3 (*) 20% p r o t e i n C a s e i n Soy 6% p r o t e i n C a s e i n Soy 79.48 + 1.44 84.38 + 5.48 58.12 + 2.06° 72.88 + 2.45 a 21.74 + 1.05 a 17.82 + 1.36 a 13.45 + 1.76 12.52 + 1.011 23.61 + 2.18 a 22.18 + 1.31 a 16.26 + 1.45° 26.76 + 1.79 a 34.12 + 3.54 44.38 + 4.39 ab a 28.41 + 2.89 33.61 + 2.71 ab 68.40 + 3.00 c 72.52 + 2.24 £ 70.23 + 3.56 a 62.54 + 3.50 a 1 Data a r e expressed 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). J 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 Intake (mg/day)} x 100. 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 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 weight and m i n e r a l i z a t i o n parameters were s i g n i f i c a n t l y a f f e c t e d by both d i e t a r y p r o t e i n source and l e v e l of p r o t e i n i n t a k e ( T a b l e 6.7). Animals f e d the 6% p r o t e i n d i e t s had s i g n i f i c a n t l y (p<0.05) d e c r e a s e d femur ash w e i g h t s , and c a l c i u m and magnesium c o n t e n t s , i n comparison t o those f e d 20% p r o t e i n . Femur Ca/P r a t i o was not a f f e c t e d by d i e t a r y p r o t e i n l e v e l . Bone P h y s i c a l and B i o m e c h a n i c a l Parameters: Femur d r y weight and l e n g t h were s i g n i f i c a n t l y (p<0.05) de c r e a s e d i n ani m a l s f e d the 6% p r o t e i n d i e t s compared to those f e d 20% p r o t e i n d i e t s ( T a b l e 6.8). The o v e r a l l r e d u c t i o n i n bone s i z e observed i n a n i m a l s f e d the 6% p r o t e i n d i e t s was a r e s u l t of r e t a r d e d a n i m a l growth c h a r a c t e r i s t i c s as i n f l u e n c e d by the reduced p r o t e i n i n t a k e of these a n i m a l s . T h i s was observed when femur ash w e i g h t , e x p r e s s e d as a per cent of f i n a l body weight (range 0.087 +. 0.003 to 0.092 ±_ 0.002 % F i n a l body w e i g h t ) showed no d i f f e r e n c e between l e v e l s of d i e t a r y p r o t e i n i n t a k e . Bone biomechanics were i n f l u e n c e d by both the d i e t a r y p r o t e i n source as w e l l as the p r o t e i n l e v e l i n the d i e t ( T a b l e 6.8). At both the 20% and 6% d i e t a r y p r o t e i n l e v e l s , a n i m a l s f e d soy p r o t e i n had lower femur bending f a i l u r e energy v a l u e s than those of c a s e i n f e d c o u n t e r p a r t s . Moreover, an i m a l s f e d the 6% p r o t e i n d i e t s had s i g n i f i c a n t l y (p<0.05) d e c r e a s e d femur bending f a i l u r e e n e r g i e s compared t o 20% p r o t e i n f e d a n i m a l s . Femur maximum bending s t r e s s was reduced i n 6% soy p r o t e i n f e d animals 189 moreso than 6% c a s e i n f e d a n i m a l s when compared t o the 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 E f f e c t of p r o t e i n l e v e l on femur m i n e r a l i z a t i o n 1 . D i e t Ash wt. Ca Ca/P Mg (g) (mg/bone) r a t i o (mg/bone) 20% p r o t e i n C a s e i n 0.237 +. 0.006* 94.60 +_ 3.85* 2.00 ± 0.01* 1.84 +_ 0.06* Soy 0.206 +_ 0.006«> 78 . 75 +. 3.21«> 1.97 +. 0.05* 1.62 + 0.07* 6% p r o t e i n C a s e i n 0.158 + 0.004° 63.90 +_ 2.22" 2.10 + 0.05* 1.33 +_ 0.03» Soy 0.146 +. 0.008" 58.03 +_ 4.58« 2.00 +_ 0.03* 1.20 +_ 0.09»> 1 Data are ex p r e s s e d as mean +_ SEM. 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 d i f f e r e n t at p<0.05. 191 TABLE 6.8 E f f e c t of p r o t e i n l e v e l on b i o m e c h a n i c a l p a r a m e t e r s 1 . femur p h y s i c a l and Bending F a i l u r e Maximum D i e t Dry wt. Length Energy Bending S t r e s s (g) (mm) (x 10 -2 j ) (N/mm2) 20% p r o t e i n C a s e i n 0.387 +_ 0.010° 30.61 +_ 0.47 a 9.24 + 0.93° 68.94 +_ 3.06 a Soy 0.360 +_ 0.009 a 30.49 +_ 0.21 a 6.12 +_ 0.60 b 65.82 +_ 1.45 a 6% p r o t e i n C a s e i n 0.295 +_ 0.008 b 27.82 +. 0.33 b 4.68 + 0.44 b c 63.96 +. 3.70 a Soy 0.266 ±_ 0.017 b 27.07 +_ 0.58 b 3.56 +. 0.34« 56.37 +_ 1.38 b 1 Data are e x p r e s s e d as mean +_ SEM. 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 d i f f e r e n t at p<0.05. 192 D i s c u s s i o n A d i e t which i s low i n p r o t e i n i s c o n s e q u e n t l y a l s o low i n phosphorus, i f the phosphorus l e v e l i s not compensated f o r . The poor p a l a t a b i l i t y of s y n t h e t i c d i e t s d e f i c i e n t i n phosphorus, r e p o r t e d by o t h e r s ( L o t z e_t_ a i . , 1968), l i k e l y c o n t r i b u t e d to the reduced f e e d i n t a k e , body weight g a i n e d and FER, observed i n r a t s f e d the 6% p r o t e i n d i e t s . D e s p i t e b a l a n c i n g the d i e t s f o r c a l c i u m c o n t e n t , the l o n g t e r m reduced fe e d i n t a k e of a n i m a l s f e d the 6% p r o t e i n d i e t s , r e s u l t e d i n an e x p e r i m e n t a l l y induced c a l c i u m d e f i c i e n c y , as demonstrated i n these a n i m a l s , by reduced plasma t o t a l c a l c i u m and i n c r e a s e d plasma phosphorus l e v e l s ( P a t t and L u c k h a r d t , 1942). C a l c i u m d e f i c i e n c y i s compensated f o r , by a l t e r a t i o n s i n the r e g u l a t i o n of c a l c i u m m e t a b o l i s m i n the k i d n e y s , bone and i n t e s t i n e , as i n f l u e n c e d by p a r a t h y r o i d hormone (PTH) s e c r e t i o n and v i t a m i n D m etabolism (Aurbach, 1988). Thus, i t i s s u s p e c t e d t h a t PTH mediated bone r e s o r p t i o n i n these a n i m a l s , r e s u l t e d i n a r e l e a s e of both c a l c i u m and phosphorus i n t o plasma, l e a d i n g t o a h i g h e r plasma phosphate c o n c e n t r a t i o n . The m o b i l i z a t i o n of phosphate, c o u p l e d w i t h c o n s e r v a t i o n of absorbed d i e t a r y phosphate, r e f l e c t e d the f a c t t h a t u r i n a r y and f e c a l e x c r e t i o n of phosphate were reduced i n a n i m a l s f e d the 6% p r o t e i n d i e t s . N o r m a l l y , an excess of plasma phosphate i s e v e n t u a l l y compensated f o r by an i n c r e a s e d r e n a l phosphorus e x c r e t i o n mediated by PTH (Agus e_t al_. , 1981). D e s p i t e d i f f e r e n c e s i n c a l c i u m h o m e o s t a s i s , i l e a l ( p a r a -c e l l u l a r ) a b s o r p t i o n of 4 5 C a was not d i f f e r e n t between animals 193 f e d the two d i f f e r e n t l e v e l s of d i e t a r y p r o t e i n . T h i s r e s u l t was c o n t r a r y t o the g r e a t e r apparent a b s o r p t i o n of c a l c i u m i n anim a l s f e d the 6% p r o t e i n d i e t s i n the ba l a n c e s t u d y , which r e f l e c t e d a b s o r p t i o n a l o n g the e n t i r e i n t e s t i n a l t r a c t . The absence of an i n c r e a s e i n 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 e f f i c i e n c y by the 6% p r o t e i n f e d a n i m a l s , was l i k e l y due to a decrease i n c i r c u l a t i n g c a l c i t r i o l l e v e l s i n c h r o n i c c a l c i u m d e f i c i e n c y (Rader et a1. . 1979). P r e v i o u s workers have suggested t h a t c y c l i c AMP (cAMP) mediates the PTH s t i m u l a t i o n of r e n a l 1 - a - h y d r o x y l a s e . F u r t h e r , the i n f l u e n c e of PTH on cAMP was shown to be i m p a i r e d i n c a l c i u m d e f i c i e n t r a t s due to a depre s s e d response of t a r g e t c e l l s exposed to e l e v a t e d l e v e l s of PTH, hy p o c a l c e m i a f o r an extended p e r i o d of t i m e , or an i n t e r a c t i o n between these two c o n d i t i o n s ( H o r i u c h i et al_. , 1977 ; Carnes e_t a l . , 1978). Rader et a l . (1979) h y p o t h e s i z e d t h a t reduced r e n a l f o r m a t i o n of cAMP by s e v e r e l y h y p o c a l c e m i c r a t s may decrease 1-a-hydroxylase a c t i v i t y and t h e r e f o r e i m p a i r c a l c i t r i o l s y n t h e s i s . These e f f e c t s , i n t u r n , would i n h i b i t the u s u a l i n c r e a s e i n e f f i c i e n c y of the a c t i v e , t r a n s c e l l u l a r v i t a m i n D-dependent component of 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 r e p o r t e d i n c a l c i u m d e f i c i e n t s u b j e c t s ( B r o n n e r , 1987). Thus, as a r e s u l t of s i m i l a r l u m i n a l c a l c i u m c o n c e n t r a t i o n s , a n i m a l s f e d the 6% p r o t e i n d i e t s absorbed e q u i v a l e n t p r o p o r t i o n s of c a l c i u m from the lower s m a l l i n t e s t i n e compared to the 20% p r o t e i n f e d a n i m a l s . D e s p i t e the s i m i l a r f e e d , and t h e r e f o r e c a l c i u m , i n t a k e s of an i m a l s f e d the 6% and 20% p r o t e i n d i e t s , r e s p e c t i v e l y , d u r i n g 194 the 24 hr b a l a n c e s t u d y p e r i o d , the d a t a r e f l e c t the l o n g t e r m i n t a k e d e f i c i e n c i e s of the 6% p r o t e i n groups. The decrease i n u r i n a r y c a l c i u m e x c r e t i o n of a n i m a l s f e d the 6% p r o t e i n d i e t s i n d i c a t e s a p o s s i b l e PTH mediated enhancement of r e n a l t u b u l a r c a l c i u m r e a b s o r p t i o n r e p o r t e d w i t h a c a l c i u m d e f i c i e n c y (Agus et  a1 . . 1981). Renal c o n s e r v a t i o n of e x t r a c e l l u l a r c a l c i u m o c c u r s because PTH a c t s on the d i s t a l t u b u l e s to i n c r e a s e r e a b s o r p t i o n of c a l c i u m from the g l o m e r u l a r f i l t r a t e . The g r e a t e r c a l c i u m apparent a b s o r p t i o n and s i m i l a r c a l c i u m b a l a n c e of a n i m a l s f e d 6% p r o t e i n i n comparison to 20% p r o t e i n , suggests r e t e n t i o n of a g r e a t e r p r o p o r t i o n of absorbed c a l c i u m due to r e n a l c o n s e r v a t i o n . The c a l c i u m b a l a n c e s t u d y extends the 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 d a t a , which showed no i n c r e a s e i n p a r a c e l l u l a r c a l c i u m t r a n s p o r t i n c a l c i u m d e f i c i e n c y . A l t e r n a t i v e l y , o t h e r workers have i n d i c a t e d t h a t the c o l o n may have an i n c r e a s e d r o l e i n c a l c i u m a b s o r p t i o n i n c a l c i u m d e f i c i e n c y (Favus et a l . . 1980), which was not d e t e c t e d h e r e i n u s i n g a l i g a t e d i l e a l l o o p t e c h n i q u e . The presence of a p r o t e i n source e f f e c t on i l e a l c a l c i u m a b s o r p t i o n i n the 6% p r o t e i n f e d a n i m a l s i n d i c a t e d t h a t an enhancing e f f e c t of the c a s e i n p o s t - d i g e s t i o n phosphopeptides (CPP) was s t i l l a p p a r e n t . Thus, the a b i l i t y of CPP to enhance 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 by i n c r e a s i n g the p r o p o r t i o n of s o l u b l e c a l c i u m i n the l u m i n a l c o n t e n t s (Sato et a1. . 1983) was r e t a i n e d by f e e d i n g a lower (6%) c a s e i n p r o t e i n d i e t . The t r a n s l o c a t i o n of absorbed 4 5 C a to the femora was however, 195 i n v e r s e l y r e l a t e d t o the i n t e s t i n a l a b s o r p t i o n d a t a . The 4 5 C a s p e c i f i c a c t i v i t y of the femora was i n c r e a s e d i n a n i m a l s f e d the 20% soy d i e t as w e l l those f e d the 6% p r o t e i n d i e t s , secondary to the d e c r e a s e d bone c a l c i u m c o n t e n t s of these a n i m a l s . S i n c e o n l y 1 hr was a l l o w e d f o r * 5 C a d e p o s i t i o n i n t o the femora to o c c u r , i t i s l i k e l y t h a t d e p o s i t i o n , r e f l e c t s o n l y an exchange of 4 5 C a w i t h 4 0 C a on the s u r f a c e of the bone. T h i s r e s u l t was a l s o observed i n the l a c t o s e ( e x p e r i m e n t 1) s t u d y , and f u r t h e r c o n f i r m s t h a t the 4 5 C a d e p o s i t i o n t o femur measurement s h o u l d not be c o n s i d e r e d as a t r u e index of u t i l i z a t i o n i n acute a b s o r p t i o n s t u d i e s . Magnesium b a l a n c e was not a l t e r e d s i g n i f i c a n t l y i n t h i s s t u d y , r e g a r d l e s s of the f a c t t h a t u r i n a r y Mg e x c r e t i o n was h i g h e r i n both 6% and 20% c a s e i n f e d a n i m a l s , compared to soy f e d c o u n t e r p a r t s . CPP has been observed to i n f l u e n c e the b i o a v a i l -a b i l i t y of m i n e r a l s o t h e r than c a l c i u m ( M e l l a n d e r , 1963); t h u s , CPP c o u l d f e a s i b l y enhance the b i o a v a i l a b i l i t y of magnesium from the c a s e i n d i e t s i n the lower s m a l l i n t e s t i n e . A g r e a t e r p a r a c e l l u l a r a b s o r p t i o n of magnesium i n c a s e i n f e d anim a l s would e x p l a i n the i n c r e a s e i n u r i n a r y Mg e x c r e t i o n observed i n these a n i m a l s . 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 l i m i t e d t o the lower s m a l l i n t e s t i n e , may have p r e c l u d e d d e t e c t i o n i n the b a l a n c e s t u d y . Phosphorus b a l a n c e was i n f l u e n c e d by both the source of d i e t a r y p r o t e i n as w e l l as the l e v e l i n the d i e t . I n t a k e of phosphorus was d e c r e a s e d moreso i n the anim a l s f e d 6% c a s e i n , than 6% soy, a l i k e l y r e s u l t of the h i g h e r phosphorus content of 196 soy p r o t e i n s o u r c e s (Zemel, 1988). The lower p r o t e i n l e v e l , c o u p l e d w i t h d e c r e a s e d feed i n t a k e i n the 6% p r o t e i n f e d a n i m a l s caused a r e l a t i v e phosphorus d e f i c i e n c y i n these a n i m a l s . T h i s i s r e f l e c t e d i n the h y p o p h o s p h a t u r i a , d e c r e a s e d f e c a l phosphorus e x c r e t i o n and phosphorus b a l a n c e of a n i m a l s f e d the 6% p r o t e i n d i e t s . Phosphorus apparent a b s o r p t i o n was not a f f e c t e d by d i e t a r y p r o t e i n t r e a t m e n t s however. C o n s e r v a t i o n of both c a l c i u m and phosphorus i s n e c e s s a r y f o r bone me t a b o l i s m as i n f l u e n c e d by c o l l a g e n s y n t h e s i s and bone m i n e r a l i z a t i o n (Grey, 1974). I t i s noteworthy t h e r e f o r e , t h a t the p h y s i c a l parameters and m i n e r a l i z a t i o n of the femora from a n i m a l s f e d the 6% p r o t e i n d i e t s were low compared to the 20% p r o t e i n f e d a n i m a l s . P r o t e i n d e f i c i e n c y has a l s o been r e p o r t e d to c o n t r i b u t e to d e c r e a s e d bone l e n g t h i n s t u d i e s w i t h humans (Adams and B e r r i d g e , 1969; P a r f i t t , 1983); s i m i l a r to the r e s u l t s o b t a i n e d h e r e i n w i t h a r a t animal model. Moreover, o t h e r workers have observed the acute i n h i b i t i o n of bone m i n e r a l i z a t i o n i n phosphorus d e f i c i e n c y ( B r u i n et a1.. 1975). In the p r e s e n t s t u d y , a n i m a l s f e d the 6% p r o t e i n d i e t s had lower femur c a l c i u m and magnesium c o n t e n t s . The Ca/P r a t i o of the femora however, was not a d v e r s e l y a f f e c t e d by d i e t a r y t r e a t m e n t l i k e l y due to d e f i c i e n c i e s of both c a l c i u m and phosphorus i n these a n i m a l s . A decrease i n femur b i o m e c h a n i c a l bending f a i l u r e energy i n a n i m a l s f e d d i e t s low i n p r o t e i n , i n d i c a t e d a reduced amount of work needed to break these bones. The reduced amount of work energy, c o u p l e d w i t h the d e c r e a s e d p h y s i c a l s i z e and m i n e r a l 197 c o n t e n t of these femora, were l i k e l y a r e s u l t of reduced bone growth as i n f l u e n c e d by the r e t a r d e d growth c h a r a c t e r i s t i c s of these a n i m a l s . Femur maximum bending s t r e s s was a l s o d e c r e a s e d i n a n i m a l s f e d the 6% p r o t e i n d i e t s , a l b e i t the d i f f e r e n c e was s i g n i f i c a n t i n o n l y the 6% soy f e d a n i m a l s . T h i s r e s u l t may be e x p l a i n e d by the s l i g h t l y reduced bone s i z e and m i n e r a l i z a t i o n of femora from the 6% soy group of a n i m a l s compared to 6% c a s e i n f e d c o u n t e r p a r t s . Taken t o g e t h e r , these d a t a support the concept t h a t a d i e t l i m i t e d i n p r o t e i n and phosphorus i s c o n t r a i n d i c a t e d f o r the young and e l d e r l y , a l i k e (Shahani and Kaup, 1989). 198 C o n c l u s i o n s C a l c i u m b i o a v a i l a b i l i t y and the subsequent u t i l i z a t i o n of c a l c i u m i n c a l c i u m homeostasis as indexed by i s o t o p i c i n t e s t i n a l a b s o r p t i o n m e t h o d o l o g i e s , b a l a n c e s t u d i e s as w e l l as endpoint measurements of bone 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 s t r e n g t h were d e t e r m i n e d . The p r e c e d i n g e xperiments have i n v e s t i g a t e d the r o l e of the c h e m i s t r y of 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 p r o d u c t s , t h e i r l e v e l i n the d i e t , as w e l l as the e f f e c t s of ther m a l p r o c e s s i n g on c a l c i u m a b s o r p t i o n and b i o a v a i l a b i l i t y . F u r t h e r , the r e l e v a n c e of these e f f e c t s on c a l c i u m h o m e o s t a s i s as i t r e l a t e s t o bone metabolism and o s t e o p o r o s i s was d i s c u s s e d . The h i g h b i o a v a i l a b i l i t y of c a l c i u m from d a i r y p r o d u c t s , and m i l k i n p a r t i c u l a r , i s due to the presence of c o - n u t r i e n t s which enhance the i n t e s t i n a l t r a n s p o r t of d i e t a r y c a l c i u m . L a c t o s e enhancement of c a l c i u m a b s o r p t i o n was shown to be due to an o p t i m a l c o n c e n t r a t i o n of l a c t o s e i n the lumen which was a c h i e v e d w i t h o n l y the h i g h (50%) l a c t o s e d i e t compared to c o n t r o l and 20% l a c t o s e c o n t a i n i n g d i e t s . F u r t h e r , the unchanged bone m i n e r a l c o n t e n t , but reduced bone s t r e n g t h observed i n a n i m a l s f e d the 50% l a c t o s e d i e t s , suggests t h a t m a l a b s o r p t i o n has a g r e a t e r e f f e c t on bone metabolism than the enhanced i n t e s t i n a l t r a n s p o r t of c a l c i u m observed i n these a n i m a l s . The r e l a t i v e l y low l a c t o s e c o n t e n t of d a i r y foods ( 5 % i n f l u i d m i l k ) would make o n l y a s m a l l c o n t r i b u t i o n t o 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 f o r humans. These f i n d i n g s are r e l e v a n t to o s t e o p o r o t i c i n d i v i d u a l s who are l a c t o s e i n t o l e r a n t . 199 The - importance of the d i e t a r y p r o t e i n component to 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 was e l u c i d a t e d i n s t u d i e s c o n c e r n i n g the e f f e c t s of f i r s t , v a r i o u s d a i r y p r o t e i n s o u r c e s ; s e c o n d l y , c a s e i n p r o t e i n s v e r s u s a soy p l a n t p r o t e i n source w i t h v a r y i n g l e v e l s of d i e t a r y c a l c i u m ; t h i r d l y , d i e t a r y f o r t i f i c a t i o n w i t h c a s e i n phosphopeptides (CPP); f o u r t h , t h e r m a l p r o c e s s i n g of d i e t a r y p r o t e i n s ; and f i n a l l y , the l e v e l of p r o t e i n i n the d i e t on c a l c i u m b i o a v a i l a b i l i t y and u t i l i z a t i o n . In s e l e c t e d s t u d i e s , the o s t e o p o r o s i s prone, 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) r a t a n i mal model was used because i t s d i s t u r b e d c a l c i u m metabolism c o u l d h i g h l i g h t changes i n c a l c i u m b i o a v a i l a b i l i t y and u t i l i z -a t i o n due to d i e t a r y t r e a t m e n t s . D i e t a r y p r o t e i n s o u r c e , and i n p a r t i c u l a r the c a s e i n f r a c t i o n of d a i r y p r o t e i n s was found to a f f e c t i l e a l c a l c i u m t r a n s p o r t i n W i s t a r r a t s , p o t e n t i a l l y due to the f o r m a t i o n of c a l c i u m s e q u e s t e r i n g b i o a c t i v e p e p t i d e s (CPP). The enhancing e f f e c t of c a s e i n p r o t e i n s on c a l c i u m a b s o r p t i o n was found t o have l i t t l e e f f e c t on the l o n g t e r m bone m i n e r a l i z a t i o n i n a n i m a l s f e d an adequate l e v e l of c a l c i u m . The l e v e l of d i e t a r y c a l c i u m was found t o i n f l u e n c e the b i o a v a i l a b i l i t y and u t i l i z a t i o n of c a l c i u m from c a s e i n and soy p r o t e i n d i e t s . Both SHR and WKY a n i m a l s f e d the adequate and low l e v e l s of c a l c i u m i n c a s e i n 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 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 . Femur c a l c i u m c o n t e n t appeared to be i n f l u e n c e d by d i e t a r y p r o t e i n source i n SHR a n i m a l s f e d the h i g h and adequate d i e t a r y l e v e l s of c a l c i u m . Femur biomechanics 200 were i n f l u e n c e d by the low l e v e l of d i e t a r y c a l c i u m moreso than d i e t a r y p r o t e i n s o u r c e . To determine i f the b i o a c t i v e d i g e s t i o n p e p t i d e s from t r y p t i c d i g e s t i o n of c a s e i n (CPP) i n f l u e n c e d c a l c i u m a b s o r p t i o n from c a s e i n d i e t s o b s e r v e d i n experiments 2 apd 3, the e f f e c t of d i e t a r y f o r t i f i c a t i o n w i t h CPP was i n v e s t i g a t e d . In p r e l i m i n a r y i n v i t r o s t u d i e s , the b i n d i n g of c a l c i u m to c a s e i n d i g e s t i o n phosphopeptides was c o n f i r m e d . D i e t a r y f o r t i f i c a t i o n w i t h CPP r e s u l t e d i n enhanced i l e a l c a l c i u m a b s o r p t i o n i n c a s e i n and soy f e d a n i m a l s . However, t h i s i n c r e a s e i n absorbed c a l c i u m by c a s e i n f e d a n i m a l s was not u t i l i z e d i n bone m i n e r a l i z a t i o n or b i o m e c h a n i c s , but r a t h e r , was e x c r e t e d . The l e v e l of CPP f o r t i f i c a t i o n was not g r e a t enough, or the p o t e n t i a l e f f e c t s u f f i c i e n t l y s u s t a i n e d , to s i g n i f i c a n t l y a f f e c t bone m i n e r a l -i z a t i o n of soy f e d a n i m a l s . The d i g e s t i v e p r o d u c t i o n of the b i o a c t i v e CPP may be i n f l u e n c e d by the t h e r m a l p r o c e s s i n g of d a i r y p r o d u c t s , t h u s , the e f f e c t s of a severe heat t r e a t m e n t on d i e t a r y c a l c i u m b i o a v a i l -a b i l i t y and u t i l i z a t i o n were i n v e s t i g a t e d . In v i t r o s t u d i e s i n d i c a t e d t h a t the d i g e s t i b i l i t y of the heat t r e a t e d d i e t a r y p r o t e i n s was markedly re d u c e d , which i n t u r n would d e c r e a s e the y i e l d of d i g e s t i v e p e p t i d e s , such as CPP. In v i v o s t u d i e s f u r t h e r showed t h a t heat d e n a t u r a t i o n of d i e t a r y p r o t e i n s r e s u l t e d i n reduced c a l c i u m a b s o r p t i o n , c a l c i u m b a l a n c e and bone m i n e r a l i z a t i o n secondary to the n u t r i e n t m a l a b s o r p t i o n e x p e r i e n c e d by these a n i m a l s . The e f f e c t s of m a l a b s o r p t i o n on 201 bone b i o m e c h a n i c a l parameters c o n f i r m e d those observed i n the l a c t o s e experiment (experiment 1) above. A f i n a l v a r i a b l e to the p r o d u c t i o n of b i o a c t i v e c a s e i n d i g e s t i o n p e p t i d e s , i s the l e v e l of p r o t e i n i n the d i e t . In t h i s s t u d y , the e f f e c t of 6% and 20% p r o t e i n d i e t s on c a l c i u m b i o a v a i l a b i l i t y was d e termined i n c a s e i n and soy f e d a n i m a l s . The p r o p o r t i o n of c a l c i u m absorbed from the d i e t s was s i m i l a r between the two p r o t e i n l e v e l s i n d i c a t i n g r e t e n t i o n of the p o s i t i v e e f f e c t of c a s e i n on c a l c i u m b i o a v a i l a b i l i t y at a lower d i e t a r y l e v e l . The r e d u c t i o n s i n amount of c a l c i u m absorbed, c a l c i u m b a l a n c e and bone m i n e r a l i z a t i o n of the 6% p r o t e i n f e d a n i m a l s were due to the c h r o n i c a l l y c a l c i u m d e f i c i e n t s t a t e of these a n i m a l s . Femur biomechanics were l i k e w i s e reduced i n a n i m a l s f e d the 6% p r o t e i n d i e t s as a r e s u l t of d e c r e a s e d d i e t a r y f a c t o r s a v a i l a b l e f o r use i n bone metabolism. Thus, d e c r e a s e d d i e t a r y p r o t e i n and phosphorus are d e t r i m e n t a l to c a l c i u m m e t a b o l i s m , as i t p e r t a i n s t o bone me t a b o l i s m . In c o n c l u s i o n , c a l c i u m b i o a v a i l a b i l i t y was shown to be enhanced by l a c t o s e at h i g h l e v e l s of d i e t a r y i n t a k e o n l y . As w e l l , i l e a l c a l c i u m a b s o r p t i o n was enhanced i n a n i m a l s f e d d i e t s c o n t a i n i n g c a s e i n . T h i s r e s u l t was l i k e l y i n f l u e n c e d by the i n t r a l u m i n a l presence of c a s e i n d i g e s t i o n phosphopeptides (CPP) which s e q u e s t e r c a l c i u m and r e t a i n i t i n a s o l u b l e s t a t e f o r a b s o r p t i o n . D i e t a r y f o r t i f i c a t i o n w i t h CPP enhanced c a l c i u m a b s o r p t i o n from a soy p r o t e i n d i e t , a l b e i t the e f f e c t was not extended to bone c a l c i f i c a t i o n at the l e v e l of f o r t i f i c a t i o n 202 used. Thermal p r o c e s s i n g of c a s e i n l e d to a r e d u c t i o n i n p r o t e i n d i g e s t i b i l i t y as w e l l as l o s s of the c a s e i n e f f e c t on c a l c i u m a b s o r p t i o n . The l e v e l 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 enhancing e f f e c t of c a s e i n on c a l c i u m b i o a v a i l a b i l i t y . The experiments h e r e i n , u s i n g both acute i n t e s t i n a l a b s o r p t i o n of i s o t o p i c c a l c i u m , as w e l l as b a l a n c e data i n d i c a t e t h a t c a l c i u m t r a n s p o r t from a s p e c i f i c segment of the i n t e s t i n e may not r e f l e c t the a b s o r p t i o n of c a l c i u m from the e n t i r e i n t e s t i n a l t r a c t . 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In s i t u s t u d i e s of c a l c i u m a b s o r p t i o n i n r a t s . Am. J . P h y s i o l . 220:1261-1265. 222 A p p e n d i x 223 POINT o f FRACTURE 4 5 TIME (seo T i m e - f o r c e d e f o r m a t i o n c u r v e o f 3 - p o i n t b e n d i n g o f b u n t i l f r a c t u r e o c c u r r e d . TABLE 1. I n t e r a c t i o n between a n i m a l s t r a i n and c a l c i u m i n t a k e i n femur u t i l i z a t i o n . Source of Sum of Degrees of Mean F-value P-value V a r i a t i o n Squares Freedom Square W i t h i n cases 6719.54 61 110 .16 Anima1 s t r a i n 1817.36 1817.36 16 . 50 0.000 C a l c i u m l e v e l 37536.43 18768.22 170.38 0.000 Animal x C a l c i u m 1613.93 806 .96 7 . 33 0 .001 225 TABLE 2. I n t e r a c t i o n between p r o t e i n source and c a l c i u m l e v e l i n femur u t i l i z a t i o n . Source of Sum of Degrees of Mean V a r i a t i o n Squares Freedom Square F-value P-value W i t h i n cases 7396 .83 61 121 .26 P r o t e i n source 36 .17 36 .17 0.30 0.587 C a l c ium l e v e l 37991.53 18995.77 156.65 0.000 P r o t e i n x Ca 1c ium 2286.36 1143.18 9.43 0.000 226 TABLE 3. I n t e r a c t i o n between animal s t r a i n and c a l c i u m l e v e l i n t i b i a magnesium c o n t e n t . Source of Sum of Degrees of Mean V a r i a t i o n Squares Freedom Square F-value P-value W i t h i n cases 4 .77 54 0.09 Animal s t r a i n 1 . 17 1 . 17 13.26 0.001 Ca1c ium l e v e l 4.36 2 . 18 24.68 0.000 Animal x Ca1c ium 2 .33 1 .16 13 .17 0.000 227 TABLE 4. I n t e r a c t i o n between animal s t r a i n and p r o t e i n heat t r e a t m e n t i n magnesium b a l a n c e . Source of Sum of Degrees of Mean V a r i a t i o n Squares Freedom Square F-value P-value W i t h i n cases 4 .00 24 0 . 17 Animal s t r a i n 0.01 0.01 0 .08 0 .774 P r o t e i n heat t r e a t m e n t 30.98 10.33 62 .04 0 .000 Animal x P r o t e i n Tmt. 2.92 0.97 5.85 0.004 228 

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